Name: an electrophysiology protocol to measure reward anticipation and processing in children
Uploaded: 2018-10-04T14:30:00.000+00:00
Duration: 5 min 4 s
Description: Watch this Scientific Journal Video about An Electrophysiology Protocol to Measure Reward Anticipation and Processing in Children at JoVE.com

<p class="jove_content">We thank all the children and families who participated in the protocols described. Publishing fees were paid by Brain Products.</p>

<p class="jove_content">Procedures involving human participants were approved by the Human Subject Research Ethics Committee/Institutional Review Board at University of California, Riverside and University of California, San Diego.</p>
<p class="jove_title">1. Stimuli Preparation</p>
<p class="jove_content">Note: The procedures described below are specific to a commercially available editing suite (see <strong>Table of Materials</strong>); however, other photo editing software can certainly be used.</p>
<ol>
	<li>Prepare a set of emotional face photographs<sup class="xref">25</sup> categorized into two facial expressions (happy and sad). Using previously collected behavioral ratings, choose photographs with highly accurate emotion ratings (in which over 80% of participants identified the emotion correctly)<sup class="xref">25</sup>.<br />
	NOTE: In our protocol, there were 33 chosen adult faces (18 female, 15 male). Of the 18 females, 9 are Caucasian, 4 are African American, and 5 are Asian American. Of the 15 males, 9 are Caucasian, 6 are African American, and none are Asian American. In this case, photos have already been cropped and placed on a white background. It is important to note that the NimStim set of facial expressions was rated and normed by adults<sup class="xref">25</sup>. Thus, it is possible that children or adolescents may perceive facial expressions differently than the adults who normed the images.</li>
	<li><strong>Standardizing the emotional expression stimuli</strong>
	<ol>
		<li>Using a photo editing suite, edit the photographs to be in grayscale, then save them (in the software used here, this is done by selecting <strong>Image | Mode | Greyscale</strong>).</li>
		<li>Make sure that the resolution is 72 pixels/inch and that the file width is 8.5 inches and height is 11 inches.</li>
		<li>Using a ruler, change the width of the photograph (in pixels) until the face measures 11 cm from the outside of the hair on one side to the outside of hair on the other side, and 14 cm from the start of hairline to the bottom of the chin.</li>
	</ol>
	</li>
	<li><strong>Creating the scrambled face arrow stimuli</strong>
	<ol>
		<li>Download the &#34;scramble&#34; plugin (http://telegraphics.com.au/sw/product/Scramble).</li>
		<li>Extract the ZIP and move it into &#34;plug-ins&#34; inside the editing suite&#39;s application folder.</li>
		<li>In the photo editing program, create a &#34;custom shape&#34; in the <strong>shape</strong> options. Make sure that the shape is an arrow.</li>
		<li>Open the emotional face images one at a time. Use the selection tool to select only the face and not the background.</li>
		<li>Select <strong>Filter | Telegraphics | Scramble | OK</strong>.</li>
		<li>Select <strong>Window | Layers</strong> (this should produce a &#34;layers&#34; window to the side).</li>
		<li>Double click <strong>background</strong>, then click <strong>ok</strong> and rename it to <strong>layer 0</strong> (any name is fine).</li>
		<li>Right click on the <strong>shape</strong> tool in the left-hand menu bar and choose the custom arrow shape.</li>
		<li>Drag the arrow shape into the middle of the scrambled image.</li>
		<li>Use <strong>Ctrl + T</strong> to drag the arrow to the middle of the image and adjust the size so it is the same as the face image (<em>e.g., </em>11 x 14 cm).</li>
		<li>Use the angle tool to ensure that for happy faces the arrow faces upwards and for sad faces the arrow faces downwards.</li>
		<li>Click <strong>layer 0</strong> or the name given in step 1.3.7.</li>
		<li>Select <strong>Layers | Group</strong> with previous.</li>
		<li>Click <strong>Select-all | Edit | Copy merged</strong>.</li>
		<li>Create a new file with the arrow on a white background by selecting <strong>File | New</strong>. The new file should be 8.5 x 11 inches at a resolution of 72 pixels/inch.</li>
		<li>Save the files.<br />
		NOTE: After these are saved, there should be photographs of individuals with sad expressions, happy expressions, and the scrambled versions. The scrambled photograph with the upwards pointing arrow is the nonsocial image for happy, and the scrambled photograph with the downwards pointing arrow is the nonsocial image for sad.</li>
	</ol>
	</li>
	<li><strong>Creating the reward images</strong>
	<ol>
		<li>Find an image of a goldfish cracker (without anything else in the frame) and download it to the computer.</li>
		<li>Open the goldfish cracker image in a photo editing suite and edit it to be in grayscale.</li>
		<li>Create two goldfish cracker images: 1) one in grayscale and 2) one that is crossed out (<em>e.g., </em>has a surrounding circle/oval with a line through the middle).</li>
		<li>Using <strong>Copy | Paste</strong>, place images of the intact goldfish cracker around the happy images (<em>e.g., </em>both the upwards pointing arrow and smiling people).</li>
		<li>Using <strong>Copy | Paste</strong>, place images of the crossed-out goldfish cracker around the sad images (<em>e.g., </em>both the downwards pointing arrow and frowning people).</li>
	</ol>
	</li>
	<li><strong>Preparing the stimuli in electrophysiology (EEG) presentation software</strong>
	<ol>
		<li>Create two blocks of stimuli in an EEG presentation software package. One block will be social (<em>e.g., </em>pictures of smiling and frowning faces) and the other will be nonsocial (<em>e.g., </em>pictures of upwards and downwards facing arrows).</li>
		<li>Pseudo-randomize the order of stimuli in each block such that no image occurs more than once in a row, and such that the participant does not see more than three sad/downwards facing arrows or happy/upwards facing arrows in a row.</li>
		<li>Set up each trial to contain the following: a fixation cross; 2 boxes with question marks (the participant will use a button press to indicate a choice between the left and right box); an arrow pointing to the box the participant choses via the button press; and feedback (the stimuli created above).<br />
		NOTE: Although participants are choosing the left or right box, whether correct (<em>e.g., </em>happy face or upwards facing arrow surrounded by intact goldfish) or incorrect (<em>e.g., </em>sad face or downwards facing arrow surrounded by crossed out goldfish) feedback shown is pre-programmed by the randomization described in step 1.5.2. Thus, participants may feel that they are guessing correctly or incorrectly, but in reality the choice does not affect which feedback image is shown.</li>
		<li>Display each trial based on the following durations: 1) fixation cross for 500 ms, 2) two boxes with question marks inside for 3000 ms, 3) two boxes with question marks inside, with an arrow pointing towards the box chosen by the participant for 2000 ms, and 4) feedback (<em>e.g., </em>face or scrambled face images) for 1000 ms. See <strong class="xfig">Figure 1</strong>.<br />
		NOTE: If participants fail to respond (via the button press) within 3000 ms, the trial ends and the next trial begins. The anticipated visual angle is a horizontal visual angle of 14.5&#176; and vertical visual angle of 10.67&#176;.</li>
	</ol>
	</li>
</ol>
<p class="jove_title">2. Behavioral Procedures</p>
<ol>
	<li>Recruit participants with and without ASD diagnoses based on the Institutional Review Board guidelines.</li>
	<li>Administer cognitive tests (<em>e.g., </em>Wechsler Abbreviated Scale of Intelligence, WASI<sup class="xref">26</sup>) to all participants to confirm that children have cognitive scores within the low-average to average range (<em>e.g., </em>a full-scale IQ of at least 70).<br />
	NOTE: It was determined that children with full-scale IQs below 70 would likely have difficulty understanding and remembering task directions. Therefore, an IQ cut-off of 70 was chosen as an exclusionary criterion for participation.</li>
	<li>For participants with a previous diagnosis of ASD, administer the autism diagnostic observation schedule (second edition, ADOS-2)<sup class="xref">27</sup> to confirm their eligibility.</li>
</ol>
<p class="jove_title">3. EEG Recording</p>
<ol>
	<li>Set up the participants.
	<ol>
		<li>Ensure that each participant sits in a comfortable chair in a dimly lit room and adjust the chair so individuals are 72 cm away from the computer screen. Provide a brief tutorial about the procedure.<br />
		NOTE: In this study, participants were told the following: &#34;You will be playing a guessing game - just like pick-a-hand, except on the computer. You will see 2 boxes with question marks in them, and then you will use this button box to choose whether you think the right or left box is the right one. If you think the left box is the right one, press the left button. If you think the right box is the right one, press the right button. Once you pick, you&#39;ll see the boxes with question marks and an arrow in the middle showing which one you picked. Then you&#39;ll see if you got it right. For each one you get right, you&#39;ll get 1 goldfish cracker. If you don&#39;t like goldfish, you can trade for fruit snacks. When you get it right, you&#39;ll see a ring of goldfish crackers. That means you get a goldfish cracker! When you get it wrong, you&#39;ll see a ring of crossed out goldfish crackers. When you get it wrong, you won&#39;t lose any goldfish, you just won&#39;t gain any that time. The computer will keep track of how many goldfish you get, and then I&#39;ll give you that many after we are all done.&#34; After the tutorial, ask participants, &#34;Ok, so what are you going to do?&#34; followed by, &#34;What will you see when you get it right?&#34; and &#34;What will you see when you get it wrong?&#34; to confirm that they understand the task. If they do not seem to understand, explain it again until they are able to answer those questions correctly.</li>
	</ol>
	</li>
	<li>Use an EEG cap with 32 Ag/AgCl electrodes in the international 10-20 system, with additional vertical (VEOG) and horizontal (HEOG) electrodes to capture eye movement.</li>
	<li>Measure the participant&#39;s head to determine which size cap is appropriate for the head size.</li>
	<li>Using a blunt needle, pre-gel the cap by injecting conductive gel into the electrodes.</li>
	<li>Connect the EEG cap to the amplifier with a low-pass filter at 70 Hz, a directly coupled high pass (DC) filter, a 60Hz notch filter, and 500 Hz sampling rate.</li>
	<li>Fit the EEG cap to the participant&#39;s head such that the &#34;Cz&#34; electrode is placed at the middle of the scalp (<em>e.g., </em>middle of nasion to inion) as per the 10-20 system.</li>
	<li>Using a blunted needle or sterile wooden stick, swirl inside the electrode to move any hair and allow the gel to contact the scalp.</li>
	<li>Use an impedance meter (or the EEG computer) to ensure that impedance is below 10 K&#937;&#160;for a low impedance system and below 50 K&#937; for a high impedance system.</li>
	<li>Once all the electrodes on the cap show acceptable impedance levels, place the HEOG and VEOG electrodes. Place the HEOG electrodes at the canthus of each eye, and VEOG electrodes above and below the eye.</li>
	<li>If any electrodes have impedance levels above the acceptable thresholds noted above, record them in a notebook or on the computer.</li>
	<li>Begin the experimental blocks, counterbalancing the order of blocks (<em>e.g., </em>social and nonsocial) between participants. Ensure that the EEG computer is recording, the EEG computer and recording computer are synchronized, and the events are being sent correctly.</li>
	<li>Give the participants 30-second breaks after every 15 trials (approximately every 2-3 minutes) to allow them to move around if needed.<br />
	NOTE: Although no explicit directions were given to children regarding movement, participants were told to use breaks to &#34;get the wiggles out&#34;, &#34;get some energy out&#34;, or to perform any other movements they wanted to.</li>
	<li>Between blocks, give participants a longer break (about 5 minutes). After each block, have participants fill out a 4-question Likert scale about how much they enjoyed the guessing game and how often they felt they could get correct answers.</li>
	<li>After both blocks are completed and participants have filled out both Likert questionnaires, take off the EEG cap and allow them to wash their hair.
	<ol>
		<li>Provide payment (or an equivalent &#34;prize&#34;) to the participants and their families.</li>
		<li>Clean and sterilize the EEG cap.</li>
	</ol>
	</li>
</ol>
<p class="jove_title">4. Processing EEG Data</p>
<p class="jove_content">Note: The procedures and commands described in this section are specific to EEGLAB and ERPlab toolboxes<sup class="xref">28</sup>.</p>
<ol>
	<li>In ERPlab, filter the EEG data using a high-pass filter of .01 Hz and low-pass filter of 30 Hz.</li>
	<li>Discard (in ERPlab) or interpolate (in EEGLAB) bad channels that appear to have high impedance and/or were problematic during recording (<em>e.g., </em>lost contact with the skin during recording).</li>
	<li>Using the <strong>EEG channel operations</strong> GUI (in ERPlab), re-reference the EEG data using the average of the left and right mastoid electrodes (mastoid electrodes were chosen as the reference due to not having a dense electrode array; in which case, experts sometimes suggest using the average reference<sup class="xref">29</sup>, and because using the average of both mastoid electrodes is less problematic for laterality effects versus a single mastoid<sup class="xref">30</sup>).</li>
	<li>If using the average of mastoid electrodes for re-referencing, those two electrodes must provide a clear signal. If either mastoid electrode has a poor-quality signal (<em>e.g., </em>with high impedence or lost contact with skin during recording), do not use the participant&#39;s data for analysis.</li>
	<li>Use the <strong>EVENTLIST</strong> dropdown menu in ERPlab to create an <strong>EEG Eventlist</strong>, and assign codes from the stimulus computer to bins using <strong>BINLISTER</strong>.</li>
	<li>Using the <strong>extract bin-based epochs</strong> dropdown menu in ERPlab, segment the continuous EEG data into stimulus-locked epochs and baseline correct. To measure the stimulus-preceding negativity (SPN), use epoch from -2200 to 100 ms (baseline of -2200 to -2000 ms). To measure reward processing or post-stimulus brain activity, use epoch from -200 to 800 ms (baseline of -200 to 0 ms).</li>
	<li>Plot the data in EEGlab, and mark and discard epochs that appear to contain non-eye blink artifacts (<em>e.g., </em>excessive noise or motor movement).</li>
	<li>Choose <strong>Run ICA</strong> on the epoched data.</li>
	<li>Plot the independent components (select <strong>Plot | Component activations</strong>) and identify any artifacts from eye movement and blinks.</li>
	<li>Remove the components identified as eye movements or eye blink artifacts. To check whether the components marked for removal are responsible for eye movement, choose <strong>plot single trial data</strong> to visualize the data with the identified components removed. Once the blinks and eye movements are removed, accept the removal of the identified components.</li>
	<li>In ERPlab tools, select <strong>Artifact rejection in epoched data | Moving window peak-to-peak procedure</strong>. In the current study, a 200 ms moving window, 100 ms window step, and 150 mV voltage threshold were utilized.</li>
	<li>Compute the average ERPs. Make sure to use the default settings, which state that all epochs marked for removal will be discarded from the averaged ERP.</li>
	<li>To analyze average brain activity occurring prior to the onset of the stimuli, extraction (in step 4.11) should occur during the last 200 ms prior to stimulus onset (<em>e.g., </em>-210 to -10 ms) from the electrodes of interest.
	<ol>
		<li>In the case of stimulus-preceding negativity (SPN), electrodes of interest include: F3/F4, C3/C4, P3/P4, and T5/T6 (note that in some systems, temporal electrodes in this region are labeled T6/T7 or T3/T4).<br />
		NOTE: -210 to -10 ms was chosen in this protocol rather than -200 to 0 ms to avoid contamination by brain activity not related to the SPN (<em>e.g., </em>the beginning of neural activity when the feedback stimuli is shown at 0 ms).</li>
	</ol>
	</li>
	<li>To export numeric data for analysis, use the <strong>ERP measurement tool</strong>.<br />
	NOTE: This tool allows researchers to specify latency or amplitude information, the time windows of interest, and the electrodes of interest. Amplitude can be calculated either as the local peak in a given time window, or as the mean amplitude of a given time window.</li>
	<li>Download the numeric data as a .txt file. As needed, export the data into excel or copy and paste it into a statistical analysis program (<em>e.g., </em>SPSS or JMP).</li>
</ol>
<p class="jove_title">5. Processing Differences for ERSP Analysis</p>
<ol>
	<li>Begin with the file created in step 4.11 (<em>e.g., </em>the epoched file with all artifact rejection procedures complete).</li>
	<li>Utilize the EEGlab &#34;newtimef&#34; plug-in to obtain a time x frequency transform with values for each time point, frequency, and trial.</li>
	<li>To measure anticipatory alpha band activity, calculate the average values from 8 to 12 Hz.</li>
	<li>Calculate the average activation prior to feedback onset (<em>e.g., </em>-2200 to -100 ms with baseline of -2200 to -2000 ms) in the same electrodes used for the SPN.</li>
	<li>To calculate alpha asymmetry prior to feedback, subtract the log power in the left hemisphere from the right hemisphere.<br />
	NOTE: To calculate ERSP activation after feedback onset, data would need to be re-analyzed and re-grouped into epochs using a different time window (<em>e.g., </em>-200 to 800 ms with baseline of -200 to 0 ms).</li>
</ol>
<p class="jove_title">6. Statistical Analysis</p>
<ol>
	<li>Paste the numeric data extracted in section 4 (steps 4.11 and 4.12) into a statistical program (<em>e.g., </em>SPSS or JMP).</li>
	<li>Conduct repeated measures ANOVA in the statistical software to the mean values of ERPs to compare brain activity between hemispheres (left, right), electrode positions (frontal, central, temporal, parietal), conditions (face, arrow) and groups (autism spectrum disorder, typically developing).<br />
	NOTE: Hemisphere, electrode position, and condition are within-subjects factors, and group is a between-subjects factor.
	<ol>
		<li>If hemisphere or electrode position is not statistically significant, collapse across for future analysis.</li>
		<li>If the relationship between behavioral measures (<em>e.g., </em>ADOS severity score) and ERPs is of interest, correlation analyses can be run.</li>
	</ol>
	</li>
</ol>

