Last summer, researchers demonstrated that non-invasive imaging combined with a staining technique enables the fast comparison and study of earthworm species and other animals in unprecedented detail.

In the first comparative morphology study of its kind, the research team produced three-dimensional images of individual muscle fibers and single blood cells in earthworms. The technique allowed them to compare the internal organs of freshly dug worms with those of 60-year-old museum specimens in their natural anatomical context, generating rich 2D and 3D data.

A two-dimensional section of an earthworm created from 3D imaging data set the mouth is located to the right. The segmented internal anatomy of the earthworm and the sediment grains inside the gut are clearly visible. (Courtesy of Alexander Ziegler)

“The main advantages in this scenario are scale—more animals can be analyzed—and speed—specimens can be analyzed faster,” said Dr. Alexander Ziegler, an assistant professor at the Institute of Zoology at the University of Bonn in Germany, who is a co-author on the paper. “This approach could be extended to other soft-bodied animal groups such as mollusks or leeches. Data can be easily shared with others too, including the interested public (citizen science).”

Ziegler hopes that the high-resolution images, videos and interactive models afforded by the new method will reinvigorate the field of comparative zoology. Anatomical studies generally rely on conventional dissection, he says, and the field has suffered in recent years from criticism that it relies on centuries-old observations and descriptive practices.

In an effort to change that, Ziegler and his colleagues analyzed two earthworm species with micro-computed tomography (microCT), plus a staining method that uses iodine, lead or tungsten solutions. While microCT and staining are already used for imaging diverse animal tissues, however, only a few techniques have appeared for imaging preserved soft animal tissues and structures in museum specimens. The research team’s innovation was to combine a range of existing techniques and fine-tuning them to analyze high-resolution, 3D imagery of earthworm specimens simultaneously.

Staining the internal structures enabled the earthworms’ anatomy to be scanned in rich detail. The derived images, videos and 3D interactive models of the earthworms were produced at a scale of about 10 micrometers resolution – or 10 millionths of a meter, which is about a tenth the thickness of a human hair. In the study, a researcher could rotate the internal structure of the earthworm fossil in real-time 3D, allowing for better understanding of its complex morphology.

A 3D rendering of an earthworm made from a micro-computed tomography imagery dataset. This specimen was virtually dissected using the ‘wedge dissect’ tool in the open-source visualization software Drishti. (Courtesy of Alexander Ziegler)

In comparison to conventional dissection techniques used in zoology or palaeontology today, the new method leaves the specimen intact and is relatively quick, taking only one or two hours of scan time. It avoids the need for dissection because an observer can manipulate a 3D dataset on a computer, rotating or panning as needed. Also, two or more datasets can be viewed in parallel, enabling direct comparison of animal morphology and anatomy.

“This potentially constitutes an eye-opener for colleagues in our field,” Zielger says. He and his colleagues published their paper in the journal PLOS ONE.

According to Ziegler, new imaging technologies make hypothesis-driven science easier. The methods used in the earthworm study could be employed to produce high-resolution imagery of hundreds of specimens in a much shorter timeframe than traditional protocols would permit. The scale of data that could be generated across all organisms could be as high as one zettabyte, equivalent to about 250 billion DVDs. Ziegler’s earthworm study alone produced 45 gigabytes of data, which were made freely available in an online data repository that is part of the open access and open data journal GigaScience.

“The amount of potential data is infinite,” Ziegler says. “If you expand the scanning approach to other living species—there are millions—we are looking at exa- or even zettabytes of data.”

Better understanding of the intricate interplay between internal structures or the growth of a single specimen would lead to larger studies to help resolve wider biodiversity issues of habitat loss and the long-term impact of how species interact with their environments. Moreover, raw image data can be uploaded to data repositories, resulting in a drastic increase in transparency for further data mining. Future robotic, data processing and control software techniques could be used to perform rapid throughput screening of specimens to build on existing knowledge.

A composite of a two-dimensional section of a sea urchin (grey) and 3D rendering of selected internal organs (colored). The modeled organs are the gonads (yellow), the digestive tract (blue) and so-called Stewart's organs (red). (Courtesy of Alexander Ziegler)

For instance, earthworms were chosen for Ziegler’s study because of their ecological significance. They can be environmental engineers that sculpt the soil, encourage hydration, oxygenation and the breakdown of organic matter, which help feed plant and insect life within the soil-food web. But they are also an invasive species in parts of North America, threatening the diversity of plant and animal life by breaking up the organic layer of material and reducing nutrients and vegetation.

“The world is changing rapidly. We need to better understand the complex interactions between organisms and their environment; but in particular, if we want to conserve biodiversity,” said Dr. Elizabeth Shea, an expert in cephalopod ecology and systematics at the Delaware Museum of Natural History who was not involved in the PLOS ONE study. “There are so many species and so few people to do the work that we need new approaches that will move the work along faster.”

With few students entering comparative zoology and a lack of funding, future discoveries could be gathering dust on the shelves of museum collections across the globe, according Shea. But scientific exploration is not the only important point – it's also about having fun when doing the science. Open access featuring stunning high-resolution images, videos and interactive models could complement teaching aids to engage students in classrooms.

“Strangely, although exploration is the prime essence for scientific discovery, there is a high reluctance to fund exploratory morphological studies,” said Ziegler. “This is in stark contrast to the massive funding of genomic studies. But what original hypothesis can you seriously have when you sequence an entire genome? Probably none – you first want to explore the data and ask questions later. I dare to make the prognosis this approach will become common practice in morphology as well.”

Shea has three ideas to build upon the best-practice application that Ziegler and his colleagues demonstrated in their paper. First is to transform morphological analysis from its current position as a data-collecting intensive practice to an analysis-intensive practice. Second is to ensure specimen data in museums around the world is made available to more scientists so data can be mined for biodiversity research. “The U.S. National Science Foundation has made this a priority in its Advancing Digitization of Biodiversity Collections initiative,” said Shea.

Third is the need for automated ways to sift through high-definition images or videos of scanned specimens and to develop pattern recognition software to help identify imaged specimens. Current software is not capable of performing complex shape and pattern recognition tasks that a taxonomist is trained to do, but this may change in the coming years.

The contribution of enthusiastic students, experienced researchers and better pattern recognition software are already benefiting mass sequencing DNA studies. Further international collaboration, ideas for larger-scale studies—or even big data approaches—and updated student courses will improve interest and speed up exploration in animal morphology, biodiversity and zoology research.