Name: assays to detect uv-reflecting structures and determine their importance in mate preference using the sailfin molly poecilia latipinna
Uploaded: 2016-09-14T12:30:00
Description: 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

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
<ol>
	<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...

<ol>
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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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E., Witzany, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+in+the+Ultraviolet:+Unravelling+the+Secret+Language+of+Fish.">Communication in the Ultraviolet: Unravelling the Secret Language of Fish.</a> Biocommunication of Animals. , 299-320 (2014).</li><li>Modarressie, R., 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=UV+matters+in+shoaling+decisions.">UV matters in shoaling decisions.</a> Proc. Biol. Sci. 273 (1588), 849-854 (2006).</li><li>Lim, M. L. 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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.

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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>
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		<tr height="21">
			<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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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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Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

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Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

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The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

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Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

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Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

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Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...