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H., van Boxtel, G. J. M., B&#246;cker, K. B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>The Oxford Handbook of Event-Related Potential Components</em>. , (2011).</li><li>van Boxtel, G. J. M., B&#246;cker, K. B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+Measures+of+Anticipation.">Cortical Measures of Anticipation.</a> <em style='font-style: italic'>Journal of Psychophysiology</em>. <b>18</b> (2-3), 61-76 (2004).</li><li>Ohgami, Y., Kotani, Y., Hiraku, S., Aihara, Y., Ishii, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reward+and+stimulus+modality+on+stimulus-preceding+negativity.">Effects of reward and stimulus modality on stimulus-preceding negativity.</a> <em style='font-style: italic'>Psychophysiology</em>. <b>41</b> (5), 729-738 (2004).</li><li>Takeuchi, S., Mochizuki, Y., Masaki, H., Takasawa, N., Yamazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+preceding+negativity+represents+arousal+induced+by+affective+picture.">Stimulus preceding negativity represents arousal induced by affective picture.</a> <em style='font-style: italic'>International Congress Series</em>.  (1278), (2005).</li><li>Parker, A. B., Gilbert, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activity+during+anticipation+of+smoking-related+and+emotionally+positive+pictures+in+smokers+and+nonsmokers:+a+new+measure+of+cue+reactivity.">Brain activity during anticipation of smoking-related and emotionally positive pictures in smokers and nonsmokers: a new measure of cue reactivity.</a> <em style='font-style: italic'>Nicotine &amp;amp; Tobacco Research</em>. <b>10</b> (11), 1627-1631 (2008).</li><li>Poli, S., Sarlo, M., Bortoletto, M., Buodo, G., Palomba, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-preceding+negativity+and+heart+rate+changes+in+anticipation+of+affective+pictures.">Stimulus-preceding negativity and heart rate changes in anticipation of affective pictures.</a> <em style='font-style: italic'>International Journal of Psychophysiology</em>. <b>65</b> (1), 32-39 (2007).</li><li>Tottenham, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NimStim+set+of+facial+expressions:+judgments+from+untrained+research+participants.">The NimStim set of facial expressions: judgments from untrained research participants.</a> <em style='font-style: italic'>Psychiatry Research</em>. <b>168</b> (3), 242-249 (2009).</li><li>Wechsler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wechlser+Abbreviated+Scale+of+Intelligence+(WASI).">Wechlser Abbreviated Scale of Intelligence (WASI).</a> <em style='font-style: italic'>The Psychological Corporation</em>. , (1999).</li><li>Lord, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autism+Diagnostic+Observation+Schedule:+ADOS-2.">Autism Diagnostic Observation Schedule: ADOS-2.</a> <em style='font-style: italic'>Western Psychological Services</em>. , (2012).</li><li>Lopez-Calderon, J., Luck, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERPLAB:+an+open-source+toolbox+for+the+analysis+of+event-related+potentials.">ERPLAB: an open-source toolbox for the analysis of event-related potentials.</a> <em style='font-style: italic'>Frontiers in Human Neuroscience</em>. <b>8</b>, 213 (2014).</li><li>Luck, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>An introduction to the event-related potential technique</em>. , (2005).</li><li>Bruder, G. E., Kayser, J., Tenke, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Oxford handbook of event-related potential components</em>. , (2012).</li><li>Yau, S. H., McArthur, G., Badcock, N. A., Brock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Case+study:+auditory+brain+responses+in+a+minimally+verbal+child+with+autism+and+cerebral+palsy.">Case study: auditory brain responses in a minimally verbal child with autism and cerebral palsy.</a> <em style='font-style: italic'>Frontiers in Neuroscience</em>. <b>9</b>, 208 (2015).</li></ol>

