Session Two (Part 2): Case Studies and Examples

Moderator: Sharon Shriver, Public Responsibility in Medicine and Research

In Session Two, Part 2, senior director Sharon Shriver introduced speakers who presented case studies and examples related to the laws, regulations and permits associated with fish and wildlife discussed in Part 1. Presentations included unexpected complications with capture of some non-target animals, such as exotic species not under study and endangered species; venomous and dangerous species; concerns associated with capture; and ownership of the animals and data from them (e.g., the Kyoto Protocol).

UNIQUE CHALLENGES WHEN WORKING WITH FREE-RANGING WILDLIFE: HUMMINGBIRDS AS A CASE STUDY

Lisa A. Tell, veterinarian and faculty member with the University of California (UC), Davis, School of Veterinary Medicine highlighted challenges researchers face when working with wildlife compared to traditional laboratory animals. Tell, who has worked with wildlife for more than 30 years, was trained clinically at the National Zoological Park, is boarded in avian and zoological medicine, and oversees the UC Davis Hummingbird Health and Conservation Program. Tell commented that, although working with wildlife is a wonderful experience, unique challenges arise from the substantial diversity in anatomy, behavior, and physiology. Investigators find themselves working in an uncontrolled environment having to balance the needs of the animals and the investigative team while still trying to accomplish research objectives, she said.

Tell explained that as a veterinarian who has worked with birds throughout her career, she realized that there is a steep learning curve when working with wildlife. When working with free-ranging wildlife, Tell finds that it pays to plan ahead, because permits and Institutional Animal Care and Use Committee (IACUC) approval must be obtained before work can even start. Ingenuity is also helpful, she added, because there are fewer commercial products for wildlife compared to laboratory animals. Due to the unpredictability of wildlife and the working environment, meticulous planning is important for protecting animal and human health. Lastly, a mental commitment to spending more time on tasks such as data reporting, developing field protocols, and planning for emergency situations is indispensable when working with wildlife, she said. Tell has written a monograph, published by Texas Tech University, to help people navigate the process and it highlights the importance of having a welfare conscious approach when working with wildlife. Although her monograph is hummingbird-specific, it does include information on permits and other general concepts that might be helpful for other animal species (Tell et al. 2021).

Obtaining permits and IACUC approval for wildlife research can be difficult, Tell explained. Depending on the type and location of the study, multiple federal permits may be necessary, as well as potentially state and land use permits. Given that the IACUC committee members might not be as familiar with wildlife species, getting institutional approval might require educating committee members, which may delay the process. A common dilemma is that often the IACUC may want to see approved permits, while the permitting agency may want IACUC approval before it will issue the permits. It is important that investigators understand the conditions of their permits, so they can ensure that all performed activities are authorized. For example, holding a hummingbird in captivity for less than 24 hours for research purposes is only covered by a U.S. Fish & Wildlife Service (FWS) permit. Meanwhile, a Bird Banding Laboratory permit allows a researcher to hold a hummingbird in captivity for less than 24 hours only to ensure its safety and well-being, but not for collecting research samples such as urine and feces. It is important that researchers understand these nuanced distinctions so that they can remain compliant with the various rules and regulations, Tell said.

Working with free-ranging wildlife presents many new situations and special considerations, Tell explained, and researchers do not have the advantage of the commercial products, a controlled environment, and standardized protocols found in the laboratory environment. The training necessary to properly handle wildlife is often extensive and species-specific, she said. In the case of hummingbirds, knowledge of their anatomy is fundamental for safe handling. Because birds do not have a diaphragm, the keel has to be able to freely move up and down so that air can move in and out of the respiratory tract. To demonstrate the complexities of hummingbird restraint, Tell provided an image depicting a method of restraint for eye examination, emphasizing that there was trial and error and ingenuity involved to balance the needs of both the bird and the investigator. When working with some species of free-ranging wildlife, researchers need to determine the age, sex, and species of the animal, whereas with laboratory animals, this information is commonly known. In the case of hummingbirds, it is easy to identify the age, sex, and species of adult males, but it takes specialized training to be proficient at identifying the species of adult females and immature males and females. Inspecting a bird’s beak for corrugations can help researchers to age the bird, while investigating tail feathers can help with identification of the sex and species.

