Name: assays to detect uv-reflecting structures and determine their importance in mate preference using the sailfin molly poecilia latipinna
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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 equal quantity of sodium bicarbonate until fish becomes unresponsive. Once unresponsive, immediately place the fish on a black, non-reflective background. Note: MS-222 is potentially toxic, and gloves should be worn at all times.</li>
	<li>Take all measurements in a darkened room to minimize excess light beyond that of the spectrometer light source.</li>
	<li>Measure reflectance using a fiber optic probe held against the body of the organism. Make sure to form a consistent 45&#176; angle between the fiber optics and the body, as is built into some probe designs. Note: For measurement consistency, it may be desirable to average across multiple scans (2 - 5) per body region.</li>
	<li>Record reflectance data with appropriate reflectance analysis software. Note: The software used here was set to: integration time = 2,000 sec, average scans = 2, and pixel smoothing = 5. Data was copied and pasted to a spreadsheet.</li>
	<li>Repeat on multiple body areas to determine presence or absence of UV reflectance and range of phenotypic variation across organisms. Note: Preliminary scans may be necessary to determine which, if any, body areas consistently show UV markings.</li>
	<li>Return the fish to a well-oxygenated holding aquarium until fully recovered, as evidenced by normal operculum beats and swimming behaviors. Note: A commercial protectant to restore the mucous coating to the body may be added to aid in skin recovery.</li>
	<li>Repeat across multiple individuals, including both sexes if desired.</li>
	<li>On spreadsheet where all data have been copied, plot data using a scatterplot, with reflectance on the Y-axis and wavelength on the X-axis. Amplitude peaks in the UV range (300 - 400 nm) indicate the presence of UV reflectance on the fish.</li>
</ol>
2. Dichotomous Mating Trials
NOTE: Perform control observations, in which focal fish, UV filters, and opaque filters are in place. In these trials, when the opaque filters are removed, focal fish will be exposed to parts of the tank displaying the presence and absence of UV light, but lacking potential mating partners. For details on the validity of dichotomous choice tests in this experiment see Palmer and Hankison (2015)10, although note that these tests also have drawbacks11.
<ol>
	<li>Use sexually receptive individuals as focal organisms. Use female P. latipinna &#60; 48 hr postpartum5 and males isolated for at least 24 hr prior to testing to improve the likelihood of sexual motivation.</li>
	<li>Place a single focal individual into a neutral area adjacent to two choice compartments. For P. latipinna, use a 75.7 L test aquarium (30.5 cm x 30.5 cm x 75 cm; Figure 1) divided into three sections by transparent Plexiglas partitions, forming a central area for the focal individual flanked by two equal-sized choice compartments. Ensure that no water or olfactory cues are shared between individual tank sections. Repeat for each individual to be observed.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54453/54453fig1.jpg" /> 
Figure 1. Experimental Aquarium Setup. A rectangular aquarium divided into three sections: a central area that held the focal individual and two end compartments that held object pairs (the figure shows a female in the middle with object males at the ends). Interchangeable filters that either blocked or permitted UV reflectance could be placed over the glass partitions dividing the male and female sections of the aquarium. This figure is modified from Palmer and Hankison (2015) 10. <a href="http://cloudfront.jove.com/files/ftp_upload/54453/54453fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Under full-spectrum lighting conditions (lighting whose spectrum extends into the UV-A range), isolate one potential mating partner in each end compartment (choice compartment) of the test aquarium. Ensure that potential mating partners resemble one another as closely as possible in size, coloration and display characteristics, so that the main difference between the two isolated mating partners is whether they appear behind a filter that blocks or transmits UV light. 
	NOTE: It may be preferable to record reflectance in the visible spectrum to enable the best possible color matching, although here matching was done by eye.</li>
	<li>Acclimate individuals under visual isolation (opaque filters) for 15 min. Ensure that UV + and - filters are in place at this time. Following acclimation, remove the opaque filters. To ensure that side biases are not present (inherent preference for one side of the tank regardless of object), randomly assign the UV+ and UV- sides of the tank for each each trial.</li>
	<li>Record the amount of time that the focal individual spends within specific preference zones (areas near the choice compartments, in this case approximately two standard body lengths) by using stopwatches. For female fish, this is the amount of time it spends near each of two males, and vise versa for male focal fish. 
	NOTE: Event recorder software can also be used for recording. Fifteen minutes trials were used in this experiment and fish that did not visit each preference zone at least once were excluded from the results as being unresponsive.</li>
	<li>Record and analyze the time that fish spend near empty UV+ and UV- compartments to ensure that there is not an inherent bias towards a particular lighting environment that may influence the mating choice of the fish. Use paired t-tests to determine if individuals prefer a particular UV condition.</li>
</ol>

