From Water Bears to Grizzly Bears: Unusual Animal Models

From Water Bears to Grizzly Bears: Unusual Animal Models 

Getting Creative with an Extreme Animal Model

The tardigrade’s unusual body plan and resistance to harsh conditions provide researchers like Bob Goldstein with a creative perspective on animal biology.

Interviewed by Niki Spahich, PhD

Interviewee Affiliations 

Bob Goldstein, PhD

James L. Peacock III Distinguished Professor of Biology

Adjunct Professor of Art

University of North Carolina at Chapel Hill

Image Caption 

In the laboratory, Bob Goldstein and his research group stain the tardigrade Hypsibius exemplaris with fluorescent dyes. This tardigrade was stained with LysoTracker Green (cyan), TMRE (magenta), and NucBlue (green). Scale bar: 20μm 

Tardigrades are eight-legged, translucent, microscopic animals. They crawl within dirt, moss, and lichen, and also live in the world’s harshest regions such as mountaintops, the deep sea, and the Antarctic. Also known as water bears, tardigrades survive extreme conditions, including desiccation, extreme heat and cold, and radiation exposure, thanks to their unique biology. Bob Goldstein, a biologist at the University of North Carolina at Chapel Hill, uses tardigrades as a model system to understand body plan evolution and their extraordinary survival skills.

What inspired you to work on tardigrades?

Evolution alters animal shapes by tinkering with genes that control development and cellular functions. When I first trained as a developmental biologist, I was mostly using Caenorhabditis elegans as a model system, and I wondered how it evolved. In the late 1990s, researchers found that C. elegans and Drosophila belong to a clade called Ecdysozoa.¹ That is when my obsession began. I wanted to find something in this group that was outside of the arthropods and nematodes, so that I could have a good comparative organism for studying C. elegans and Drosophila evolution and development.

Tardigrades are members of Ecdysozoa, so I decided to collect different species from the outdoors and from biological supply companies. I identified ones that had small cells and rapid cell cycles—those things might roughly correlate with small genome size. I also wanted ones that were optically clear like C. elegans, so that I could view their development without injecting individual embryonic cells to trace cell lineage. That narrowed it down to the species that we typically use, which is called Hypsibius exemplaris. 

What aspects of tardigrade biology do you research?

Tardigrades have a different body form than other models such as Drosophila, which have many segments. We found that the tardigrade had only one head segment and four body segments.² They are missing the parts that would correspond in Drosophila to the entire thorax and almost the entire abdomen. We think that the origin of tardigrades came with losing a big part of their ancestor’s body.

We also study how tardigrades survive extremes that animal life should not be compatible with. They are loaded with protein protectants and exceptional repair mechanisms. We have published papers on desiccation and radiation tolerance mechanisms, and for each of those, I am sure we just scratched the surface.^3,4 Animal life should not survive desiccation, and the level of radiation we can give them is 1,000 times more than what humans can survive. We are fascinated to understand these mechanisms, and the protein protectants that they make could be useful. For example, medicines come with expiration dates—tardigrades do not. We think they make protectants that could protect biomedical materials. 

What techniques do you employ to study tardigrades?

Tardigrades are easy to work with. We keep them in spring water, feed them unicellular algae, and they live indefinitely in the laboratory. However, compared to using C. elegans, there are constant challenges because fewer methods are worked out. Because of this, tardigrade research demands creativity. 

The genome of H. exemplaris is sequenced and we developed an RNA interference (RNAi) method, which we can use to study protectants. Since then, other scientists have developed CRISPR and transgenic methods. 

We started soaking the tardigrades in fluorescently tagged molecules simply to know what can get into them. We exposed the tardigrades to conditions, such as electroporation, to see if that helped the dyes get in. That project provided us with a collection of markers that can label sub-cellular compartments in an organism that was taken from the wild. This is also important for CRISPR and RNAi to avoid injecting individual animals, which is laborious.

We have also come up with some unusual ways to identify protectants. Jonathan Hibshman, a former lab member who studied desiccation, made a cDNA library so that each bacterium was expressing a tardigrade gene. Then he would torture tubes of bacteria like they were tardigrades, killing almost all the cells, and sequence the survivors. This was a clever way to identify protectants that did not depend on doing forward genetic screens within the animal. So, our research is like a hard-fought battle and we are proud when we win it.

This interview has been condensed and edited for clarity.

References

1. Aguinaldo AMA, et al. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature. 1997;387(6632):489-493. 
2. Smith FW, et al. The compact body plan of tardigrades evolved by the loss of a large body region. Curr Biol. 2016;26(2). 
3. Hibshman JD, et al. Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation. Commun Biol. 2023;6(1):121. 
4. Clark-Hachtel CM, et al. The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation. Curr Biol. 2024;34(9):1819-1830.e6. 

