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. J., Dacke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+navigation+in+nocturnal+Insects.">Visual navigation in nocturnal Insects.</a> Physiology. 31, 182-192 (2016).</li><li>Taylor, G. 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=The+dual+function+of+Orchid+bee+ocelli+as+revealed+by+x-ray+microtomography.">The dual function of Orchid bee ocelli as revealed by x-ray microtomography.</a> Curr. Biol. 26, 1-6 (2016).</li><li>H&#246;lldobler, B., Wilson, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Ants. , (1990).</li><li>Ali, T. M. M., Urbani, C. 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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. B. <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+and+light/dark+adaptation+in+the+compound+eyes+of+male+and+female+Acentria+ephemerella+(Lepidoptera:+Pyraloidea:+Crambidae).">Sexual dimorphism and light/dark adaptation in the compound eyes of male and female Acentria ephemerella (Lepidoptera: Pyraloidea: Crambidae).</a> Eur. J. Entomol. 104, 459-470 (2007).</li><li>Wipfler, B., Pohl, H., Yavorskaya, M. I., Beutel, R. 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+review+of+methods+for+analysing+insect+structures+-+the+role+of+morphology+in+the+age+of+phylogenomics.">A review of methods for analysing insect structures - the role of morphology in the age of phylogenomics.</a> Curr. Opin. 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 ...

The overall goal of this suite of microscopy techniques is to obtain detailed anatomical information of the ant visual system. This method can help address key questions in the field of visual ecology, to estimate various visual parameters, such as optical sensitivity, visual acuity, and size of the visual field. The main advantage of this technique is that it gives the comprehensive overview of a given visual system.It can also be adapted to use with many different species. To make the cornea replicas, use ants preserved in ethanol or mounted on a pin, or in Plasticine. If the head is relatively large, the remaining body parts can be removed.To begin, quickly spread fast-drying, colorless nail polish over the eye, using a toothpick, without scratching the eye. Let the polish set at room temperature. Then, use a fine insect pin to gently lift the replica from the head capsule surrounding the eye.And using forceps grasp the part of the replica that covers the head, but not the eye. Next, place the replica on a glass slide. There, use a razor blade to carefully trim excess material around the eye, while securing the replica.Before placing a cover slip over the replica, it may be necessary to make three to four fine partial radial slices to help flatten it. When placing the cover slip, document the replica's orientation on the slide. Then, seal the cover slip, using small dabs of nail polish on the corners, making certain that the liquid does not touch the replica.The slide is then ready to image using a compound microscope. To begin, anesthetize the ant and place it in a petri dish. If dye needs to be adapted for bright or dot conditions this should be done for a few hours before the dissection.If the dot conditions seem to be maintained, the dissection should be carried out in the red light. First, remove the antennae using forceps. Then, remove the mouth parts using a sharp razor blade.In large specimens, cut through the anterior part of the eye. In a small specimen, cut as close to the eyes as possible, without tugging on the brain, as this may tear out the retina. Next, orient the head and make a transverse incision, that removes the ventral portion of the head, which may include part of the ventral eye.Then, detach the head capsule with a coronal incision, just posterior to the compound eye. In this example, the transverse and coronal incisions are made simultaneously. Finally, transfer the dissected head capsule with the compound eyes to ice cold fixative.It's important to perform these steps quickly, to prevent tissue from degrading, so the tissue should go into the fixative within two minutes of the first incision. Following the text protocol, prepare the specimen and drain the excess resin. Then, place the specimen on the block in a vertical position, and if needed, use a little excess resin to secure it there.Label the block with paper labels embedded in the resin, or attached to the block. Then, cure the resin in a 60 degree Celsius oven, for 12 to 14 hours. Once cured, load the specimen into a clean envelope.Next, mount the block onto a microtome chuck. Before sectioning, first make certain that the specimen's orientation is appropriate for the sectioning plane. Under a dissecting microscope, trim the resin block using a razor blade.Then, remount the chuck onto the microtome arm, and angle the specimen. Next, fill the knife boat with distilled water using a syringe with a 0.45 micron filter. Load the water up to the edge of the knife.In some places the meniscus may be convex. Then, drain the boat until the meniscus is very slightly concave, but still reaches the edge of the knife. Now, slowly and carefully bring the knife towards the specimen, and align the block to the knife.Make frequent checks via the ocular. When the knife is close but not yet cutting the specimen start cranking the microtome wheel to complete the approach. Then, when approaching the region of interest in the block adjust the section collecting thickness.In this case, a diamond knife is used to make one micron sections. Then, from the filter equipped syringe place a series of small droplets of distilled water on a slide. To collect a section of interest, pick it up using an eyelash tool.And carefully, float the collected section onto a water droplet. Continue loading as many sections onto each slide as can fit. When a slide gets fully loaded transfer it to a hot plate at 60 degrees Celsius to evaporate the water, and adhere the sections to the slide.This takes about 15 to 20 minutes. Next, use another filter equipped syringe to deposit a drop of toluidine blue dye onto the slide. Place the drop on one end of the slide and spread it using the edge of a needle without touching or scraping the sections.Then, place the slide on the hot plate for 20 to 60 seconds. Afterwards, spray the slide with distilled water from a wash bottle, and use the hot plate to dry it off. Later, check the slides under the compound microscope and then image them.The described techniques enable a detailed study of the simple and compound eyes of ants. Imaging the dorsal view of the head using Z-Stack photomicrography provides an overview of the layout of the visual system useful for making gross measurements. SEM imaging provides the detail needed to resolve small eyes and the detail needed to identify the shape of each lens.Semi-thin sections imaged using light microscopy show the internal anatomy of an eye. Lens, crystalline cone, and rhabdomere dimensions can be measured at this resolution and magnification. Pigment cells, cone tract presence and rhabdomere shape distributions can also be studied.Ultra-thin sections imaged by TEM provide ultrastructure information, such as microvillar orientation, and for example, the width of the constricted crystalline cone tract. While attempting this technique, it's really important to be patient, to use clean instruments, and to use freshly made solutions. Following these experiments, additional behavioral or physiological experiments can then be carried out.These experiments will inform us about the functional visual abilities of the animal, relative to the eye anatomy. Using these techniques allows us to explore the diversity of visual systems, and how they have evolved to suit different lifestyles and different ecologies, in different species of ants. Though this method can provide insight into the ant visual system, it can also be used to study the visual system of other insects, as well as, other nervous tissues.Don't forget that working with fixatives and resins is extremely hazardous and precautions such as, working in the fume hood and wearing nitrile gloves, should always be taken.

techniques-for-investigating-the-anatomy-of-the-ant-visual-system

Research

JoVE Journal

Environment

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...