<p class="jove_content">Open Access fees for this article were provided by Brain Vision LLC.</p>

<p class="jove_content">The current article describes the stimuli, data collection process, and analysis of ERP data in a reward paradigm for children. In this paradigm, children play a guessing game similar to pick-a-hand on the computer and see feedback about whether their guess is correct or incorrect. ERP results for reward anticipation (brain activity prior to the onset of feedback) were consistent with the stimulus-preceding negativity (SPN). Between conditions, the results suggest that TD children anticipate reward stimuli accompanied by faces more strongly than reward stimuli accompanied by non-face images<sup class='xref'>6</sup>. Between groups of children, the results suggest that TD children anticipate face stimuli significantly more than children with autism do. These results are exciting, as they provide important information about how social and nonsocial information are anticipated in children with autism. This is particularly important for furthering the understanding of neural mechanisms of autism and providing support for the social motivation hypothesis. These findings provide useful information for the creation and refinement of interventions, as it underscores the importance of social motivation for children with ASD; for example, it may be important for interventions to explicitly attempt to increase the reward value of social partners to directly affect social motivation in this population. </p>
<p class="jove_content"> This protocol is useful for measuring anticipatory brain activity in children with and without ASD, and the data provides evidence that this type of brain activity can be reliably and successfully elicited in children over 6 years old. Furthermore, this method allows social and nonsocial conditions to be directly compared without the presence of confounds related to reward properties (since the reward for correct responses was goldfish in both conditions). In the current protocol, faces were scrambled and an arrow shape was created. This procedure preserves the physical stimulus properties of faces in the nonsocial (non-face) condition. This protocol may be useful for future investigations into sub-groups of ASD (<em>e.g., </em>some children with ASD are more socially motivated than others), and could be utilized to better understand why some children respond more effectively than others to certain interventions. </p>
<p class="jove_content">There are limitations to the current approach that must be taken into consideration. First, the paradigm described above is useful for children between 6 and 11 years old with and without ASD who have cognitive abilities in the average range. Pilot data of typically developing children younger than 6 was not successful, as children were confused by the directions and did not understand the game's instructions. In the current protocol, exclusionary criteria included a full-scale IQ score below 70. Therefore, the current paradigm may not be appropriate for children with a mental or chronological age below 6. However, it may be possible to modify the current protocol so it is appropriate for individuals with lower IQs and younger children. Some modifications to make it more appropriate for young children such as toddlers are currently being investigated. Such modifications include changing the task to be passive (<em>e.g., </em>having children watch stimuli that appear at predicable intervals in a block design) and using an S1/S2 paradigm<sup class='xref'>24</sup>. In such a design, the content of S1 reliably provides information about the content of S2 (<em>e.g., </em>if S1 is a square, then S2 will be a face; if S1 is a circle, then S2 will be an arrow). Alternatively, the timing structure of the current paradigm could be used to create an anticipatory auditory protocol. </p>
<p class="jove_content">In ASD, it would be of interest to use speech versus non-speech groups and measure brain activity in children with ASD who are non-verbal and have difficulty responding to instructions or attending to visual stimuli<sup class='xref'>31</sup>. Related to the first limitation, it should be noted that results from children with ASD who have cognitive abilities in the average range are likely not representative of the entire autism spectrum - which, by definition, captures a broad range of functioning levels. Therefore, these representative results cannot be extrapolated to all children with ASD. Finally, it is important to note that the stimuli used in the current protocol were normed by adults rather than children. Therefore, future studies should consider using a stimulus set of facial expressions normed by children. </p>

<p class="jove_content">Autism spectrum disorder (ASD) is a developmental disability characterized by impairments in social communication (verbal and non-verbal) and the presence of restricted interest and/or repetitive behaviors<sup class="xref">1</sup>. Given that ASD is hypothesized to be neurologically-based<sup class="xref">2</sup><sup>,</sup><sup class="xref">3</sup>, it is unsurprising that neuroscience research involving children with ASD has become highly prevalent over the past decade. Though many theories about the brain basis of ASD have been proposed, one in particular that has garnered considerable research attention is the social motivation hypothesis<sup class="xref">4</sup>. Briefly, the social motivation hypothesis states that children with ASD engage in less social interaction than their typically developing (TD) peers because social interaction is not as rewarding for them. Chevallier <em>et al.</em> provide a review of the social motivation hypothesis<sup class="xref">5</sup>. Because this hypothesis directly relates to the reward system, specifically whether or not the system in ASD is responsive to social information, multiple studies have investigated the social reward system in ASD<sup class="xref">6</sup><sup>,</sup><sup class="xref">7</sup><sup>,</sup><sup class="xref">8</sup><sup>,</sup><sup class="xref">9</sup><sup>,</sup><sup class="xref">10</sup><sup>,</sup><sup class="xref">11</sup><sup>,</sup><sup class="xref">12</sup>. Results from these studies have differed, with some providing evidence that the reward system in ASD is hypoactive to both social and nonsocial information, and others suggesting that the reward system functions typically for nonsocial information but is hypoactive to social stimuli. One potential reason for these inconsistent results relates to the stimuli and methodology used in the protocols. It is difficult to match social and nonsocial rewards in an experimental context; for example, multiple studies have used a picture of a smiling face as the social reward, and the nonsocial reward is monetary (<em>e.g., </em>getting money after the experiment is complete<sup class="xref">7</sup><sup>,</sup><sup class="xref">8</sup><sup>,</sup><sup class="xref">11</sup>). Though these studies provide an important foundation for future research, it is difficult to determine whether or not the findings relate to differences in social versus nonsocial reward responsiveness in ASD or if they are due to differences between reward conditions.</p>
<p class="jove_content">The current protocol is designed to investigate the reward system in high-functioning children with ASD using electrophysiology. To explore differences between children with and without ASD based on reward anticipation, the stimulus-preceding negativity (SPN) was measured. The SPN is a slow-wave component that reflects an expectation of a reward stimulus<sup class="xref">13</sup>. The significance of the SPN is typically conceptualized as emotional anticipation<sup class="xref">14</sup><sup>,</sup><sup class="xref">15</sup><sup>,</sup><sup class="xref">16</sup> and is thought to be reflected by activity in the insula<sup class="xref">17</sup><sup>,</sup><sup class="xref">18</sup>. The SPN is often measured after participants perform a motor response and before feedback onset during a decision-making task<sup class="xref">19</sup><sup>,</sup><sup class="xref">20</sup>. The SPN is sensitive to reward magnitude and is consistently larger in reward versus no-reward conditions<sup class="xref">15</sup><sup>,</sup><sup class="xref">16</sup><sup>,</sup><sup class="xref">21</sup>. Though the SPN is typically measured during decision-making tasks, researchers have reported that the SPN can be observed when anticipating affective upcoming stimuli without any task<sup class="xref">22</sup><sup>,</sup><sup class="xref">23</sup><sup>,</sup><sup class="xref">24</sup>. One critical aim of the current protocol is to perform an experimental task in which the rewards between social and nonsocial conditions are matched to eliminate potential confounds. Another goal is to test young children between 6 and 11 years old. Therefore, the protocol can serve as an age-appropriate reward task that children may find engaging without becoming frustrated.</p>