Teaching Old Flies New Tricks

Robert J. Wessells is using Drosophila to discover key molecular interactions and signaling pathways modulating how the body responds to exercise as it ages.

Interviewed by Nathan Ni, PhD

Interviewee Affiliations 

Robert J. Wessells, PhD

Associate Professor 

Department of Physiology 

Wayne State University

Image Caption 

MD/PhD student Maryam Safdar places vials of Drosophila on their exercise machine for a training run. Inset: Drosophila on the fly exercise machine respond to a downward drop by running up the sides of the vial.

Drosophila melanogaster is no stranger to biologists. But while the humble fruit fly is commonly associated with studies on genetic inheritance and mutagenesis, it is also becoming more popular as a human disease model. Robert J. Wessells, a physiologist at Wayne State University, has used Drosophila to study physiological responses to exercise for over a decade. Many of his discoveries involve gene and molecular interactions that are conserved in humans, with implications for our understanding of metabolism, neuroscience, and the cardiovascular system. 

How did you become interested in Drosophila as a research model?

I was lucky to have a chance to do research using Drosophila as an undergraduate, and I just fell in love with the organism as a model. At first, I was interested in developmental biology, which Drosophila is a fantastic model for. Later, as I became more curious about adult physiology, I thought, “Why not use Drosophila genetics for that as well?”

My team wants to understand the mechanisms by which exercise protects against functional decline during aging or age-related diseases. Drosophila helps us identify genetic pathways that mediate and execute these benefits, and we could potentially leverage these as pharmaceutical targets to help protect the aging population and lower the risk of various diseases associated with aging. 

We like to focus on whole animal physiological outputs because they can be tracked longitudinally, focusing specifically on improved speed and endurance. We also see if there is a correlation between whole animal outputs and the performance of isolated hearts or muscle tissue. 

What advantages are there to using Drosophila for systems-level disease research?

With flies, you have an accumulation of widely available genetic tools. The fly community is exceptionally generous and has set up enormous stock centers that freely distribute everything that the community makes. We have access to many different reagents that we do not have to make ourselves, and those kinds of support and distribution systems are not available for rodents because of the economics. That roadblock forces people to go on their own, and it slows things down.

Now, we look at how function is maintained across a lifespan, and it is much easier to longitudinally track Drosophila across their whole lifespan because it is so much shorter than rodents. From adulthood on, one day for a Drosophila roughly correlates to one year in a human. Beyond that, it is easier to work with Drosophila in large numbers, whereas getting large sample sizes is one of the biggest obstacles in rodent research. This lets us look at effects that might be more subtle or better account for human genetic diversity. For example, testing something across ten different mouse strains is prohibitively expensive, but working with ten different isolated Drosophila genotypes is much easier. 

How do you place Drosophila-derived results in context with data from murine models or human studies?

I think all model systems—including humans—have their pluses and minuses, so it is important to look at as many different organisms as possible, using the experimental tools that are particularly advantageous in each system. This is the mindset we should have, rather than placing models in a hierarchy based on how genetically close they are to humans.

Most pathways or mechanisms that we have identified so far have turned out to be conserved across species. Sometimes, the things we looked at were first discovered in rodent models. Other times, we first identified them in the fly and then worked with collaborators to show that they were also required for exercise adaptations in rodents as well. The ultimate aim is to move to humans—we are hoping to start our first human clinical trial in January 2025. 

Is Drosophila becoming more popular as a systems-level disease research model?

I am expecting a real explosion, and we are already beginning to see it. When I was a student, researchers used Drosophila to look at broad genetic questions about inheritance and study mechanisms of developmental patterning. But between the fly and C. elegans communities, so many of those problems have been solved. Now, these people are pivoting to disease modeling. Rather than looking to replicate phenotypes and functions, researchers can use Drosophila to model molecular relationships that may be important for health and disease. These models can help us narrow hypotheses and find these key interactions that can be further tested in rodent models and humans.

This interview has been condensed and edited for clarity.
 

Finding Bio-Inspiration in Slug-Made Hydrogels

Andrew Smith’s research on slug secretions yields new insights into synthetic adhesives that may one day replace medical stitches and staples.

Interviewed by Deanna MacNeil, PhD

Interviewee Affiliations 

Andrew Smith, PhD

Dana Professor

Biology

Ithaca College

Image Caption 

Slugs in the species complex Arion subfuscus are invasive gastropods commonly found across the northeastern US. These slugs produce a unique glue-like secretion as a defense mechanism and the chemistry of this naturally produced tough hydrogel is the basis for Andrew Smith’s research at Ithaca College.