For research studies, animals are often tagged or marked to identify individual animals. One of the factors to consider with free-ranging wildlife is how marking or tagging will impact the animal, which can then be balanced with the research objectives. Tell explained that she uses bands to identify individual hummingbirds, and these bands must be extremely small due to the small size of hummingbirds, which range anywhere from 2.5 to 4.5 grams of body weight. Tell utilizes a band holder when working in the field to secure the bands so that she does not lose them. Additionally, she uses pit tags to identify individual birds, track their presence at research sites, and reliably get tag reads. To ensure she obtains the necessary amount of data, Tell designed a system that requires birds to fly through an antenna to get access to the hummingbird feeder. Once a hummingbird is inside the enclosure, the bird’s pit tag is read every 10 seconds, and because hummingbirds tend to fly to the highest point, the top of the enclosure is open for birds to exit. Even a simple task of animal identification may require extra efforts when working with wildlife, Tell commented.

Blood sampling is routine in a laboratory animal facility, but according to Tell, it requires special considerations when working with free-ranging wildlife. If possible, and if it does not disadvantage the animal, she marks individual animals so that they are not resampled inadvertently. She also tends to reduce the traditional blood sampling volume to maximize fitness after release, because the animal’s health status is unknown, or it may lose a little bit more blood than expected. Cutting a bird’s toenail is one method for obtaining blood samples, but Tell explained that because they have bones in their toenails, it is important to make sure the bone is not amputated when taking a blood sample. Therefore, as a best practice, she makes sure to bleed them when ambient temperature is warm, so they have good blood flow to their toes. Additionally, she cuts only the tip of the toenail to ensure that she is not cutting at the level of the bone. Taking feathers from birds is another common sampling technique, but because free-ranging wildlife are not in temperature-controlled rooms, it is important to ensure proper thermoregulation. Thus, she makes sure to minimize the number of feathers taken when sampling feathers from hummingbirds and take them from different areas, making sure there is never any exposed skin. When sampling tail feathers, she is mindful of the fact that for some species of birds, the males’ tail feathers make chirping sounds that attract females during the breeding season. Sampling primary feathers is extremely rare, and they should never be sampled when the hummingbirds are migrating, she said.

When working in the field with free-ranging wildlife, another added responsibility is to ensure the safety of the research team. Tell mentioned that researchers have to constantly watch for weather conditions, and in California, where she and her team work, active fires are an issue. Tell emphasized that mapping out the most efficient route to the nearest hospital could save someone’s life. In her case, the research team commonly encounters bees when working in the field, and because epinephrine cannot be administered to someone unless they have a prescription for an epinephrine auto-injector (EpiPen®), she advises people to bring fast-acting diphenhydramine, even if they do not have a known allergy to bee stings. In addition, when working in areas with rattlesnakes, she makes sure to know which hospitals have

anti-venom emergency protocols. If possible, Tell commented that it can be helpful to have team members take specialized training for emergency support measures.

Tell also talked about the challenges of euthanizing animals in the field. If an overdose of inhaled anesthetic is going to be used, researchers must be careful to avoid any fluid leaks so that the person driving and transporting the anesthetic is not endangered. Tell prefers not to have controlled substances in the field, and she therefore has a mini carbon dioxide chamber available that she designed for hummingbirds in case they are permanently injured and have to be euthanized. The chamber has an outlet hole at the top and a small carbon dioxide cartridge connected to a regulator that helps control the release of the carbon dioxide. Tell noted that an animal euthanized in the field with an overdose of drugs should be removed to avoid secondary poisoning of another animal.

In closing, Tell emphasized that working with free-ranging wildlife takes commitment, an ability to think outside the box, strong organizational support, and good time management skills in order to be successful in achieving research goals and spending grant money within the funding period. Overall, however, she has found that working with free-ranging wildlife is a privilege and can be extremely rewarding.¹

CONDUCTING BIODIVERSITY SURVEYS IN THE NEW AGE OF WILDLIFE DISCOVERY

Lawrence R. Heaney, the Negaunee Curator of Mammals at the Field Museum of Natural History in Chicago, discussed issues that come up with conducting biodiversity surveys in the current age of discovery of new species; he focused on mammals in particular, but stated that the issues are broadly applicable to many other groups of organisms. Heaney challenged the widespread perception that mammals as a group are relatively well known, and that scientists have a basic understanding of how many species there are, approximately where they live, and their ecology. Research performed over recent years has demonstrated that this is not the case, he said. A 2018 review by Burgin et al. examining the number of recognized species of mammals from 1750 through the 2010s revealed that the number of increases in the species of mammals recognized had increased steadily in the 2010s, with approximately 418 species added to the list, at least half of them new discoveries of mammals (Burgin et al. 2018).

