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.
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Method Article
Techniques for Investigating the Anatomy of the Ant Visual System
In This Article
Summary
Abstract
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.
Introduction
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 s....
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Protocol
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.
- Specimen collection
- Collect and store specimens directly into 70% ethanol. Collect different castes whenever possible.
- Label specimens with time, date, and place as well as any other relevant observations (e.g., collected whi....
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Results
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 .......
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Discussion
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.......
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Disclosures
The authors declare no competing interests.
Acknowledgements
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).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Ant | Myrmecia midas | ||
| Stereomicroscope | Leica M205 FA | ||
| Sputter coater | Pro Sci Tech | ||
| Ethanol | Sigma Aldrich | ||
| Petri dish | ProSciTech | ||
| Dissecting microscope | Leica MZ6 | ||
| Insect Pin | ProSciTech | ||
| Colourless nail polish | Non branded: from any cosmetic store | ||
| Glass slide | ProSciTech | ||
| Razor blade | ProSciTech | ||
| Foreceps | ProSciTech | ||
| Cover slip | ProSciTech | ||
| Compound microscope | Leica DM5000 B | ||
| Glutaraldehyde | Sigma Aldrich | ||
| Paraformalydehyde | Sigma Aldrich | ||
| Potassium Chloride (KCl) | Sigma Aldrich | ||
| di-Sodium Hydrogen phosphate (Na2HPO4) | Sigma Aldrich | ||
| Potassium di-Hydrogen Phosphate (KH2PO4) | Sigma Aldrich | ||
| Sodium Chloride (NaCl) | Sigma Aldrich | ||
| Osmium tetroxide | Sigma Aldrich | ||
| Acetone | Sigma Aldrich | ||
| Araldite Epoxy Resin | Sigma Aldrich | ||
| Pasteur pipette | Sigma Aldrich | ||
| Toluidie Blue | Sigma Aldrich | ||
| Hotplate | Riechert HK120 |
References
- Zeil, J. Visual homing: an insect perspective. Curr. Opin. Neurobiol. 22, 285-293 (2012).
- Wehner, R. Desert ant navigation: how miniature brains solve complex tasks. J. Comp. Physiol. A. 189, 579-588 (2003).
- Fent, K., Wehner, R.
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