<ol>
	<li>Endler, J. A., Zaret, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+sexual+selection+on+color+patterns+in+poeciliid+fishes.">Natural and sexual selection on color patterns in poeciliid fishes.</a> Evolutionary ecology of neotropical freshwater fishes. , 95-111 (1984).</li><li>Cummings, M. E., Rosenthal, G. G., Ryan, M. 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+private+ultraviolet+channel+in+visual+communication.">A private ultraviolet channel in visual communication.</a> Proc. R. Soc. Lond. [Biol]. 270 (1518), 897-904 (2003).</li><li>Siebeck, U. E., Parker, A. N., Sprenger, D., M&#228;thger, L. M., Wallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Species+of+Reef+Fish+that+Uses+Ultraviolet+Patterns+for+Covert+Face+Recognition.">A Species of Reef Fish that Uses Ultraviolet Patterns for Covert Face Recognition.</a> Curr. Biol. 20 (5), 407-410 (2010).</li><li>Hrbek, T., Seckinger, J., Meyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phylogenetic+and+biogeographic+perspective+on+the+evolution+of+poeciliid+fishes.">A phylogenetic and biogeographic perspective on the evolution of poeciliid fishes.</a> Mol. Phylogenet. Evol. 43 (3), 986-998 (2007).</li><li>K&#246;rner, K. E., Schlupp, I., Plath, M., Loew, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+sensitivity+of+mollies:+comparing+surface-+and+cave-dwelling+Atlantic+mollies,+Poecilia+mexicana.">Spectral sensitivity of mollies: comparing surface- and cave-dwelling Atlantic mollies, Poecilia mexicana.</a> J. Fish Biol. 69 (1), 54-65 (2006).</li><li>Farr, J. A., Travis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+Advertisement+by+Female+Sailfin+Mollies,+Poecilia+latipinna+(Pisces:+Poeciliidae).">Fertility Advertisement by Female Sailfin Mollies, Poecilia latipinna (Pisces: Poeciliidae).</a> Copeia. 1986 (2), 467-472 (1986).</li><li>Ptacek, M. B., Travis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+Choice+in+the+Sailfin+Molly,+Poecilia+latipinna.">Mate Choice in the Sailfin Molly, Poecilia latipinna.</a> Evolution. 51 (4), 1217-1231 (1997).</li><li>Ptacek, M., Grier, H. J., Uribe, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+signal+divergence,+sexual+selection+and+species+recognition+in+mollies+(Poeciliidae:+Poecilia:+Mollienesia).">Mating signal divergence, sexual selection and species recognition in mollies (Poeciliidae: Poecilia: Mollienesia).</a> Proceedings from the Second International Symposium on Livebearing Fishes. , 71-87 (2005).</li><li>MacLaren, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+male+proximity,+apparent+size,+and+absolute+size+on+female+preference+in+the+sailfin+molly,+Poecilia+latipinna.">The effects of male proximity, apparent size, and absolute size on female preference in the sailfin molly, Poecilia latipinna.</a> Behavior. 143 (12), 1457-1472 (2006).</li><li>Palmer, M. S., Hankison, 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=Use+of+ultraviolet+cues+in+female+mate+preference+in+the+sailfin+molly,+Poecilia+latipinna.">Use of ultraviolet cues in female mate preference in the sailfin molly, Poecilia latipinna.</a> Acta Ethol. 18 (2), 153-160 (2014).</li><li>Wagner, W. 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. C., Marletta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Wing+Pigmentation,+UV+and+Fluorescence+as+Signals+in+a+Neotropical+Damselfly.">The Role of Wing Pigmentation, UV and Fluorescence as Signals in a Neotropical Damselfly.</a> J. Insect Behav. 27 (1), 67-80 (2013).</li><li>Ord, T. J., Stamps, J. A., Losos, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+evolution+in+the+territorial+communication+of+a+classic+adaptive+radiation:+Caribbean+Anolis+lizards.">Convergent evolution in the territorial communication of a classic adaptive radiation: Caribbean Anolis lizards.</a> Anim. Behav. 85 (6), 1415-1426 (2013).</li><li>Siebeck, U. 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. 