Heaney said that humans have a long way to go before understanding mammalian diversity sufficiently and provided an example from his research that he and his collaborators conducted in the Philippines. Like many tropical countries, the Philippines has experienced a tremendous amount of habitat destruction throughout the country. In 1900, it is estimated that 70% of the country was covered by old-growth rainforest, with some additional second-growth. By 1992, less than 8% of the original old-growth still remained, accompanied by roughly 12% second-growth. Since then, the amount of remaining old-growth forest has declined slightly to around 6%, but the amount of second-growth has actually increased to approximately 18%. Heaney explained that this loss of old-growth forest due to agricultural expansion and logging activity has triggered erosion and periodic flooding, leading to devastating economic and social consequences for the country.

While in the Philippines, Heaney and his colleagues studied mammals such as cloud rats, which live at high elevations in the cloud forests. Ranging in body size from around 6 pounds to only about 18 grams, cloud rats are arboreal animals that feed on only plant material and are sometimes described as the Philippine equivalent of Madagascar lemurs. Heaney also studied a group referred to as earthworm mice, which live on the surface of the ground and feed mostly on various sorts of invertebrates, particularly earthworms. This is a morphologically diverse set of animals, ranging less in body size than the cloud rats, but much more diverse in their foraging ecology. Heaney said, it is known that the common ancestor of each of these two groups arrived in the Philippines a long time ago. In each case, these entire branches on the Tree of Life occur only within oceanic portions of the Philippines. In the case of cloud rats,

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¹ Tell’s video showing a successful release of a hummingbird can be viewed at https://www.nationalacademies.org/event/02-09-2022/animal-welfare-challenges-in-research-and-education-on-wildlife-non-model-animal-species-andbiodiversity.

Heaney says it is estimated their common ancestor arrived 14 or 15 million years ago and has been diversifying within the Philippines, mostly within Luzon Island, ever since. Similarly, earthworm mice comprise an entire branch on the Tree of Life that occurs nowhere else in the world, not even elsewhere in Southeast Asia, as most of them are living on Luzon Island. Their common ancestor arrived about 8 million years ago, and they have been evolving and diversifying there ever since.

Heaney said that research he and his collaborators conducted in the year 2000 revealed that the mammals known from Luzon Island, the largest island in the Philippines, revealed 28 recognized species of native, non-flying mammals (excluding bats), 20 of which occurred nowhere else in the world, not even elsewhere in the Philippines. One of their immediate questions was Why aren’t more endemic species there? Why are there some places where we know of no endemic species of mammals? Looking at the data, the answer became immediately obvious: “literally no one had gone to look,” Heaney said. Thus, they began a program of comprehensive biological surveys concentrated particularly in these areas where no mammal biodiversity surveys had been conducted previously. Heaney and his colleagues continued these surveys from 2000 to 2012, accumulating a total of 37 team-months in the field collecting specimens and voucher specimens for taxonomic studies that Heaney said allowed them to document the extent of habitat and use for animals in these areas. Heaney added the result of that field-based biological inventory was even more dramatic than expected at the outset in 2000 and what is now known. The number of known native mammal species living on Luzon Island doubled as a result of the 28 previously unknown species of mammals discovered in the course of the project (Balete et al. 2011; Heaney et al. 2016), with 93% of those species occurring nowhere else in the world, not even elsewhere in the Philippines.

These discoveries have also had significant impacts on animal conservation in a country where habitat destruction and environmental degradation are critical issues, Heaney said. He mentioned that the locals and media in the Philippines were excited to learn about these unique species. As part of their efforts to promote conservation in the areas where they worked, the team produced posters depicting the animals; furthermore, he said they printed hundreds of copies, distributing them to universities, museums, conservation organizations, and government agencies in particular. When the government updates the Philippine Red List of Threatened Wild Fauna and produces action plans for protected areas, data on centers of endemism will be important for planning (Mendoza and Malari 1997; Ong et al. 2002). Heaney shared that his work has therefore aided conservation efforts by promoting the development of new national parks. Heaney commented that, although the creation of protected areas by the government is a complex process with many individuals and issues involved, the discoveries of these mammals has had a significant impact. In addition to encouraging the development of new national parks, the team’s survey data brought increased attention to “paper parks” that were previously declared but largely ignored, while also supporting the declaration of areas previously under consideration as national parks (Tello 2006).