<table><tbody><tr><td>EEG cap</td><td>ElectroCap International</td><td>E1-SM/ E1-S/XSM</td><td>Electro-Cap - Small (50-54 cm)/Electro-Cap -Small/Extra Small</td></tr><tr><td>NeuroScan 4.5</td><td>Neuro Scan</td><td>https://compumedicsneuroscan.com/tag/scan/</td><td>EEG Recording Software</td></tr><tr><td>Stim2</td><td>Neuro Scan</td><td>https://compumedicsneuroscan.com/product/stim2-precise-stimulus-presentation/</td><td>Stimulus Presentation Software</td></tr><tr><td>JMP Pro 11&amp;nbsp;</td><td>SAS</td><td>https://www.jmp.com/en_us/software/buy-jmp.html</td><td>Statistical analysis software</td></tr><tr><td>NimStim Face Stimulus Set&amp;nbsp;</td><td>N/A, open source images&amp;nbsp;</td><td>Open source, Available at https://www.macbrain.org/resources.htm&amp;nbsp;</td><td>Face Images&amp;nbsp;</td></tr><tr><td>EEGlab</td><td>N/A, free software</td><td>N/A, free software</td><td>EEG analysis software (free download)</td></tr><tr><td>ERPlab</td><td>N/A, free software</td><td>N/A, free software</td><td>EEG analysis software (free download)</td></tr><tr><td>Photoshop</td><td>Photoshop</td><td>https://www.photoshop.com</td><td>Adobe Photoshop, image editing software</td></tr><tr><td>Photoshop &#039;scramble&#039; plug-in</td><td>Telegraphics</td><td>http://telegraphics.com.au/sw/product/Scramble</td><td>photoshop plug-in to scramble images&amp;nbsp;</td></tr><tr><td>NUAMPS EEG AMPLIFIERd</td><td>Neuro Scan</td><td>http://compumedicsneuroscan.com/wp-content/uploads/NuAmps-Brochure.pdf</td><td>EEG amplifier&amp;nbsp;</td></tr></tbody></table>

an electrophysiology protocol to measure reward anticipation and processing in children

<p class="jove_content">We present a protocol designed to measure the neural correlates of reward in children. The protocol allows researchers to measure both reward anticipation and processing. Its purpose is to create a reward task that is appropriate for young children with and without autism while controlling reward properties between two conditions: social and nonsocial. The current protocol allows for comparisons of brain activity between social and nonsocial reward conditions while keeping the reward itself identical between conditions. Using this protocol, we found evidence that neurotypical children demonstrate enhanced anticipatory brain activity during the social condition. Furthermore, we found that neurotypical children anticipate social reward more robustly than children with autism diagnoses. As the task uses snacks as a reward, it is most appropriate for young children. However, the protocol may be adapted for use with adolescent or adult populations if snacks are replaced by monetary incentives. The protocol is designed to measure electrophysiological events (event-related potentials), but it may be customized for use with eye-tracking or fMRI. </p>

<p class="jove_content" fo:keep-together.within-page="1">Designing experiments to systematically compare brain activity with social versus non-social reward stimuli is complex, due to the inherent difficulty in equating social and non-social rewards. <strong class="xfig">Figure 1</strong> represents stimuli from an experimental protocol designed for investigating neural responses to reward while controlling for reward properties. Specifically, this paradigm was designed to (i) keep rewards consistent between social and nonsocial trials, (ii) control for physical stimulus properties between social and nonsocial trials, and (iii) be age-appropriate for 6 to 11-year-old children with and without autism.</p>
<p class="jove_content" fo:keep-together.within-page="1"><strong class="xfig">Figure 2</strong>&#160;depicts ERP responses as participants anticipate social and non-social stimuli. It should be noted that because the current protocol was designed to measure reward anticipation (the SPN), the displayed epochs are largely prior to feedback onset (which occur at 0 ms in the figures). These results suggest that typically developing (TD) children anticipate reward stimuli accompanied by faces more robustly than children with ASD. Furthermore, though TD children anticipate face stimuli significantly more than non-face stimuli, children with ASD do not evidently show significant differences in brain activity between conditions.&#160;</p>
<p class="jove_content" fo:keep-together.within-page="1"></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58348/58348fig1.jpg" /><br />
<strong class="xfig">Figure 1</strong><strong>:</strong> <strong>Schematic of stimulus presentation and timing. </strong>Feedback for the social (face) condition is shown in the left column. Feedback for the nonsocial (non-face) condition is shown in the right column. Feedback for &#34;correct&#34; answers is shown on top, and feedback for &#34;incorrect&#34; answers is shown below. This figure is re-printed with permission<sup class="xref">12</sup>. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58348/58348fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58348/58348fig2.jpg" /><br />
<strong class="xfig">Figure 2</strong><strong>:</strong>&#160;<strong>Grand averaged waveforms for TD children and those with ASD from the SPN in response to social/faces (left) and nonsocial/arrows (right).</strong> TD children are represented by a solid line and children with ASD by a dashed line. The area between&#160;-210 and -10 ms, used for statistical analysis, is highlighted with a gray box. This figure is modified from a previous publication<sup class="xref">6</sup>.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58348/58348fig2large.jpg">Please click here to view a larger version of this figure.</a></p>

Watch this Scientific Journal Video about An Electrophysiology Protocol to Measure Reward Anticipation and Processing in Children at JoVE.com

An Electrophysiology Protocol to Measure Reward Anticipation and Processing in Children

<p class="jove_content">This protocol is designed to measure reward anticipation and processing in young children with and without autism. Specifically, the protocol is designed to study the neural correlates of reward during social and nonsocial conditions while controlling for reward between conditions. </p>

We present a protocol designed to measure the neural correlates of reward in children. The protocol allows researchers to measure both reward anticipation ...

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6.9K Views.  University of California, Riverside. This protocol is designed to measure reward anticipation and processing in young children with and without autism. Specifically, the protocol is designed to study the neural correlates of reward during social and nonsocial conditions while controlling for reward between conditions. 

Video: An Electrophysiology Protocol to Measure Reward Anticipation and Processing in Children

Electrolytic Cells

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Electrolytic Cells<button class="expand">Expand</button>
	<p>In this experiment, you will be assembling an electrolytic cell to perform electroplating. An electrolytic cell contains an anode and cathode connected by a power source, which drives the nonspontaneous redox reaction in the cell. When the anode material is oxidized, the ions travel to the cathode, where they are reduced and plated.</p>
	<p>In the case of this experiment, the electrolytic cell contains a copper anode and a brass key for the cathode. Oxidation of the anode creates copper(2+) ions, which are then reduced on the brass key, forming a thin plating of solid copper.</p>
	<ul class="lab-items">
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		<div class="lab-step">Put on gloves, safety goggles, and a lab apron, which must be worn at all times.</div>
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		<div class="lab-step">Obtain one 10-ohm resistor, one copper sheet electrode, one copper wire, and one piece of emery paper.</div>
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		<div class="lab-step">Use the emery paper to polish the brass key and the copper sheet. Rinse the key and the copper electrode with water. The brass key will be the cathode, and the copper sheet will be the anode.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Take one 50-mL beaker and a watch glass to the instructor&#39;s hood and obtain approximately 20 mL of acetone. Place the watch glass on top of the beaker to prevent evaporation and bring it back to your bench.</div>
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		<li data-sub-note-item="1-7">
		<div class="lab-step">Label the other 50-mL beaker as &#39;organic waste&#39;.</div>
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		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a disposable pipette to rinse the copper electrode and key with acetone. Collect excess in the waste beaker. Place the key and copper electrode on a paper towel to dry.</div>
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		<li data-sub-note-item="1-9">
		<div class="lab-step">Measure the initial masses of the key and copper electrode and record their values in your lab notebook.
		<h4><strong>Table 1: Moles of Cu Transferred</strong></h4>
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					<td style="text-align: center;"><strong>Moles Cu plated </strong></td>
					<td style="text-align: center;"><strong>Mass of Cu electrode (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu lost </strong></td>
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			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/53/Table_1._Moles_of_Cu_Transferred.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set up the electrolytic cell. Use one electrical wire with alligator clips to connect the negative terminal on the battery to the current probe. Wrap the 10-ohm resistor around the other end of the current probe.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use another wire to connect the positive terminal on the battery to the copper electrode.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Wrap the copper wire around the brass key and use an electrical wire to connect the resistor on the current probe to the copper wire on the brass key.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Obtain about 200 mL of concentrated copper sulfate in a 250-mL beaker. The amount does not need to be exact. Bring the beaker back to your bench and set it on the stir plate. Carefully, place a magnetic stir bar in the beaker and turn the stir setting on to high.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Connect the data acquisition system to the current probe and allow the data acquisition system to sense the probe. It should return a reading in amps. Change the rate to two scans per min and the duration to 5 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Place the brass key in the beaker. Hang it off the side of the beaker so that the key is almost completely submerged but does not touch the magnetic stir bar. <strong>Note: </strong>The copper wire should not be in the solution.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Place the copper electrode in the beaker, bending it so that it hangs over the edge of the beaker. Ensure that it is not touching the stir bar or the brass key.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Start the scan and let it collect data for 5 min. This will act as a test run to make sure plating occurs on the brass key.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">After the scan ends, remove the key and copper electrode. The copper plating on the brass key should be evident. If it is not visible, check your circuit to make sure that the battery is connected in the correct orientation.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Once you have completed a successful 5-min plating, disconnect the key and copper electrode from the wiring. Remove the copper wire from the key and rinse the key and copper electrode with water and then acetone. Lay them on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Measure the masses of the key and the copper electrode and record them in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Reconnect the key and copper electrode and place them back in the solution like before. Restart the run on the data acquisition system, making sure that the rate is set to two scans per min. With the duration set to 30 min, allow the electroplating process to run.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">After the scan is complete, remove the electrode and key from the solution, disconnect them from the wires, and rinse them with water and then acetone. Allow them to dry on a paper towel, then weigh them and record their masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Reassemble the setup and repeat the electroplating for a second 30-min trial. When the scan is complete, remove the key and copper electrode, rinse them like before, and record their mass.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Save the recorded data as a text file on a flash drive.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">To clean up, turn the stir setting off and remove the stir bar from the solution using forceps. Dispose of the acetone in the organic waste container provided by your instructor. The copper sulfate solution can be reused for future labs, so pour the concentrated copper sulfate back into the carboy.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Rinse all of your glassware with water and return it to the instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">First, calculate the total charge Q that flowed through the system during the different trials. One coulomb is the quantity of electricity flowing through a wire carrying 1 ampere of current in 1 s, thus Q = I x t.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Review the current probe data for the first 5-min electroplating trial.<strong> Note: </strong>If the current dropped to 0 or close to 0 at the end of the trial, then the electrons stopped flowing from the copper electrode to the key. Only use the values recorded before the drop in current occurred. If the current drop to 0 early in the plating process, repeat the trial.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The current varies over time, so calculate the average current, I.
		<h4><strong>Table 2: Moles of Electrons Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>Time (s) </strong></td>
					<td style="text-align: center;"><strong>Current<sub>average</sub> (A) </strong></td>
					<td style="text-align: center;"><strong>Total charge (C) </strong></td>
					<td style="text-align: center;"><strong>Moles (e?) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">Total</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/53/Table_2._Moles_of_Electrons_Transferred.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Use the average current over the total time to determine the total charge transferred during electroplating, Q. About 20 coulombs were transferred during the 5-min trial, and between 110 and 120 coulombs were transferred during each 30-min trial. The total charge transferred over the entire 65-min electroplating process was about 250 coulombs.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Use the total charge transferred to calculate the moles of electrons plated during each trial. The moles of electrons plated equals the total charge transferred divided by the Faraday constant. The Faraday constant represents the magnitude of electric charge per mole of electrons and is determined by multiplying the charge of an electron by Avogadro&#39;s constant.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Determine the moles of electrons that were transferred for the 5-min and 30-min trials.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the moles of copper that were plated on the key after each trial. Similarly, determine the moles of copper lost from the copper sheet electrode.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the moles of copper that were lost from the electrode to the amount that was plated to the brass key. The numbers should be close.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check to see if these values match what is expected based on the number of moles of electrons that were transferred. The reduction of 1 mole of copper(2+) ions requires 2 moles of electrons to produce 1 mole of solid copper.</div>
		</li>
	</ul>
	</li>
</ol>

