By Jiri Hulcr, Ph.D., and Jackson Landers

When we study insects, we constantly struggle to observe extremely small organs. In the course of our ongoing research into ambrosia beetles at the Ambrosia Symbiosis Lab at the University of Florida’s Institute of Food and Agricultural Sciences (UF/IFAS), we decided to compare the results from several different imaging systems.

In a study published last week in the open-access Journal of Insect Science, we found that old technology is still valuable, but the newer methods of micro-CT scanning and laser ablation tomography offer some unique advantages.

We wanted to better understand the structure of the mycangia of ambrosia beetles. Mycangia are hollow structures within the bodies of ambrosia beetles that carry the fungus that pioneering beetles need to grow their food in a newly established gallery. As the beetles chew tunnels through wood, the fungus they carried begins to grow within the wood, and this is what the beetles eat.

Because some ambrosia beetles can become serious pests of native trees when they appear in a new location as an invasive species, it is critical that we learn more about how the beetles carry and nurture their fungus. For example, Raffaelea lauricola, the fungus that causes laurel wilt, is transmitted by the redbay ambrosia beetle (Xyleborus glabratus). Laurel wilt has wiped out redbay trees throughout the American south and currently threatens Florida’s $65 million annual avocado crop.

However, a simple challenge in studying ambrosia beetles is their size: At most they grow to just a few millimeters in length, and thus a mycangium may measure just a few tenths of a millimeter across.

Researchers studying fungus-farming ambrosia beetles have compared a variety of of techniques for imaging the mycangia (fungus-storing internal organs) within the beetles. In part A, the red red dotted line illustrates the location of where the cross-sections below. Images B and E via micro-CT scanning. Images C and F via laser ablation tomography. Images D and G via microtome sectioning. White and black arrows point out mycangial membranes. Scales bar in A: 1 mm. Scale bar in B-G: 0.1 mm. (Image originally published in Li et al 2018, Journal of Insect Science)

Researchers studying ambrosia beetles used micro-CT scanning to develop this 3-D reconstruction of the mycangia and other internal organs within the prothorax and mesonotum of an Xylosandrus amputatus beetle. A: lateral side; B: frontal view; C: ventral view; D: mycangium with prothorax musculature nearby. (Image originally published in Li et al 2018, Journal of Insect Science)

The traditional standard for imaging the organs of insects is microtome sectioning. Tissue is soaked in paraffin wax before being sliced into razor thin sections, prepared for slides, and photographed under a microscope. It is a two-dimensional imaging system that scientists have been using for over a century.

We compared microtome sectioning with laser ablation tomography and micro-CT scanning.

Laser ablation tomography was invented in 2012 at Penn State’s Roots Lab as a system for imaging the roots of plants. The basic premise is similar to microtome sectioning, except that instead of using a manually operated blade a laser is used to vaporize a very small and very precise amount of tissue with each pass. A photograph is taken of the tissue before each pass of the laser, and then a proprietary software package builds the successive images into a computerized three-dimensional (3D) model.

Micro-CT uses X-rays to repeatedly scan a tissue sample as it rotates within a small chamber. It has been used extensively by archaeologists and medical researchers since the 1980’s. Like laser ablation tomography, it produces a 3D computer model. Various software packages are available for building and processing the 3D models. Unlike either laser ablation tomography or microtome sectioning, micro-CT does not require the destruction or disassembly of the sample.

After comparing the results of each method on an array of eight different species of ambrosia beetles, we found that all of these technologies have a place in an entomologist’s tool kit.

While microtome sectioning requires skill and time and doesn’t create a 3D image, it was the only tested method that allowed us to see individual cells of the fungus within the mycangium. Microtome sectioning allows structures to be viewed in contrasting colors. Laser ablation tomography also does this but in 3D.

However, micro-CT stood out for several reasons. It is a very fast process that allows us to see three-dimensional structures in reference to other internal structures. It also left our specimens intact and available for other forms of study. Micro-CT does not produce real color images, but the colors of individual structures may be added after the fact for illustrative purposes.

The beauty of these beetles’ mycangia became very clear through micro-CT imaging. It is one thing to see basic two-dimensional slices of the organs in a microtome section. To see the individual organ and its supporting muscles in three dimensions, and rotate them in any direction, has added a whole new depth to our understanding.

Jiri Hulcr, Ph.D., is an associate professor of forest entomology at the University of Florida’s Institute of Food and Agricultural Sciences (UF/IFAS). Website: www.ambrosiasymbiosis.org. Email: hulcr@ufl.edu. Jackson Landers is strategic science communicator at the UF/IFAS Emerging Threats to Forests Research Group. Email: jack.landers@gmail.com