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DOE OSTI2021

Tribal Revegetation Project Final Project Report: 92-Acre Area, Area 5 Radioactive Waste Management Complex, Nevada National Security Site, Nevada

Nuwu (Southern Paiute), Newe (Western Shoshone), and Nuumu (Owens Valley Paiute) are linguistically related, Numic-speaking peoples who are part of the broader Uto-Aztecan language group. Numic peoples view the land as a holistic, living, sentient being with feelings and purpose. The land is personified with human characteristics and it needs to be experienced to be understood through “learning by doing.” Numic peoples do not support ground disturbing activities within their ancestral lands, including activities tied to the storage of low-level radioactive waste or classified materials on the NNSS, which they view as culturally inappropriate. These deep-rooted ancestral connections are the impetus for reinforcing Numic responsibility for healing disturbed areas by integrating respect and patience with consistent Tribal interaction. Tribal Ecological Knowledge (TEK) is the science of describing Tribal approaches for understanding natural resources. Numic TEK is embedded in traditional teachings learned incrementally over time though experience and it evolves through lessons learned and responses to environmental changes over millennia. Therefore, TEK can broaden and enhance Western scientific knowledge associated with revegetation, especially in highly disturbed areas. The project blended TEK with Western scientific ecological methods to create a vegetative cover within test plots on the 92-Acre Area located at the Radioactive Waste Management Complex (RWMC) located in Area 5 on the Nevada National Security Site (NNSS). The vegetated test plots were systematically created for the Department of Energy (DOE) in tandem with the existing Federal Facilities Agreement and Consent Order (FFACO) with the Nevada Department of Environmental Protection (NDEP). Three previous contractor-lead attempts at revegetation, one targeting full cover revegetation and two targeting test plot revegetation, did not achieve the anticipated results at this location. When presented to the 16 American Indian Tribal nations and affiliated groups with cultural and historical ties to the NNSS, the group appointed a Tribal Revegetation Committee (TRC) that included six expert Tribal knowledge holders to collaborate with an ethnoecologist/cultural anthropologist and two biologists. Project design, planning, seed and outplant selection, spiritual land preparation, and methodology were guided by the TRC and an ethnoecologist/cultural anthropologist from Portland State University (PSU) and biologists from Desert Research Institute (DRI). Using TEK, the TRC recommended a specific seed mixture that contained nine native plant species and three species of outplants. The revegetation effort included preparing and planting thirty 10 m × 10 m (32.8 ft × 32.8 ft) seeded plots, twelve of which also included outplants; and eight 10 m × 100 m (32.8 ft × 328 ft) plots that only received outplants, all atop a waste cell cover. The TRC and the project team creatively adapted TEK with Western scientific methods so that the planned revegetation efforts could occur within the safety and security parameters of the RWMC. Test plots were subjected to one of five soil treatments with varying combinations of straw or mulch applications, soil amendment, and/or outplant planting and one of two watering treatments (watered or unwatered). Planting was divided into two events: one in the fall season and another during the subsequent spring season based on TEK and a corresponding recommendation from the TRC. Monitoring and spiritual management was conducted by the TRC to evaluate plant progress on a monthly basis (in conjunction with the biologist and anthropologist) during each respective growing season for a period of three years after planting. This approach allowed Tribal members the opportunity to conduct traditional blessings and other culturally appropriate activities to restore balance to the land in accordance with Tribal protocols. Following TEK-guided methods, successful plant establishment from seed stock and outplants was observed in many plots. Overall, plots planted in the spring, as recommended by the TRC, showed higher rates of outplant survival and native seedling emergence than those planted in the fall. This finding is significant because it is contrary to the original guidance and previous approaches provided for planting in this region. The TRC believes the frequent co-occurrence of native seedlings near surviving outplants indicates an important symbiotic relationship understood by Tribal communities and overlooked by others. Watered outplants displayed much higher survivability than unwatered plants, even after watering was reduced after the plants were established. Soil amendments and mulch created higher densities of native plants from seed. Many native seedlings showed significant delays in germination, which is considered a normal adaptation to desert climates. Some native plants did not germinate until the third growing year, whereas others germinated during the first growing year, which demonstrates the complexity of the desert environment. Evidence of native insects, reptiles, mammals, and birds, as well as native plants that were not part of the planted species, were noted and considered culturally significant. Despite the presence of non-native plants, native outplants continued to thrive and the incidence of native plant germination from seed increased over time. These successful revegetation results where previous efforts were unsuccessful reinforce the importance of integrating regionally appropriate, TEK-guided methodology with Western science to achieve positive results and the necessity of integrating Tribal involvement in all stages of the revegetation effort. Expanded approaches coupled with Tribal knowledge and tools from Western science addressed a complex problem tied to revegetating atop a low-level radioactive waste cell. The level of Tribal participation serves as a progressive model for building collaborative relationships and addressing ecological challenges on the NNSS.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Environmental Effects Assessment for Proposed Offshore Wind Farm off the Coast of Grays Harbor, Washington