Electrolytic Cells: Construction and Electroplating - Procedure

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Electrolytic CellsExpand
	In this experiment, you will be assembling an electrolytic cell to ...

Education

JoVE Lab Manual

Chemistry

Lab Manual Sub Articles

Galvanic Cells

Galvanic Cells: Construction and Reduction Potential Measurement - Procedure

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Constructing a Lead-Copper Galvanic CellExpand
	In this experiment, you will construct a ...

Energy Dynamics

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Caterpillar Energy Dynamics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this lab you will grow caterpillars in conditions with and without food to examine the transfer of biomass in a typical trophic system.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Weigh and record the wet mass of enough leaves of cabbage to line the bottom of a plastic tub.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, collect two batches of five caterpillars from their cabbage habitat, and weigh and record the wet mass of each batch separately in individual weigh boats. Make sure to tare the balance before each measurement. HYPOTHESES: The experimental hypothesis is that only a fraction of energy will be transferred between the trophic levels of the cabbage and the caterpillars and a significant amount will be lost as waste and heat energy. The null hypothesis is that there will be no significant difference between the energy input from the cabbage, and the obtained energy stored as biomass in the caterpillars.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place the weighed cabbage and five caterpillars in a plastic tub lined with damp paper towels, leaving the lid slightly cracked open for ventilation, but not wide enough for the animals to escape.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Place the other five caterpillars in a second clean container prepared the same way, but with no cabbage.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After three days, transfer the caterpillars to new weigh boats, and weigh and record the final wet mass of both groups of caterpillars and the cabbage.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then place the caterpillar waste, or frass, and cabbage into two separate dishes, and put both dishes into a 37 &#176;C incubator to dry for 48 hours.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Euthanize the weighed caterpillars by putting them into a minus 20 &#176;C freezer for 24 hours.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, transfer the dishes to a 37 &#176;C incubator for 48 hours.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">After drying, weigh and record the dry mass of the uneaten cabbage, the two groups of caterpillars, and the frass, and use these data to calculate the percent biomass of the caterpillars.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Finally, discard all of the dried organisms and plates into the hazardous waste bin, and wipe down your workspace with 70% ethanol.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Take a look at the data you collected. Ensure the initial wet mass, the final wet mass, and the final dry mass columns are accurately filled out in your table. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/13/Table_1_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">NOTE: Remember an object&#39;s biomass is equal to its dry mass. Therefore, percent biomass for each group is the final dry mass divided by the final wet mass times 100. For example, obtain the percent biomass of the cabbage by dividing the final dry mass by the final wet mass and multiplying by 100. This means that approximately 15% of the cabbage&#39;s total weight is biomass.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">After this, estimate the initial biomass of the cabbage and caterpillars by multiplying the percent biomass by the initial wet mass, and put the values in this new table. NOTE: Remember, biomass equals dry mass. Therefore, the final dry mass is equal to the final biomass of each group.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate the approximate initial and final energy in the form of kilocalories within the cabbage, caterpillars, and frass, multiply the biomass values recorded in your table by the conversions, and record the calculated values in the table. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/13/Table_2_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-5"><div class="lab-step">After this, subtract the initial energy from the final energy to obtain the change in energy for each group.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">NOTE: The negative sign denotes loss in energy, and positive indicates gain.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using your data table, calculate the energy in kilocalories that the fed caterpillars lost to respiration. The formula is the energy gained from cabbage, which is the positive of the energy lost by cabbage, minus the biomass gained from cabbage, minus the energy lost via frass. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/13/Table_3_Energy_Dynamics.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Then, repeat the same calculation for the unfed group of caterpillars. Make another column in the table and note this down.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Finally, add up all of the energy gained via biomass and cabbage consumption, and the energy lost via frass and respiration for each group of caterpillars. NOTE: For both groups of caterpillars the net energy is zero, meaning that this experiment obeyed the first law of thermodynamics.
</div></li></ul></li></ol>

Studying Energy Dynamics and Productivity - Procedure

Caterpillar Energy Dynamics
ExpandNOTE: In this lab you will grow caterpillars in conditions with and without food to examine the transfer of biomass in a ...

Biology

Ecology

Extinction

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Diversity and Extinction during the Paleogene Epoch
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin this procedure by reading the extinction reading supplement that has been printed out for you. <br /><strong>NOTE:</strong> Alternatively, you may download the reading supplement at the link below.&nbsp; <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/24/Lab_20_Reading Supplement.docx" style="display:block;margin:20px;" target="_blank">Click here to download the extinction reading supplement</a>
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Using the information contained in the extinction reading supplements pertaining to the taxa of interest and the Chicxulub impact event, make predictions for each taxon listed in Table 1. Consider whether diversity in each taxon increased, decreased, or stayed the same immediately following the Cretaceous-Paleogene boundary and later on in the Paleogene epoch. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/24/Table_1_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, fill out the reasons for each of your predictions in the Contributing Factors column. HYPOTHESES: The experimental hypothesis is that many of the different taxa will show changes in diversity after the Cretaceous-Paleogene event, and again at the end of the Paleogene, with some increasing in diversity, while others decrease. The null hypothesis is that there will be no changes in diversity between taxa following the Cretaceous-Paleogene event, or at the end of the Paleogene.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Gather diversity data by going to the Paleobiology Database: <a href="http://www.paleobiodb.org" target="_blank">www.paleobiodb.org</a>.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">In the center of the screen, click the yellow box labeled Download Data. Under the italicized heading, &#8220;What do you want to download?&#8221;, select Diversity over time on the left side and Comma-separated values on the right side.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Scroll down to the heading &#8220;Select by taxonomy&#8221;.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the first box labeled 'Taxon or taxa to include', type in the name of the taxon that data is being gathered on.  NOTE: Ensure the spelling is correct.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Now, go to the dropdown menu right below that box labeled &#8220;Taxonomic resolution&#8221;, and select genera.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Under the next heading, Select by time, type Maastrichtian into the first box and Chattian in the box directly below. Make sure the dropdown menu labeled Time rule is set to major and the Temporal resolution is set to stage.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Select &#8220;Assume extant taxa range through to present&#8221;. Scroll back up to the middle of the page and click download. A save window will open.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Save the .csv file to a safe and accessible place and rename it to make it easy to identify.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Open the file and scroll down to the main dataset under Records.  NOTE: The data are arranged in reverse time order by geological stage. The second column, interval_name, gives the accepted name of the stage in question. The next two columns give the appropriate dates of the stage in millions of years before the present. The final column, number of occurrences, gives a total count of the genera extant during that geological stage as determined from occurrence of fossils.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To more easily work with this data, copy and paste each column into a new sheet. Be sure to label this sheet with the taxon in question, the data and time the data were obtained, and the data source.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Repeat this process for each of the taxa in Table 1 (steps 4 &#8211; 12). NOTE: Data from multiple taxa can be kept on the same sheet, so long as they are kept distinct and labeled appropriately.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To create line plots for each of the taxa showing the change in diversity over time, select the max_ma column and the number of occurrences column simultaneously.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">In Excel, select Insert from the top toolbar and find the section labeled Charts. Select the bottom center icon, Insert Scatter Chart.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Then, select the Scatter with Straight Lines and Markers and a graph should appear. NOTE: Since the axis of this graph is in units of millions of years ago, the axis is in the reverse direction of time when put in numerical order.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">To fix this, click on the green plus sign in the top-right corner of the graph and hover over Axes. This should reveal a small black arrow. Click the arrow to open up a small menu and select More Options.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">This will open the Axis Options menu. Under Axis Options, click the box next to Values in reverse order. NOTE: It is also possible to change the axis scales from this menu, which can be used to eliminate the excess time that is not being analyzed in this lab. Keep any changes consistent between different graphs.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Double-click on the title at the top of the graph and change it to the taxon being represented.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Make a separate graph for each of the taxa in Table 1. Keep them organized and in one location. A separate sheet for graphs is recommended.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">For each graph, identify the location of the Cretaceous-Paleogene boundary and draw a vertical line through the graph at this point.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using the information from the graphs, fill out Table 2. In some cases, it may be helpful to go back to the original dataset and compare actual values, particularly when they are small. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/24/Table_2_Extinction.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare these values to the predictions made in Table 1 and discuss if the original contributing factors are still likely candidates.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Using the dataset, calculate the percentage of total taxa that went extinct over the Cretaceous-Paleogene boundary using this equation.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">There is an event in the middle of the Paleogene known as the Paleocene-Eocene Thermal Maximum about 55.5 million years ago. This represents a high point in temperature during the Paleogene epoch at approximately 8 &#176;C higher than today&#39;s average temperature. Does this point seem to have an effect on diversity for any of the taxa? If yes, then which ones?
</div></li></ul></li></ol>

