We are grateful to anonymous reviewers for comments and suggestions that greatly improved this manuscript. We thank R. Bowes, R. Carreno, and T. Panhuis for assistance in collecting the fish. We also thank M. Lee for assistance with male preference trials. We are grateful to the Ohio Wesleyan University Department of Zoology for helpful advice and suggestions throughout this study and Arizona State University (McGraw Lab) for software advice.

All experiments were conducted with the approval of Ohio Wesleyan University&#39;s Institutional Animal Care and Use Committee.
1. Recording UV Reflectance of Fish using a Spectrophotometer
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	<li>Calibrate a spectrophotometer and light source with a known white standard over the range of wavelengths to be measured according the recommendations of the instrument or software.</li>
	<li>Anesthetize fish by placing in a 0.5% solution of ethyl 3-aminobenzoate methane sulfonic acid salt (MS-222) buffered with an e...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+female+mating+preferences.">Measuring female mating preferences.</a> Anim. Behav. 55 (4), 1029-1042 (1998).</li><li>R&#233;my, A., Gr&#233;goire, A., Perret, P., Doutrelant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediating+male-male+interactions:+the+role+of+the+UV+blue+crest+coloration+in+blue+tits.">Mediating male-male interactions: the role of the UV blue crest coloration in blue tits.</a> Behav. Ecol. Sociobiol. 64 (11), 1839-1847 (2010).</li><li>Guillermo-Ferreira, R., Ther&#233;zio, E. M., Gehlen, M. H., Bispo, P. 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M., Li, J., Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+UV-reflecting+markings+on+female+mate-choice+decisions+in+Cosmophasis+umbratica,+a+jumping+spider+from+Singapore.">Effect of UV-reflecting markings on female mate-choice decisions in Cosmophasis umbratica, a jumping spider from Singapore.</a> Behav. Ecol. 19, 61-66 (2008).</li><li>Rick, I. P., Bakker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+signaling+in+conspicuous+red+sticklebacks:+do+ultraviolet+signals+surpass+others?.">Color signaling in conspicuous red sticklebacks: do ultraviolet signals surpass others?.</a> BMC Evol. Biol. 8, 189 (2008).</li><li>Siebeck, U. 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Spectrophotometry was successful in identifying UV markings on P. latipinna. Both sexes of P. latipinna possess UV markings along their sides. In addition, some males had UV markings on their dorsal fins, traits previously found to be important in female mating preferences7.
We recommend using UV spectrophotometry as a mechanism to detect the presence of UV markings. Further testing could determine its role in social interactions, including mating preference (as de...

Understanding the cues and signals used in animal social interactions allows us to comprehend the phenotypic variation both within and among species. This variation plays an important role in evolutionary processes such as population divergence, sexual selection, and speciation. Often, however, researchers are limited to exploring the cues most obvious to human sensory systems, especially those within the visual or auditory realms. Use of spectrophotometry, however, allows us to expand our investigations beyond the human visible spectrum and into wavelengths that may be important in social interactions in other species.
In particular, the short-range communication afforded by ultraviolet (UV; 300 - 400 nm) sensitivity has the potential to be highly advantageous during mate choice1. Many visually-hunting predators of birds and fishes, for example, are unable to detect UV radiation. In systems where males display elaborately to females, these males would reduce their risk of predation while maintaining their ability to attract mates by exploiting the UV spectrum rather than developing cues detectable in the visible spectrum2,3.. If one fails to consider the possibility that organisms are communicating with each other using these &#34;private communication channels&#34;, significant drivers of behavior and evolution may be missed.
This protocol outlines an investigation into the use of UV cues for mate choice in the sailfin molly, Poecilia latipinna, a polygamous fish that has no previously known ability to detect UV or utilize UV markings. This fish species has a close phylogenetic proximity to other UV-sensitive live-bearing fishes4 and there is microspectroscopic evidence that P. latipinna, along with other molly species such as P. mexicana and P. formosa, possess a class of cones (photoreceptor cells responsible for color vision) that are most sensitive for UV wavelengths5. In this sexually dimorphic species, female choice has played a strong role in the evolution of the males&#39; brightly colored and enlarged fins6-9. This methodology allows us to explore whether UV is an additional medium by which females assess male quality.
The detection and measurement of UV markings on P. latipinna using a spectrophotometer with a fiber optic probe is detailed here. Further, whether receptive female mollies differentially associate with males viewed through an optical filter transmitting full-spectrum light including UV-A ([UV+]; 320 - 700 nm) and males viewed through an UV-blocking filter ([UV-]; 400 - 700 nm) is discussed. This method has broad applications for discovering UV sensitivity and color patterns in fishes and other organisms, allowing research into a variety of questions involving UV and its role in behavior.