Grays Harbor Wind LLC (GHW) is proposing to develop a floating offshore wind farm offshore of west Grays Harbor County, Washington (Grays Harbor). The proposed GHW Offshore Wind Project (Project) would entail construction, installation and operation of a 1,000-megawatt (MW) offshore wind farm consisting of approximately 75 floating units, each containing a floating foundation and wind turbine generator (WTG). The Project location is approximately 25 miles (21.7 nautical miles [nmi]) offshore west of Grays Harbor, at waters depths of 360 to 700 feet. The Pacific Northwest National Laboratory (PNNL) was contracted by Herrera Environmental Consultants, Inc. on behalf of GHW to carry out this preliminary scoping study to evaluate baseline conditions and potential effects on fish and marine mammals from development and operation of a floating offshore wind farm installed within a designated area off the coast of Washington1. Floating offshore wind units installed in an ocean environment as part of the Project would interact with marine wildlife. This Study report provides an initial data aggregation and analysis, using publicly available data, of the Project effects, both negative and positive, on the marine environment. The scope of this assessment is limited by the fact that the Project development is presently at the conceptual level. Data on marine organisms were aggregated and evaluated; however seabirds were evaluated by Herrera Environmental Consulting and, along with bats, were not included in the scope of this study. Significant additional work is necessary to characterize ocean, seafloor, and environmental conditions; select appropriate floating offshore wind technologies; identify construction methods and locations; and assess facility locations, including electrical interconnection. Data aggregation and analysis of seabirds and bats is also needed. Evaluation of the full range of potential environmental effects would be conducted following an award of a lease from the Bureau of Ocean Energy Management (BOEM) as part of the leasing, National Environmental Policy Act (NEPA)/State Environmental Policy Act (SEPA) environmental review and permitting processes. While this initial data aggregation and analysis uses best available public scientific information and current assumptions about the Project configuration, the effects discussed herein are based on the status of review to-date and may change as Project-specific details are developed.

17 WIND ENERGY↗

DOE OSTI2021

Brain Connectivity Workshop Series Report

The National Institutes of Health (NIH) BRAIN Initiative aims to accelerate development and application of technologies that show how individual brain cells in complex neural circuits interact at the speed of thought and action. Complete wiring diagrams of the mammalian brain will revolutionize the capabilities of researchers to formulate and test models of how activity within brain circuits drives coordinated function and behavior. A top priority for the BRAIN Initiative, motivated by the strategic guidance of the BRAIN 2.0 Working Group report, is to muster the resources and foster the collaborations needed to generate these wiring diagrams at the level of long-range projections (“projectomes”) and synapses (“connectomes”) in whole mammalian brains, including those from rodents, humans, and other large-brained mammals. Achieving the scale and resolution necessary to characterize and use these projectomes and connectomes will not only push the boundaries of current imaging and computational capabilities to generate faster, cheaper, and more scalable technologies, but also drive broad innovation in data science, artificial intelligence, and machine learning.

42 ENGINEERING↗

DOE OSTI2021

A Miniaturized Long-Life, Low-Frequency Acoustic Transmitter for Fish Tracking in Marine Environment - CRADA 447

Advance state-of-art technologies for tracking marine mammals, fish, sea turtles, and other protected species, based on acoustic telemetry. Objectives include: (1) develop a new acoustic transmitter that has a greatly improved detection range and service life while being significantly smaller and lighter than the existing commercial counterparts; and (2) reach a TRL of 5 so the technology is ready for organizations interested in small- or medium-scale field trials.

42 ENGINEERING↗

DOE OSTI2022

TEAMER: DAISY Flow-noise Testing (Abstract)

Evaluating impacts of marine energy devices is generally difficult given the dynamic environment where these devices need to be placed. The University of Washington’s (UW) DAISY (The Drifting Acoustic Instrumentation SYstem) is designed to measure radiated noise around marine energy converters operating in energetic waves and currents. In currents, a primary limitation for measurement fidelity at low frequencies (< 100 Hz) is the potential for non-propagating “flow-noise” to mask propagating sound and inflate estimates of the radiated noise from marine energy converters at frequencies that overlap with hearing sensitivities of fish and some marine mammals. While free-drifting measurements help to minimize the relative velocity that produces flow-noise, significant levels were still observed during initial DAISY tests. This motivated the development of a fabric “flow shield” around the hydrophone that disrupts both flow-noise generation mechanisms proposed by the initial tests. First, the flow shield is a source of substantial drag which keeps the hydrophone package moving with the approximate velocity of the surrounding water, compensating for differential wind or current forcing on the surface expression. By minimizing relative velocity around the hydrophone, turbulence shed by the hydrophone is also minimized. Second, the flow shield creates a largely quiescent pocket around the hydrophone, minimizing advection of free stream turbulence over the hydrophone element. Field data collected in these experiments will test effectiveness of these flow shields and provide quantitative data use and deployment.