Extinction: Diversity and Extinction during the Paleogene Epoch - Procedure

Diversity and Extinction during the Paleogene Epoch
ExpandBegin this procedure by reading the extinction reading supplement that has been printed out for ...

Group Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">The Hawk-Dove Game
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe hypothetical costs and benefits of the antagonist or cooperative behavior respectively. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Before the activity starts, review the cost-benefit table so that you understand how to score the game. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/33/Table_1_Group_behavior.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-3"><div class="lab-step">HYPOTHESES: The experimental hypothesis for this exercise could be that the dove strategy will become more frequent between repeated partners, and the hawk strategy will become more frequent when interactions are with a variety of partners. The null hypothesis could be that there will be no change in strategy over time with repeat or different partner combinations. NOTE: Benefit can be determined by looking at the left column and choosing the row that corresponds to the card you played. The box that corresponds to the choice of your partner can be found in the top row. For the purposes of this game, B equals four and C equals three. So depending on the cards played, player one here, which is you, would gain a score of two, four, zero or one half. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Collect a set of two game cards. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Take out the cards labeled hawk and dove and place them face down on the table or conceal them in your hands. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">When the instructor gives the signal show either of the two cards to the student sitting across from you. Try to strategize how to receive the highest benefit from every interaction on your own but do not communicate with your partner. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">In the Single Partner Recording Table (Table 2) provided by your instructor, note the cards played as well as the benefit you received for that round.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Repeat showing cards to the same partner and determining benefit on your instructor&#39;s signal for a total of 15 rounds.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">At the end of those rounds, the student with the highest total benefit value was determined to be the &#8220;winner&#8221;, or the most fit.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">As a class, determine the percentages of hawks and doves played as well as the average benefit received for each round, using this formula: Class average benefit=(sum total benefit)/(number of players)
</div></li><li data-sub-note-item="1-11"><div class="lab-step">For the second game, and then for each round of play, one row of students should rotate to their right, leaving all players with a new partner.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Continue playing for 15 rounds or until students end up back in their starting positions, recording the data in the Multiple Partner Recording Table (Table 3) given to you by your instructor.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">For each round, determine the percentages of hawks and doves played that round, as well as the average benefit received.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Cheating and Cooperation Exercise
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: In this activity, you will exchange benefits, represented by colored or similar items, with your classmates. In this simulation, a cheater is defined as a person who gives fewer benefits than they receive. During this activity, you will observe the frequency of cheating behavior over time. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">HYPOTHESES: The experimental hypothesis is that cheaters will receive fewer benefits over time and cooperators will continue to provide equal benefits to one another. The null hypothesis is that the frequency of cheaters and cooperators does not change over time. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Each person should collect beads of one color to represent a tangible benefit, as well as one paper bag and a three-ring binder.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Get into a group of three so that all members have objects of the same color.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now find another group with a different color to pair with. In each round, partners will exchange benefits without knowing what they are receiving from their partner and without discussion within their group.  NOTE: Benefits of a different color from your starting color are worth twice as much.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Turn face to face with a member from the other group while still being able to conceal the objects in your possession.  TIP:<strong> </strong>Setting up a screen using a notebook or sitting behind a desk can be helpful here.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When given a signal from the instructor, place zero to two benefits into the paper bag and pass it to your partner.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Simultaneously, receive the bag from your partner and then remove the benefits from the bag.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Make a note of how many benefits you gave away and how many you received in the Cheating and Cooperation Table (Table 4) provided by your instructor.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Turn to a new member from the other group and repeat the exchange.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Rotate through all of the members of the other group for as many rounds as the instructor determines - which will not be revealed until the game ends so as not to alter player behavior.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Once the game has ended, note for each round in the cheating and cooperation table whether you gave more than your partner, received more from them or if you gave the same amount.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Now total up all of the benefits in your possession, with benefits you&#39;ve received from the other group being worth twice as much.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">For each round, the whole class should total up the number of cheaters - with cheaters defined as those who gave less than they received.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">For both the single partner hawk-dove game and the multiple partners hawk-dove game, use the whole class data to plot the percentages as two separate lines - one for the percentage of hawks, and one for the percentage of doves, for each of the 15 rounds.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Take note of whether the cards played changed as more rounds were played.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Manipulate the values for B and C to see if you can find the point at which values of B and C would render the hawk strategy no longer effective (this would be the point when there is no benefit to choosing the hawk strategy over always choosing the dove strategy).
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As a class, use the data from the cheating and cooperation activity to make a graph showing the percentage of rounds each individual player cheated versus the amount of benefits they ended with.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Consider whether it is better to be a cheater or a cooperator in this scenario and discuss as a class what makes this scenario different from the hawk-dove game.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Now, use the class data to graph the percentage of cheaters against the number of rounds, and look for any trends over time.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Consider which type of strategy would most likely evolve in a setting where partners interact repeatedly.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Then, note which tactic might be beneficial for a species that relies on cooperation. 
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Discuss as a class what would likely happen if you increased or decreased the relative value of the benefits you receive.
</div></li></ul></li></ol>

Group Behavior: Hawk-Dove Game and Trading Exercise - Procedure

The Hawk-Dove Game
ExpandNOTE: In this activity, you will play the hawk and dove game with a single partner and then with multiple partners to observe ...

Optimal Foraging

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Foraging with Artificial Habitats and Prey
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with varying prey densities. Without knowing what these densities are, foragers must obtain as many prey items as possible in as little time as possible. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">HYPOTHESES: In this experiment, the experimental hypothesis could be that foragers will catch the most prey in the higher prey density bucket and also spend the most time foraging there.  The null hypothesis would then be that foragers will catch an equal number of prey in each bucket and that foragers will spend an equal amount of time foraging in each patch. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To begin the foraging activity, record the names and role of each participant at the top of the student data collection sheet.  NOTE: Before starting the experiment, the forager is allowed to become familiar with the foraging process &#8211; the following steps are a practice round. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">The forager should kneel or sit in front of the average density bucket. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Once the timer says start, the forager may begin looking for the pinto beans representing the prey. The forager can spend as much time as they like foraging in this bucket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once a prey item is located, the forager must signal that one has been found and place the prey in the cup next to the bucket.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once they believe they have acquired most of the prey in the patch, they should tell the timer to stop the time and the practice round is over. 
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To begin the simulation proper, the forager should sit or kneel in front of bucket A. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">The timer must be ready to start the stopwatch. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">The recorder should be ready to record the time each prey item is located, in addition to the time that the forager arrived at and left each bucket. 
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When all parties are ready, the timer should start the stopwatch and say &#8220;start&#8221;,  and the forager should begin foraging. NOTE: The stopwatch will run through the rest of the round for buckets B and C.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">As the forager signals finding pinto beans and places them in the cup next to the bucket, the timer should tell the recorder the time.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">The recorder should then write the time in the student data collection sheet.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/34/Table_1_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Once the forager has decided to move on to the next bucket, they should tell the timer &#8220;done&#8221;, and begin moving to the next patch. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">The recorder should record the time the forager left and the time they arrived at bucket B in the student data collection sheet.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Repeat the same procedure for buckets B and C. Once the forager has completed the activity, it is time for the next forager to begin using the same procedure. 
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Before performing any data analysis, review the data collection sheet and compile your individual data for captured prey, foraging time, and travel time for each bucket in your student calculations table. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/34/Table_2_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-2"><div class="lab-step">After this, calculate your individual capture rate for each bucket by dividing the number of prey captured by the sum of the foraging time and travel time and record these values in your table. Individual capture rate =  Number of prey captured / (foraging time + travel time)
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To calculate your Giving-Up-Time or a GUT for each bucket, subtract the time that the last prey item was acquired from the time you left the bucket.  GUT = time forager left bucket  -  time last prey item acquired
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, calculate the mean GUT for your foraging group and record the values in your table.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Finally, calculate your scaled GUT for each bucket by dividing the bucket GUT by your group&#39;s mean GUT. Scaled GUT =  Bucket GUT / Group mean GUT NOTE: The scaled GUT will allow class data to be pooled later on without differences in individual foragers skewing the data. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">The instructor should now compile the data into the classroom spreadsheet.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/34/Table_3_Optimal_foraging.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Plot a bar graph of the average number of prey collected in each bucket. On the x-axis, A should represent the low prey density bucket, B should represent the medium prey density bucket, and C the high prey density bucket. Plot the y-axis as the number of prey items caught. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Next, plot the graph of the class average scaled GUT, where the x-axis represents the buckets with varying levels of prey density and the y-axis shows the values for the average scaled GUT. 
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Plot a third graph of the average time spent foraging in each bucket. 
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Now review the Marginal Value Theorem (MVT) from the concepts video and examine the three graphs that you generated from your class data. Take note of whether the graphs conform to the predictions of MVT. 
</div></li></ul></li></ol>