With a background in comparative biomechanics, Andrew Smith’s research on adhesive biomaterials has become more interdisciplinary over the course of his research career, intermingling biochemistry and mechanical assays to investigate sticky organisms through an engineering lens. In his laboratory at Ithaca College, Smith takes inspiration from a particularly unique slug, Arion subfuscus, which oozes an abundance of glue-like material as a defense mechanism. The slug’s unique hydrogel production serves as a remarkable model for designing synthetic adhesives with medical potential.

What are the properties of good medical adhesives?

The classic medical adhesive that people use has the same chemistry as super glue. It is strong but it is a hard plastic that is not super flexible and is not suited for internal use in the human body. Ideally, a medical adhesive would be made of a material that is more flexible to match the properties of the skin, like a tough hydrogel that is strong and flexible.

Another drawback to super glue-type adhesives is that if the injury is not sterile underneath, bacteria can be trapped under the adhesive and cause a bad infection. In an emergency situation, you may want an adhesive that can easily spread over an injury, set and seal to stop the bleeding, and then can be removed at the hospital. That is the most recent thing that we figured out from the slug, how to get a gel that is tough, sets fast, and has a reversible adhesion. 

The slug secretes a material that, within seconds, turns into this rubbery, elastic mass that sticks everything together. Some slugs can climb walls and they have a little bit of stickiness, but Arion subfuscus secretes roughly five to 10 percent of its body mass in glue at a time, which spreads, sticks, and sets quickly. But if you soak the glue in a high salt solution, it will peel away.

How do you study slug hydrogels?

We used classic and modern biochemistry and molecular biology to determine the glue components, including spectroscopy and enzymatic digestion assays to determine the important ingredients, such as proteins, carbohydrates, and metal ions. We sequenced all of the slug’s mRNA transcripts and matched every protein in the glue with its transcript. 

We identified which glue proteins could stick to different surfaces. They are all modified lectins, which is a common type of protein that typically binds to carbohydrates and can be modified to bind different molecules.¹ They have many aromatic side chains, which stick to wet surfaces well. We think these lectins are involved in adhesion through both hydrophobic and charge-based interactions. That means they stick to just about anything, but high salt concentrations wash most of them off. 

How do the slugs create tough hydrogels?

Usually, tough hydrogels take a long time to set because the toughening mechanisms are a multi-step process during ingredient mixing. We investigated the slug’s secretion glands to see how the components of the glue could mix and set so quickly. 

We identified two main glands involved in secretion, but the real shock was when we looked at the gland that was producing the protein components.² We were confident that the glue’s stiff properties came from proteins and that the long carbohydrates provided stretchiness. When we looked at the protein glands, they were not homogeneous. In parts of the gland, the material looked very granular like little spots, and then in another part it looked solid. We realized that the glue was setting before it was released. That gets around the problem of making a tough hydrogel, because the slug makes microscopic bits of gel in advance and assembles them afterwards, gluing them together like bricks and mortar. It is a complex mechanism and it is hard to duplicate. But if scientists can duplicate it, it will be easy to make a tough hydrogel that will adhere to anything.

What are the future applications for bio-inspired adhesives?

I would love to make a medical adhesive that could replace stitches. I have studied gastropods enough to see that snails and slugs can produce different forms of these gels with different properties―they have a whole toolkit. The long-term goal is to make designer gels with whatever properties we want; fast setting, slow setting, more reversible, less reversible, tougher, matching the mechanics of different tissues. If you make a really cool material, who knows how it could be used.

This interview has been condensed and edited for clarity.

References

1. Smith AM, et al. Strong, non-specific adhesion using C-lectin heterotrimers in a molluscan defensive secretion. Integr Comp Biol. 2021;61(4):1440-1449.
2. Smith AM, Flammang P. Analysis of the adhesive secreting cells of Arion subfuscus: insights into the role of microgels in a tough, fast-setting hydrogel glue. Soft Matter. 2024;20(24):4669-4680.

Bear Necessities: Insights from Grizzly Bears

For biologists like Heiko Jansen, grizzly bears offer a fascinating opportunity to analyze seasonal metabolic changes relevant to human health and disease.

Interviewed by Charlene Lancaster, PhD

Interviewee Affiliations 

Heiko Jansen, PhD 

Professor

Department of Integrative Physiology and Neuroscience

Washington State University College of Veterinary Medicine

Image Caption 

Despite accumulating large amounts of fat before hibernation, grizzly bears do not experience the health complications exhibited by people with obesity. Researchers hope to leverage the knowledge gained by examining the animal’s physiology to devise new therapies to treat patients with metabolic disorders.