Heaney said that while there is value in performing this kind of research, researchers encounter many challenges along the way. For example, an IACUC typically pose questions such as Which species will be the target of the research? How many specimens will be collected? How abundant is this species typically in the area? Where is the research site? Heaney pointed out that, by definition, researchers in this field do not know what species they will encounter. The primary purpose of the research, he said, is to discover new species and obtain the first information on distribution, evolutionary history, and ecology of these animals, so that questions can be answered in the future. Heaney emphasized that once researchers leave the United States and arrive in another country, they are obligated to follow all legal requirements, which are not always consistent with those in the United States. He gave an example of one time when he was recommended to use a certain euthanasia drug to collect voucher specimens for taxonomic and anatomical studies, but if he had used that euthanasia drug or even had it in his possession, he would have been immediately subject to arrest and imprisonment. In addition to other country’s legal requirements, cultural practices must also be respected, and Heaney highlighted that this may require some flexibility. He has worked in some places, for instance, where the local people welcomed them and allowed them to conduct research, but only under the condition that no one went out into the forest at night. Therefore, flexibility was necessary in regard to when traps were checked.

Heaney closed by saying that 40 to 50 previously unknown species of mammals are discovered around the world each year and that these numbers are increasing steadily. Heaney said, in fact, there is every reason to think that during the 2020s, 500 or more species of mammals previously unknown will be formally described. The information on patterns of diversity and ecology gathered as a result of these discoveries and formal descriptions will be essential to promoting successful conservation, he said. However, in order for this research to proceed, a certain level of flexibility with regulations may be necessary, as well as simultaneous maintenance of the high standards for animal care and use.

FIELD RESEARCH INVOLVING REPTILES AND AMPHIBIANS, REMOTE STUDY SITES, AND UNDERGRADUATE STUDENTS

Heather L. Bateman, associate professor at Arizona State University, focuses on applied research, studying the relationship of amphibians and reptiles to management actions, such as controlling nonnative plants, as well as to the natural resource values of wild and scenic streams. Bateman explained that working in river systems means working across different jurisdictions. For example, she conducted a project that examined how well reptiles and amphibians respond to habitat changes brought about by biocontrol on the Virgin River in the Southwestern United States. Bateman had to acquire scientific collecting permits from two federal entities, including a national park and the Bureau of Land Management (BLM), as well as from three states in the west. In addition, IACUC protocols had to be followed. Bateman provided some examples of how field research can be complicated working across different boundaries and collecting data for 12 years for a long-term mark and recapture project.

Bateman provided a brief overview of the main live trapping method used during mark and recapture research, called the classic drift fence array, which consists of several elements. The drift fence intercepts animals as they are moving across the landscape and directs them into the central area, where a pitfall trap is located. This setup is often used in the Southwest for catching animals like lizards, smaller snakes, and anurans, although it is not effective at catching large-bodied snakes or large lizards like chuckwallas and desert iguanas, Bateman said. She added that the equipment is not something that can be easily purchased online; the elements of each trap have to be built in place, so gear has to be carried out to the site.

As part of the protocol, traps are checked every 24 hours, Bateman explained. Once an animal has been captured, it is measured, identified, and given a unique mark. Her team has made refinements to their methods over time; because some of the projects are more than a decade long, the state of knowledge can change in regard to animal handling and understanding of vertebrate pain, for example. There are a variety of different methods that researchers can use to mark animals and many factors to take into consideration in terms of pairing the method to the research question. Bateman and her team have chosen to use toe clippings to mark lizards, because these marks are permanent, and individual animals can thus be tracked over time. Additionally, the animals being caught range in age, so hatchling lizards that are 0.25 grams, for example, cannot be marked with a pit tag.