Optimal Foraging: Foraging with Artificial Habitats and Prey - Procedure

Simulating Foraging with Artificial Habitats and Prey
ExpandNOTE: The foragers will hunt for prey represented by pinto beans in four buckets of rice with ...

Sexual Selection and Mate Choice

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Sexual Selection Class Simulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Note: In this activity, you and your classmates will portray a sexually reproducing population consisting of an equal number of male and female animals. You will simulate several different mating scenarios to determine how relative quality, attractiveness, sex, and parental commitment affect mate choice and reproductive success. The goal of this activity is to maximize your progeny. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Hypotheses: There are several overall hypotheses for female and male behavior for this activity. The main experimental hypothesis for female behavior is that females will mate with males of high quality more frequently than those of low quality, yielding a higher reproductive success. The null hypothesis is that females will not demonstrate preference towards high- or low-quality males in any of the four rounds of mating. For males, one experimental hypothesis is that males with a higher visible score will have greater reproductive success. Another could be that males will be more selective for females in scenarios in which they join females to care for offspring as opposed to scenarios in which they exhibit no parental commitment. The null hypothesis for male behavior might be that males will not demonstrate preference towards high- or low-quality females in any of the four rounds of mating. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Activity Overview: To begin, each student will be assigned a sex as well as a relative quality and attractiveness score. Remember - the goal is to maximize your reproductive success in each round. You do this by mating with partners of the highest possible quality which is rated on a scale of one to three with three being the highest possible score. Attractiveness scores, which follow the previously described scoring paradigm, correlate with quality but do not exactly predict the score. Though useful, they are not a perfect way to choose the best mate.  If you are assigned a male ID holder, you will have 16 gametes. However, if you are assigned a female ID holder, you will only have four. A successful mating occurs when one sperm and one egg are paper-clipped together and deposited at the fertilization area. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Note - Rules of the activity: 
<ul>
	<li>Assigned male and female identities for these experiments are not representative of students&#39; personal gender identities. </li>
	<li>Running is not permitted. </li>
	<li>Talking is not permitted.</li>
	<li>Hidden values cannot be shared but individuals can attempt to hide their visible scores. </li>
	<li>Respect the space around each individual and remain an arm&#39;s length away. </li>
	<li>Offspring will only be counted towards an individual score if both gametes are paper-clipped together and dropped off in the designated fertilization area before the end of the two-minute round. </li>
</ul>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Round 1 &#8211; Hidden Quality Score
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin the first simulation, take the clip-on ID holder you were randomly assigned and make sure it contains four gametes if you are assigned female or sixteen gametes if male. Males should also have a strip of white paper and two paperclips. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Next, males should paperclip the white strip of paper over their quality score to hide it, but keep the attractiveness score visible. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Record your ID number for round one in table two.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/35/Table_2_sexual_selection.xlsx" style="display:block;margin:20px;" target="_blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, clip on the ID holder and move to the designated mating area. 
</div></li><li data-sub-note-item="2-5"><div class="lab-step">When the instructor says begin, you may commence the mating simulation. You will have exactly two minutes to produce as many offspring as possible. Males may freely give their sperm to females, and females may choose to use these sperm immediately after which they would walk to the fertilization area and paperclip it to one of their eggs, or they may choose to wait until they receive other sperm to do so. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Once the gametes have been deposited in the fertilization area, females may resume mating. 
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the two minutes have elapsed, all matings must stop and any gametes not in the fertilization area at this time will not be counted. 
</div></li><li data-sub-note-item="2-8"><div class="lab-step">The instructor should now tally all of the matings and record the numbers of the individuals who produced each offspring.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Now, find all the gametes that came with your ID holder and repackage them for the next round. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Round 2 &#8211; Hidden Attractiveness Score
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">For the second round, obtain a new randomly assigned ID holder and again ensure it contains the proper number of gametes. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Next, males should use the paperclip and strip of white paper to hide their attractiveness score (instead of the quality score, which was covered in round one).
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the ID number of your clip in table two under round two.  Note: The mating simulation process for round two will be the same as round one.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Move to the designated mating area and with for the instructor to signal the start of the round. As before, males may freely give away their sperm while females will choose to use it now or wait for different sperm. When a female decides to fertilize an egg, she will walk to the fertilization area and paperclip the gametes together. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Proceed with the simulation for two minutes, and at the signal from the instructor all mating activity should stop. 
</div></li><li data-sub-note-item="3-6"><div class="lab-step">The instructor should now tally all of the matings from this second round.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Now, find and return the gametes to your ID holder, and then return the holder to the instructor.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Round 3 &#8211; Visible Quality and Attractiveness Scores
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">For the third simulation, you should again obtain a new clip-on ID holder and check that it contains the appropriate number of gametes. Record the ID number of your clip in table two.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">For this round, leave both attractiveness and quality scores visible on male IDs. Note: The mating simulation process for round three will be the same as the previous two rounds.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Move to the mating area and wait for the signal to begin the simulation.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Once the instructor starts the timer and gives the signal, commence mating. As before, males are allowed to freely give away their sperm to females and females may use the sperm immediately or wait for different sperm. Again, fertilized eggs only count if placed in the fertilization area. 
</div></li><li data-sub-note-item="4-5"><div class="lab-step">After the two-minute time limit, the instructor should tally all matings. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Now, find and return the gametes to your ID holder, and then return the holder to the instructor.
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Round 4 &#8211; Increased Male Parental Care
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">In the final round, males will again leave their attractiveness and quality scores visible. 
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Obtain an ID holder and ensure it contains the appropriate number of gametes. 
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Clip on the ID holder and move to the mating area. Note: As before, males may freely give away their sperm to females. However, in this round, once a female chooses to mate with a male, both the male and female will walk to the fertilization area to paperclip their gametes together. After placing the paper-clipped gametes on the table, both male and female actors may return to the mating area. 
</div></li><li data-sub-note-item="5-4"><div class="lab-step">The instructor should signal the start of the round and begin the timer &#8211; at this point participants can begin the simulation.
</div></li><li data-sub-note-item="5-5"><div class="lab-step">Once the two-minute time limit is up, all matings must stop. 
</div></li><li data-sub-note-item="5-6"><div class="lab-step">The instructor should tally all mating events.
</div></li><li data-sub-note-item="5-7"><div class="lab-step">Find and return the gametes to your ID holder. Give your ID holder to the instructor and notify them if any gametes or supplies are missing. 
</div></li></ul></li><li data-note-item="6" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="6-1"><div class="lab-step">To analyze the data generated during the mating activities, first split up into groups of four. 
</div></li><li data-sub-note-item="6-2"><div class="lab-step">Collect the class data from the instructor for ONE of the four rounds of mating. Note: In this activity, there were several types of females and males distinguished by different letters. These letter types correspond to specific combinations of quality and attractiveness scores. In the compiled data, the average number of matings and reproductive success has already been sorted by letter type for you by the instructor. 
</div></li><li data-sub-note-item="6-3"><div class="lab-step">Use these data to generate four graphs for the round you were assigned. First plot two graphs for the average number of matings against female and male type. 
</div></li><li data-sub-note-item="6-4"><div class="lab-step">Then plot the reproductive success for males and females against male or female type, giving a total of four graphs.
</div></li><li data-sub-note-item="6-5"><div class="lab-step">Once these graphs are complete, each group should present their data to the class, making sure to state the original hypothesis for your assigned round and whether or not the hypothesis was upheld.
</div></li><li data-sub-note-item="6-6"><div class="lab-step">After each group presents, discuss the overall results of the four rounds together. 
</div></li><li data-sub-note-item="6-7"><div class="lab-step">Note the affect that hiding the quality score had on the overall quality of mates chosen.
</div></li><li data-sub-note-item="6-8"><div class="lab-step">Note how hiding or revealing the attractiveness score affected the results.
</div></li><li data-sub-note-item="6-9"><div class="lab-step">Discuss the effect of male parental investment on the number and overall quality of mating pairs.
</div></li></ul></li></ol>

Sexual Selection Class Simulation
ExpandNote: In this activity, you and your classmates will portray a sexually reproducing population consisting of an ...