When Heiko Jansen joined the Washington State University (WSU) College of Veterinary Medicine, he intended to continue his work on investigating the seasonal shifts in the sheep brain that regulate their annual reproductive cycles. However, a serendipitous opportunity to examine a grizzly bear’s brain redirected his research focus. Now Jansen's team at the WSU Bear Research, Education, and Conservation Center studies bear physiology during hibernation to gain further insights into human health and disease.

How might the study of bear physiology contribute to understanding and treating human diseases?

Grizzly bears undergo seasonal metabolic changes. Compared to the active season, bears drop their metabolic rate by approximately 75 percent during hibernation. I think analyzing how the animals facilitate these alterations could improve scientists’ comprehension of some human diseases and help uncover potential treatment options. For example, if we could find an approach that increases metabolic rate in humans, we could apply that therapy to patients with metabolic disorders, such as type 2 diabetes.

Additionally, bears accumulate a considerable amount of fat when preparing for hibernation. But the animals do not exhibit the pathological characteristics observed in humans with obesity, even though the animals become insulin resistant during hibernation.¹ Unlike people with type 2 diabetes, grizzly bears can reverse this resistance in the active season. Using cell culture, we determined that adipocytes collected from hibernating bears become sensitive to insulin when cultured in bear serum obtained during the active season. This indicates that there are circulating factors that resensitize the cells to insulin. We are still tracking down at the cellular level what genes and proteins are responsible for making this transition, and we have discovered thousands of candidate genes and several potential circulating proteins.² 

How do grizzly bears compare to more traditional animal models?

During hibernation, bears do not decrease their body temperature as drastically as rodent hibernators, such as marmots and ground squirrels. This is an advantage for us, as bears experience body temperatures that are more similar to those of humans. Consequently, I think bears are a more tractable model to translate our findings to humans. 

Due to their large size, bears also provide us with ample blood and tissue samples. The grizzly bears at the WSU Bear Center voluntarily provide blood, and we reward them with honey as a positive reinforcement. Moreover, we acquire adipose, liver, and muscle tissue through anesthetized biopsies. 

However, we only have 11 bears at the facility. As a result, we have much less flexibility in terms of experimental design compared to researchers using rats or mice. My team prides itself on constructing very carefully designed experiments to obtain statistically viable data. 

How do you study bear cells during hibernation? 

My team collects adipose and blood samples from the grizzly bears during the active season and hibernation.³ We then cryopreserve the preadipocytes and keep the separated serum in ultra-cold storage. When running an assay, we expand the cells and induce their differentiation into adipocytes in the presence of the serum. By freezing the cells, we do not need to perform invasive procedures on the animals continually. Additionally, this setup allows us to directly compare cells from both seasons in the same experiment, including examining differences in the adipocytes’ oxygen consumption and extracellular acidification rates or gene expression.^4,5

What are your long-term goals for this research? 

When we started this work 10 years ago, we thought that once we pinpointed these insulin sensitizers, we could potentially devise new therapeutic approaches to treat type 2 diabetes and obesity. Nowadays, glucagon-like peptide-1 (GLP-1) drug development has taken the forefront. However, we are still very interested in understanding the mechanisms behind the seasonal insulin sensitivity switches in bears because I think this process is different from how the existing drugs work. Physicians could then use a novel drug based on this research alone or in conjunction with GLP-1 analogs. Ultimately, there is no shortage of targets that we can uncover in this work, but it is going to take time to develop a therapy.

Despite the reduction in metabolic rate, we also observed that hibernating grizzly bears exhibit active circadian rhythms.⁵ This was surprising because they are energetically expensive to produce. As a result, circadian rhythms must be important to the animal’s physiology, and we plan to continue exploring how these cycles relate to their metabolism. 

This interview has been condensed and edited for clarity.

References

1.    Rigano KS, et al. Life in the fat lane: Seasonal regulation of insulin sensitivity, food intake, and adipose biology in brown bears. J Comp Physiol B. 2017;187(4):649-676.

2.    Saxton MW, et al. Serum plays an important role in reprogramming the seasonal transcriptional profile of brown bear adipocytes. iScience. 2022;25(10):105084.

3.    Gehring JL, et al. A protocol for the isolation and cultivation of brown bear (Ursus arctos) adipocytes. Cytotechnology. 2016;68(5):2177-2191.

4.    Hogan HRH, et al. Changing lanes: Seasonal differences in cellular metabolism of adipocytes in grizzly bears (Ursus arctos horribilis). J Comp Physiol B. 2022;192(2):397-410.

5.           Vincent EP, et al. Circadian gene transcription plays a role in cellular metabolism in hibernating brown bears, Ursus arctos. J Comp Physiol B. 2023;193(6):699-713.