Bateman’s team has made modifications to its methods in response to the IACUC goal to reduce, refine, and replace. They started using lidocaine for pain during toe clipping and decided not to mark all taxa, she said. In another example her team refined its methods by adding polyvinyl chloride (PVC) pipes in order to reduce predation while animals are in the traps and act as a refuge, allowing the animals to space themselves out and reduce stress. The addition of PVC pipes to traps is now part of the standard method. Bateman commented that communication and making refinements during the field season were helpful.

Unexpected situations may arise when conducting fieldwork. Bateman explained that during a project on the San Pedro River, as its field technician was preparing to deploy acoustic loggers to record toads at night, the team realized the field site was on fire. She highlighted the importance of keeping everyone safe, stating that their standard operating procedures (SOPs) include a list of emergency phone numbers in case team members need to call Wildland Fire, U.S. Forest Service. The team was able to communicate with the IACUC and notify them that the team would not be able to follow the protocol of

checking traps in a 24-hour period. Another example of a potential unexpected challenge in fieldwork is a road closure. Because Bateman and her team often work in relatively rural areas, they encountered a situation in which the county was replacing a cattle guard for 3 days and had blocked the road, preventing access to their research sites. Her team made sure to notify the IACUC in this instance as well. According to Bateman, this is fairly rare, and there have been only a few occasions where they have not been able to check in a 24-hour period; however, she emphasized that it is crucial to maintain good communication with the IACUC, because sometimes the unexpected does happen. In her final example of unexpected circumstances, she illustrated issues with record heat and rain as her team was collecting Sonoran Desert toads with pitfall traps. After a record amount of rainfall during the summer, the recruitment of toads increased substantially; despite never seeing more than 8 toads throughout 12 years of collecting data, her team encountered close to 80 toads within that 3-day period. Some mortality of toads did occur due to desiccation in the field, so team members communicated with the IACUC and refined their methods by adding sponges and tubs of water to the traps.

Bateman’s laboratory includes both graduate students and also undergraduate students who require extra support in guidance and training on good practices when working with rare, threatened, or endangered species, and working under permits. Bateman noted that for many undergraduate students, this may be the first time they have had a job in the field or even one in general, so students often come without much experience. Bateman addresses topics with students such as social media given that it is important that they know what can be shared and how it needs to be worded. She said she often encourages undergraduate students to start thinking about their own inquiries and interests, as well as data they could collect while they are working on projects. Also, participating in field trips with wildlife professionals allows the students to visualize career paths that they may not have known about beforehand. Bateman said she carries with her the IACUC protocol so that students can handle animals accordingly during field trips and projects.

In addition to permits for animal handling, the team also has safety protocols in place to keep people safe in the field. Bateman described how SOPs outline protocols for activities like field trips, stating that she is becoming more explicit about what students should expect in the field. The population at Arizona State University mostly comes from Phoenix and the surrounding areas, so students do not necessarily have a hiking and camping background. Therefore, Bateman makes sure to include a checklist and information on types of gear and food to bring, how to prepare meals, and sanitation in the field.

In addition to field personal protective equipment (PPE), students conducting fieldwork must wear protective clothing to protect them from potential hazards in the field, such as the sun, thorny plants, and venomous reptiles. Bateman expressed her excitement about hosting a field fashion show, which she had seen other programs do as well. Students with field experience will be recruited to model what can be worn in the field to stay safe. She added that gear can be expensive and that a lot of shopping can be done at second-hand stores. The field fashion show will also include a gear drive. Bateman mentioned that in addition to gear gathered from her laboratory’s own collection, the Arizona Game and Fish Department had donated several backpacks for students to carry.

Bateman listed several resources for reference related to equity and inclusion in a field environment. Authentic and hands-on experiences benefit students, she said, especially students from underrepresented groups. It is important to realize, however, that some individuals may be harassed in certain settings and that being in the field may put people into uncomfortable or dangerous positions, whether they are working across private land or in an urban setting (Barber et al. 2020; Chaudhary and Berhe 2020; Cronin et al. 2021; Demery and Pipkin 2021). Strategies Bateman employs in her work in urban ecology include working in teams, so there are multiple people together, and potentially having individuals in official-looking attire to avoid harassment. She emphasized that it is important for supervisors to educate themselves about the risks of negative field environments. Wrapping up, she provided a list of recent publications talking about making science more equitable (Barber et al. 2020; Chaudhary and Berhe 2020; Cronin et al. 2021; Demery and Pipkin 2021).