<table border="0" cellpadding="0" cellspacing="0" width="1044">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Spectrophotometer, P1000</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td style="width:351px;">newer models are availabe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">DT 1000 xenon UV light source</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td>newer models are availabe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ocean Optics Overture Software</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td>newer software is available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">R200-Angle-UV bifurcated fiber-optic probe (Guided Wave)</td>
			<td style="width:217px;">Ocean Optics</td>
			<td style="width:119px;"></td>
			<td>newer models are availabe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Certified reflectance standard, white</td>
			<td style="width:217px;">labsphere</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">75.7 L Aquarium, divided</td>
			<td style="width:217px;">Experimental Builder</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:357px;">Full Spectrum Bulb</td>
			<td style="width:217px;">Nature&#39;s Sunlight</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">UV blocking sheet</td>
			<td style="width:217px;">GAM UV Sheet</td>
			<td style="width:119px;"></td>
			<td></td>
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</table>

assays to detect uv-reflecting structures and determine their importance in mate preference using the sailfin molly poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Figures 1 and 2 show the mating preference aquarium set up and UV measurement sites for our experiments.
Measuring UV reflectance allowed for the determination that P. latipinna do possess UV characteristics, especially along the sides of their bodies (Figure 3), in addition to individual variation in these traits. Once UV traits were found, testing revealed that female...

Watch this Scientific Journal Video about Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna at JoVE.com

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

The overall goal of detecting UV reflectance on fish such as Poecilia latipinna is to understand how UV reflecting structures might impact the mating behavior of the species. This method can help us understand mating preferences such as whether UV cues are important, and if so, whether they are used by both sexes. The main advantage of this technique is that it allows us to detect cues that might not be visible for us, but might be very important to the fish.For this protocol, first calibrate a spectrophotometer and a light source to a known white standard. The room lights should be low prior to all calibrations. Calibrate the instrument to all the wavelengths that will be measured which must include the UV wavelengths between 300 and 400 nanometers.Now, put on gloves and anesthetize the fish in a 5%solution of ethyl 3-aminobenzoate methane sulfonic acid salt, known as MS222 buffered with an equal quantity of sodium bircarbonate. When the fish becomes unresponsive, immediately transfer it to a black non-reflective background. Before taking measurements, dim the room lights again.To record the reflectance data, use analysis software. To match the setting here, set the integration time to 2000 seconds. Set the average number of scans to two, and set the pixel smoothing value to five.Now, measure the reflectance of the fish using a fiber optic probe. Hold the probe to make a 45 degree angle between the fiber optics and the body. Take an average of several scans per body region.Check for reflectance for several body areas and look for variation between individuals. When finished, optionally apply a commercial protectant to restore the mucus coating and then return the fish to a well oxygenated holding tank. Once recovered, the fish will show normal operculum beating and swimming.This test makes uses of a choice compartment tank outfitted with UV and opaque filters. When the plexiglass partitions are removed, a fish in the neutral compartment is able to view full spectrum or non-UV light due to filters in different parts of the tank. For P.latipinna, use a 75.7 liter test aquarium.Prior to each test, ensure that no water or olfactory cues are shared by the three tank sections. Use sexually receptive individuals for the test. For a female P.latipinna, choose fish within 48 hours postpartum and use males that have been isolated for at least 24 hours.To conduct the test, first transfer the test animal to the neutral compartment. Next, choose two potential mating partners that resemble one another as closely as possible in size, coloration, and display characteristics. Color matching can be done precisely with instruments or roughly by eye.Now, under full spectrum lighting, place one of the of the two potential mates in each of the the two choice compartments. The opaque and UV filters and the compartment dividers must all be in place. Let the animals acclimate to this condition for 15 minutes.For each trial, randomly assign which compartment has the UV filtering. Then, remove the opaque covers from the dividers. After 15 minutes, start recording the time that the focal individual spends within specific preference zones using a stop watch.The area within two body lengths of a choice compartment constitutes a zone of preference. Alternatively, use event recorder software. Record the activity of the fish for 15 minutes.If the fish doesn't visit each preference zone at least once, treat it as being unresponsive and exclude it from the trial data. Measuring the UV reflectance of P.latipinna show that both sexes have UV characteristics which vary between individuals. The traits are most prominent along the sides of their bodies.Once UV traits were found, the discrimination test revealed that females, but not males, used these characteristics in their mating decisions. Because both sexes possess UV traits, it is possible that they are involved in other social interactions such as shoaling or foraging. Once mastered, the UV measurement techniques can be done in less than two hours.The mate preference trials can be completed in less than an hour for each fish. While attempting the procedure, it's important to ensure that the fish are reproductively receptive. Following this procedure, additional experiments to investigate the role of UV traits in individual or species recognition might be tested.

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6.5K Views.  Ohio Wesleyan University. The overall goal of detecting UV reflectance on fish such as Poecilia latipinna is to understand how UV reflecting structures might impact the mating behavior of the species. This method can help us understand mating preferences such as whether UV cues are important, and if so, whether they are used by both sexes. The main advantage of this technique is that it allows us to detect cues that might not be visible for us, but might be very important to the fish.For this protocol, first ca...