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Making Choice Chambers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the bottom of both bottles. One line should be 1 cm from the base of the bottle and the other should be 1.5 cm from the base of the bottle.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a razor blade to make a small cut in each bottle at the mark. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, insert the scissors and carefully cut along the line until the bottoms of the bottles are removed completely.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fit one bottle bottom into the other.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Tape the bottles together to make a secure chamber.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Use the razor blade and scissors to poke a hole into the center of the chamber. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Take a piece of tape and fold the edge onto itself to make a tab and place the tape over the hole.  Note: The tab helps with easy removal and reattachment of the tape to the chamber.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Use the Sharpie to label one side of the chamber A and the other side B.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Upon receiving the colony of <em>Drosophila</em>, perform a count to make sure there are 40 living individuals in the vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Geotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To perform a geotaxis assay, first attach a clean, empty bottle cap to each end of the choice chamber and turn the fly vial upside down onto its lid.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Open the tape flap in the center of the choice chamber.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Insert the funnel into the hole and tap all of the flies into the chamber. HYPOTHESES: The alternate hypothesis is that more flies will be moving against gravity, or up, regardless of the orientation of the choice chamber. Alternatively, if the flies are positively geotactic, then more flies will move down, or with gravity. The null hypothesis is that the flies will have no preference with regard to gravity and will move randomly within the chamber. 
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Once the <em>Drosophila</em> are in the chamber, close the tape flap, rotate the chamber so that it stands vertically with side A on the bottom, and start the timer.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Immediately, count the number of <em>Drosophila</em> that are moving up and the number that are moving down. Record these data for time point 0 in the table. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After 30 seconds, rotate the chamber so that side B is on the bottom and count the number of <em>Drosophila</em> that are moving up or down. Record these data for time point 30 in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After 60 seconds, rotate the chamber so that side A is on the bottom again and record the number of moving flies for time point 60.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/15/Table_1_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Hold the chamber over the fly vial and carefully open the cap to return all of the <em>Drosophila</em> back to their empty vial. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Phototaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To perform a phototaxis analysis, first cover side A of the choice chamber with tin foil, taking care that the foil reaches to the exact center between the two sides. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Ensure the caps are secured on both ends of the chamber.  HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found in the light side of the choice chamber, if the flues are attracted to light, or on the dark side if they are repelled by light. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus. Therefore, equal numbers of flies will be found on either side of the choice chamber.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Open the tape flap in the center of the choice chamber, insert the funnel into the hole, and tap all 40 flies into the chamber as previously described.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Set the timer to sound an alarm at 10 minutes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">When the alarm sounds, stop the timer, and count the number of flies in the open side B of the chamber.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Subtract that number from the total number of flies added to the choice chamber to calculate the number of flies in the covered side A.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record these data in the table for time point 10.  <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/15/Table_2_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Carefully, return all of the flies back to their empty vial.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Chemotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found on the side of the chamber with the apple cider vinegar if the flies are attracted to the odor. Or, if the flies are repelled by the vinegar, then more of them will be found on the side of the chamber presenting the odorless water. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus and will move randomly in the chamber.  Use an individual disposable plastic pipette to dispense two drops of the apple cider vinegar onto one cotton ball for side A of the chamber. 
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Use an individual disposable plastic pipette to dispense two drops of the distilled water onto a cotton ball for side B of the chamber. 
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Fold two pieces of tape over themselves to create individual pieces of double-sided tape and use the tape to attach each cotton ball to the inside of the single bottle cap.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Screw the bottle caps with the cotton balls onto their corresponding sides of the chamber.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Transfer all of the flies into the chamber and set the timer to sound an alarm after 10 minutes. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">When the alarm sounds, stop the timer and record the number of flies in each side of the chamber. <a href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/15/Table_3_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="4-7"><div class="lab-step">At the end of the final experiment, funnel the flies into a morgue tube containing 70% ethanol. 
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Dismantle the choice chambers for recycling.  NOTE: Since all of the chemicals used in these experiments are lab safe, the liquids can be disposed of down the sink, the chambers rinsed, and the solids can be thrown away in the trash. 
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Gather all of the data collected for the geotaxis, phototaxis, and chemotaxis experiments.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Plot these values as bar graphs for different conditions and different time points.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Use these data to draw inferences about the behavior of <em>drosophila melanogaster</em>. 
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

Making Choice Chambers
ExpandBefore beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the ...

Evolutionary Relationships

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Forming an Evolutionary Hypothesis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart used to depict the hypothetical genealogical relationships between species. The tips or leaves of the chart represent specific species and the branches of the tree are different lengths. The different lengths represent the degree of change between each of the species. The common ancestor of all of the species that a specific line branches from is located at a node, where the branches intersect. Species that share a node are called a sister group.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, look at the provided blank cladogram and make a hypothesis about where the assigned animals should be placed on the tree. If a blank cladogram has not been provided please <a target="_blank" href="/files/ftp_upload/10562/Blank_Cladogram.png">download one here</a>.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, observe the image of the fossilized animal.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Using the morphology, or physical characteristics, of the fossil, make a hypothesis about where on your cladogram it should be placed. Add a new line or mark to indicate your proposed position for the fossil.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Comparing Gene Sequences Using BLAST
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Historically, cladograms have been constructed through the comparison of morphology. Now, genetic sequences can also be compared between species of interest to construct cladograms. DNA sequences are not usually preserved in fossils, so for this lab you will use the BLAST database to compare the DNA sequences of living species that are closely related to the fossil to thousands of other living species.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Before beginning this lab, your instructor will have downloaded the three preserved gene sequences to a folder on your desktop.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Navigate to the BLAST homepage, which the instructor should have left open in your browser, and click on Saved Strategies from the menu at the top of the page.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Click Open on the Evolutionary_Relationships_Gene1.txt file.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, click View to go to the Standard Nucleotide BLAST search page. You should be able to see the DNA sequence in the Query Sequence box at the top-right of the screen.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Scroll to the bottom of the screen and click the BLAST button. Note: It will take a few moments to process and analyze the data. 
</div></li><li data-sub-note-item="2-7"><div class="lab-step">When the data has been processed, a graphic summary will appear.  NOTE: Your query sequence is represented by the blue line at the top of the box. Each of the lines below the blue line represents another sequence in the BLAST database that matches your query. The length and location of these bars represent where and how much of the sequence match up to the query. The color of the bar represents its score, or how identical the sequence is to the query. Below the graphic summary is the Sequences producing significant alignments list, which contains descriptions of the DNA sequences retrieved from the database that most closely align with the query sentence. These are listed in descending order, from the most similar at the top to the least similar at the bottom. On the right, there are several statistics on how closely related each database sequence is to the experimental sequence. The higher the Max, Total, Query Coverage, and Identity scores are, the more similar the query and retrieved sequences are to each other. Similarly, the lower the E value number, the less likely the sequences match was found by random chance and more significantly the alignment is accurate. An E value of 0 indicates a highly significant match.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Click on the name of the description of the most similar alignment listed. This will take you further down the page to the exact DNA sequence alignment between the retrieved sequence and the query sequence.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Click on the Sequence ID number. This will open a new tab with more specific information on the retrieved gene sequence.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Identify and record the scientific and common names of the organism, which should be listed next to Source.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Identify and record the protein that the gene codes for, which should be listed next to Definition.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Close the tab and hit Back to return to the Sequences producing significant alignments list.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Then, repeat steps 7 &#8211; 9 for the next most similar sequences.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">After collecting several species names, return to the Sequences producing significant alignments list and click Select: All to check all of the boxes next to the names of each listed alignment sequence.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Click on Distance of tree results to create a phylogram based on the similarity of all of the BLAST result gene sequences to the query gene sequence.  Note: Your query fossil relative sequence will be highlighted in yellow.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">Record your sequence&#8217;s location in relation to the other taxon groups shown in the tree.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Finally, return to the Saved Strategies tab and upload the next saved gene sequence.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">Query the remaining two fossil relative DNA sequences as previously demonstrated (steps 1 &#8211; 16).
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Based on your gene sequence results, come up with a second hypothesis about where your fossil specimen fit in the original cladogram.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Compare this placement with your first hypothesis.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Compare your tree with your classmates&#8217;. If any of your classmates put the fossil on a different branch, ask them how they came to that decision.
</div></li></ul></li></ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Procedure

Forming an Evolutionary Hypothesis
ExpandNOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart ...

Evolution

Scientific Method

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observation and Background Research
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: The first two steps in the scientific process involve studying and observing natural phenomena of interest.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Observe the leaves and branches of the individual plant species. Each species comes from a different environment.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Compare each branch to one another and record your observations about any qualitative similarities or differences. <a target="__blank" style="display:block;padding:20px" href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/1/Table_A_Lab_1.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use leaf clippers to cut the leaves from the branches.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Make additional qualitative observations about the leaves from each species.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Hypothesis Formulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Think of some general questions that you might have after observing the different plant species.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using these general questions, create a null and an experimental hypothesis.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Experimentation and Data Collection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Weigh and record the mass of each leaf. <a target="__blank" style="padding:20px;display:block;" href="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/1/Table_B_Lab_1.xlsx">Click Here to download Table B</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Measure and record the length of each leaf from the petiole to the tip in the table.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next, carefully trace each leaf outline onto a blank piece of computer paper.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Then, cut out and weigh and 4 x 4 cm square of the same paper, weigh the cutout tracing, and record these data in the table.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Data Analysis and Conclusion
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">To calculate the leaf surface area, divide the surface area of the 4 x 4 cm paper square by the mass of the paper square, and multiply the resulting number by the mass of the leaf tracing.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis, and the surface area in square centimeters indicated on the y-axis.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Draw a trend line through the graph if a relationship is evident.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Using your qualitative observations and quantitative measurements, draw conclusions about the data you collected.
</div></li></ul></li></ol>