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. E. <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+coral+reef+fish:+the+role+of+ultraviolet+colour+patterns+in+damselfish+territorial+behaviour.">Communication in coral reef fish: the role of ultraviolet colour patterns in damselfish territorial behaviour.</a> Anim. Behav. 68 (2), 273-282 (2004).</li><li>Cummings, M. E., De Le&#242;n, F. J. G., Mollaghan, D. M., Ryan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+UV+ornamentation+an+amplifier+in+swordtails?.">Is UV ornamentation an amplifier in swordtails?.</a> Zebrafish. 3, 91-100 (2006).</li><li>Mac&#236;as Garcia, c., de Perera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet-based+female+preferences+in+a+viviparous+fish.">Ultraviolet-based female preferences in a viviparous fish.</a> Behav. Ecol. Sociobiol. 52 (1), 1-6 (2002).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>Spectrophotometer, P1000</td><td>Ocean Optics</td><td></td><td>newer models are availabe</td></tr><tr><td>DT 1000 xenon UV light source</td><td>Ocean Optics</td><td></td><td>newer models are availabe</td></tr><tr><td>Ocean Optics Overture Software</td><td>Ocean Optics</td><td></td><td>newer software is available</td></tr><tr><td>R200-Angle-UV bifurcated fiber-optic probe (Guided Wave)</td><td>Ocean Optics</td><td></td><td>newer models are availabe</td></tr><tr><td>Certified reflectance standard, white</td><td>labsphere</td><td></td><td></td></tr><tr><td>75.7 L Aquarium, divided</td><td>Experimental Builder</td><td></td><td></td></tr><tr><td>Full Spectrum Bulb</td><td>Nature&#039;s Sunlight</td><td></td><td></td></tr><tr><td>UV blocking sheet</td><td>GAM UV Sheet</td><td></td><td></td></tr></tbody></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 ...

assays-to-detect-uv-reflecting-structures-determine-their-importance

Research

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Neuroscience

6.5K Views.  Ohio Wesleyan University. 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.

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

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Environment

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate hosts into raising their young and how hosts detect parasitism provide important insights in the field of coevolutionary biology. Brood parasites, such as cuckoos and cowbirds, gain an evolutionary advantage because they do not have to pay the costs of rearing their own young. However, these costs select for host defenses against all developmental stages of parasites, including eggs, their young, and adults. Egg rejection experiments are the most common method used to study host defenses. During these experiments, a researcher places an experimental egg in a host nest and monitors how hosts respond. Color is often manipulated, and the expectation is that the likelihood of egg discrimination and the degree of dissimilarity between the host and experimental egg are positively related. This paper serves as a guide for conducting egg rejection experiments from describing methods for creating consistent egg colors to analyzing the findings of such experiments. Special attention is given to a new method involving uniquely colored eggs along color gradients that has the potential to explore color biases in host recognition. Without standardization, it is not possible to compare findings between studies in a meaningful way; a standard protocol within this field will allow for increasingly accurate and comparable results for further experiments.

This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments.

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

Behavior

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

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

Summary

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Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Swimming Performance Assessment in Fishes

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays