Name: techniques for investigating the anatomy of the ant visual system
Uploaded: 2017-11-27T15:00:00
Description: Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

We are grateful to Jochen Zeil, Paul Cooper and Birgit Greiner for sharing their knowledge in insect anatomy and for demonstrating several of the techniques we have described here. We are grateful to the talented and supportive staff at the Centre for Advanced Microscopy at ANU and The Microscopy Unit at MQU. This work was supported by a graduate scholarship to FRE and grants from the Australian Research Council (DE120100019, FT140100221, DP150101172).

1. Specimen Preparation
NOTE: It is necessary to first understand the relative location of the compound eye and ocelli to each other and on the head. This can be achieved by acquiring images of the dorsal view of the head. For this, we recommend processing samples either for photomicrography or using SEM techniques. Below are steps involved in both processes.
<ol>
	<li>Specimen collection
	<ol>
		<li>Collect and store specimens directly into 70% ethanol. Collect different ca...

<ol>
	<li>Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+homing:+an+insect+perspective.">Visual homing: an insect perspective.</a> Curr. Opin. Neurobiol. 22, 285-293 (2012).</li><li>Wehner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desert+ant+navigation:+how+miniature+brains+solve+complex+tasks.">Desert ant navigation: how miniature brains solve complex tasks.</a> J. Comp. Physiol. A. 189, 579-588 (2003).</li><li>Fent, K., Wehner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocelli:+a+celestial+compass+in+the+desert+ant+Cataglyphis.">Ocelli: a celestial compass in the desert ant Cataglyphis.</a> Science. 228, 192-194 (1985).</li><li>Warrant, E. 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Entomol. 31, 131-142 (2006).</li><li>Bulova, S., Purce, K., Khodak, P., Sulger, E., O'Donnell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Into+the+black+and+back:+the+ecology+of+brain+investment+in+Neotropical+army+ants+(Formicidae:+Dorylinae).">Into the black and back: the ecology of brain investment in Neotropical army ants (Formicidae: Dorylinae).</a> Naturwissen. 103, 3-4 (2016).</li><li>Narendra, A., Reid, S. F., Hemmi, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+twilight+zone:+ambient+light+levels+trigger+activity+in+primitive+ants.">The twilight zone: ambient light levels trigger activity in primitive ants.</a> Proc. R. Soc. B. 277, 1531-1538 (2010).</li><li>Narendra, A., 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=Caste-specific+visual+adaptations+to+distinct+daily+activity+schedules+in+Australian+Myrmecia+ants.">Caste-specific visual adaptations to distinct daily activity schedules in Australian Myrmecia ants.</a> Proc. R. Soc. B. 278, 1141-1149 (2011).</li><li>Moser, J., 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=Eye+size+and+behaviour+of+day-and+night-flying+leafcutting+ant+alates.">Eye size and behaviour of day-and night-flying leafcutting ant alates.</a> J. Zool. 264, 69-75 (2004).</li><li>St&#246;ckl, A. L., Ribi, W. A., Warrant, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptations+for+nocturnal+and+diurnal+vision+in+the+hawkmoth+lamina.">Adaptations for nocturnal and diurnal vision in the hawkmoth lamina.</a> J. Comp. Neurol. 524, 160-175 (2016).</li><li>Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+the+visual+system+of+flies:+the+compound+eyes+and+neural+superposition+in+Bibionidae+(Diptera).">Sexual dimorphism in the visual system of flies: the compound eyes and neural superposition in Bibionidae (Diptera).</a> J. Comp. Physiol. A. 150, 379-393 (1983).</li><li>Dacke, M., Nordstr&#246;m, P., Scholtz, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twilight+orientation+to+polarised+light+in+the+crepuscular+dung+beetle+Scarabaeus+zambesianus.">Twilight orientation to polarised light in the crepuscular dung beetle Scarabaeus zambesianus.</a> J. Exp. Biol. 206, 1535-1543 (2003).</li><li>Greiner, B., Ribi, W. A., Warrant, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+and+optical+adaptations+for+nocturnal+vision+in+the+halictid+bee+Megalopta+genalis.">Retinal and optical adaptations for nocturnal vision in the halictid bee Megalopta genalis.</a> Cell Tiss Res. 316, 377-390 (2004).</li><li>Warrant, E. J., 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=Nocturnal+vision+and+landmark+orientation+in+a+tropical+halictid+bee.">Nocturnal vision and landmark orientation in a tropical halictid bee.</a> Curr. Biol. 14, 1309-1318 (2004).</li><li>Lattke, J. 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> Ants Standard Methods for Measuring and Monitoring Biodiversity. , 155-171 (2000).</li><li>Ribi, W. 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> A Handbook in Biological Electron Microscopy. , 1-106 (1987).</li><li>Narendra, A., Ramirez-Esquivel, F., Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+eye+and+ocellar+structure+for+walking+and+flying+modes+of+locomotion+in+the+Australian+ant,+Camponotus+consobrinus.">Compound eye and ocellar structure for walking and flying modes of locomotion in the Australian ant, Camponotus consobrinus.</a> Sci. Rep. 6, 22331 (2016).</li><li>Narendra, A., Greiner, B., Ribi, W. A., Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+and+dark+adaptation+mechanisms+in+the+compound+eyes+of+Myrmecia+ants+that+occupy+discrete+temporal+niches.">Light and dark adaptation mechanisms in the compound eyes of Myrmecia ants that occupy discrete temporal niches.</a> J. Exp. Biol. 219, 2435-2442 (2016).</li><li>Ribi, W. A., Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+visual+system+of+the+Australian+&amp;#34;Redeye&amp;#34;+cicada+(Psaltoda+moerens).">The visual system of the Australian &amp;#34;Redeye&amp;#34; cicada (Psaltoda moerens).</a> Arthr. Struct. Dev. 44, 574-586 (2015).</li><li>Ribi, W. A., Warrant, E. J., Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+honeybee+ocelli:+regional+specializations+and+rhabdom+arrangements.">The organization of honeybee ocelli: regional specializations and rhabdom arrangements.</a> Arthr. Struct. Dev. 40, 509-520 (2011).</li><li>Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colour+receptors+in+the+eye+of+the+digger+wasp,+Sphex+cognatus+Smith:+evaluation+by+selective+adaptation.">Colour receptors in the eye of the digger wasp, Sphex cognatus Smith: evaluation by selective adaptation.</a> Cell Tiss. Res. 195, 471-483 (1978).</li><li>Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+migration+of+screening+pigments+in+the+retina+of+Pieris+rapae+L.+(Lepidoptera,+Pieridae).">Ultrastructure and migration of screening pigments in the retina of Pieris rapae L. (Lepidoptera, Pieridae).</a> Cell Tiss. Res. 191, 57-73 (1978).</li><li>Lau, T., Gross, E., Meyer-Rochow, V. 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Insect Sci. 18, 60-68 (2016).</li><li>Streinzer, M., Brockmann, A., Nagaraja, N., Spaethe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+and+caste-specific+variation+in+compound+eye+morphology+of+five+honeybee+species.">Sex and caste-specific variation in compound eye morphology of five honeybee species.</a> PLoS ONE. 8, e57702 (2013).</li><li>Somanathan, H., Warrant, E. J., Borges, R. M., Wall&#233;n, R., Kelber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+and+sensitivity+of+the+eyes+of+the+Asian+honeybees+Apis+florea,+Apis+cerana+and+Apis+dorsata.">Resolution and sensitivity of the eyes of the Asian honeybees Apis florea, Apis cerana and Apis dorsata.</a> J. Exp. Biol. 212, 2448-2453 (2009).</li></ol>

The suite of methods outlined above allow for an effective investigation into the optical system of ants and other insects. These techniques inform our understanding of sampling resolution, optical sensitivity, and potential polarization sensitivity of the eye being studied. This knowledge provides an important foundation for physiological and behavioral investigation into their visual capabilities. Furthermore, while the methods detailed here have focused on ant visual systems, these techniques can be used on other inse...

Vision is an important sensory modality for most animals. Vision is especially crucial in the context of navigation for pinpointing goals, establishing and adhering to routes, and obtaining compass information1,2. Insects detect visual information using a pair of compound eyes and, in some cases, one to three dorsally-placed simple eyes called ocelli3,4,5.
The eyes of ants are of particular interest because, while ants are wonderfully diverse, they conserve some key characteristics across species. Despite dramatic variation in anatomy, size, and ecology, the vast majority of species are eusocial and live in colonies; as a result, different species face similar visual challenges in terms of navigating back and forth between a central place and resources. Across ants the same basic eye bauplan can be observed in animals ranging from 0.5-26 mm in body length, from exclusively diurnal to strictly nocturnal species, and from slow walking subterranean to leaping visual predators6,7,8,9,10. All of these staggering differences in ecology and behavior give rise to innumerable permutations of the same basic eye structures to suit different environments, lifestyles, and body-sizes11,12. As a consequence, studying the visual ecology of ants provides a veritable treasure trove of possibilities to the determined investigator.
Understanding the visual system of insects is essential in gaining an insight into their behavioral capabilities. This is apparent from integrative studies which nicely combine anatomy with ecology and behavior to a great success in a few insect groups (e.g., references13,14,15,16,17). Though the field of ant navigation and ant behavior in general has been quite successful, very little emphasis has been placed on ant vision outside of a few selected species. Here, we will elaborate on the techniques involved in investigating eye design of ants. While we will focus on ants, these techniques can be applied, with slight modifications, to other insects, too.

<table>
	<tbody>
		<tr>
			<td>Ant</td>
			<td>Myrmecia midas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica M205 FA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Pro Sci Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Pin</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Colourless nail polish</td>
			<td>Non branded: from any cosmetic store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foreceps</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Leica DM5000 B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformalydehyde</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium Hydrogen phosphate (Na2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium di-Hydrogen Phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Araldite Epoxy Resin</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidie Blue</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Riechert HK120</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques for investigating the anatomy of the ant visual system

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

The methods described here enable detailed study of the simple and compound eyes of ants. Imaging the dorsal view of the head using Z-stack photomicrography techniques allows one to obtain an overview of the layout of the visual system (Figure 1). This is good preparation for dissections and to determine the required sectioning angle. This technique is also useful for taking measurements such as head width, eye length, and ocellar lens diameters. SEM imaging ...

Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

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