16 TIDAL AND WAVE POWER↗

DOE OSTI2024

'Omics and Big Data in Harmful Algal Bloom Research

Phytoplankton, a group including eukaryotic microalgae and cyanobacteria, play a crucial climate role converting CO 2 into organic carbon through global primary production. They support a wide range of life, both freshwater and marine, from zooplankton to fish and mammals. While they are essential in nutrient cycles, certain phytoplankton species can proliferate excessively under favorable conditions, leading to harmful algal blooms (HABs) that pose significant threats to human and ecosystem health through the toxins they produce.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

CRCNS US-France Research Proposal: Collaborative Research: Encoding reward expectation in Drosophilia

The fruit fly Drosophila melanogaster has been a valuable model for investigating the genetic and neural bases that underlie learning and memory. Early and most current studies use basic behavior conditioning protocols to study learning in controlled laboratory settings. More recently, the ability to transgenically manipulate many of the brain neurons in the fruit fly with exquisite specificity, and the recent knowledge of the synaptic ‘connectome’ of the fruit fly brain, makes these animals almost unique as a comprehensive model for studies of learning, memory and motivated behavior. In fact, the connectome has revealed many types of new connections that had until now been overlooked. Within this context, the thesis of this proposal is that studies of learning and memory will be greatly enhanced by using more sophisticated means for evaluating memory representations, such as have been developed in vertebrates, and combining those studies with information from the connectome guided by computational modelling. We propose to push beyond the boundaries of existing conditioning protocols for fruit flies to investigate more complex memory representations. In particular, we will investigate the function of reinforcement pathways in relation to the absence of expected reinforcement. More specifically, we propose a series of experiments designed to investigate the memory representations in fruit flies when an expected consequence of a Conditioned Stimulus (CS) fails to occur. Although studies have evaluated how this failure can establish extinction memory for the CS, our studies will go beyond studying extinction. Specifically, we predict that in Drosophila when a CS is associated with a failed expectation of an appetitive food reinforcement it will acquire aversive value, and vice versa for a failed expectation of an aversive reinforcer. We combine these studies with manipulations of reinforcement pathways in the CNS inspired from the connectome, iteratively knitted in with established computational models. Intellectual Merit: The concept of reinforcement expectation and incentive contrast have been influential in the development of studies of associative learning in mammals. These questions are particularly challenging to answer in vertebrates because they require exquisite cellular, temporal, and genetic specificity of experimental manipulations. The recent development of work with identified neurons and their connectomes makes the larval and adult fly brains ripe as models for pushing our understanding of neural bases for these higher- order conditioning phenomena. Broader Impacts: Public health: These analyses and the conceptual framework of prediction error processing underlying them have a profound impact on our understanding of reinforcement-related behavior in humans, including monetary rewards and the mnemonic consequences of traumatic experiences, and for pathologies of the dopamine reinforcement system. Educational: This project will provide interdisciplinary training for postdoctoral researchers, Ph.D. and undergraduate students. The PIs will act as co-supervisors or mentors of students working in the different labs via face-to-face and internet-based technologies. We will also work with ASU’s award-winning Ask- A-Biologist program. This is an online science program designed to enrich the learning experiences of students of all ages and to provide classroom material for use by K-12 teachers. We will develop an extension of a game developed under a prior NSF award, and the new game will include modules to teach K-12 students about how insects learn. We will also integrate into the AAB site a program developed by a collaborator (B Gerber) at the Leibniz Institut für Neurobiologie, Magdeburg, and now in use in schools in Germany, to teach K-12 students how to train animals using the fruit fly larval learning paradigm. Underrepresented groups: All PIs will work with their university offices of Academic Diversity and Equal Opportunity for reaching underrepresented students.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Burrowing Owl Conservation Report: 2020

The Western Burrowing Owl (Athene cunicularia) is a small migratory species of owl found throughout the Western United States. Burrowing Owls are found throughout a variety of habitats including shrubsteppe; and during the breeding season the Hanford Site is situated at the center of the predicted distribution of Burrowing Owls in Washington State (Smith et al. 1997). The Western Burrowing Owl is declining over much of its range, including on the Hanford Site and throughout the Columbia Basin (HNF-59375, Conway 2006). These declines have been attributed to habitat loss and a subsequent loss in burrowing mammals that the owls depend on for nesting habitat (Conway et al. 2006, Johnson et al. 2010). The Washington State Department of Fish and Wildlife lists Western Burrowing Owls as a Candidate Species, meaning the species is a candidate for listing as Sensitive, Threatened, or Endangered in Washington (WDFW 2020). Without intervention, continued declines may result in the extirpation of Burrowing Owls from the Hanford Site and the Columbia Basin.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Summer Bat Monitoring Report for Calendar Year 2024

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Summer Bat Monitoring Report for Calendar Year 2023

Bats are mammals belonging to the order Chiroptera, which means “hand-wing.” They use a thin membrane of skin stretched between their fingers to fly. All bat species known in the state of Washington are insectivorous and each bat can consume 600-1,000 insects per hour while feeding. Bats typically give birth once per year to a single pup, making bat populations vulnerable to impacts and slow to recover. Several species of bats have been documented on the Hanford Site, with nine species identified during The Nature Conservancy surveys in 1997 and 1998, and an additional seven species listed as potentially present as documented in Biodiversity Inventory and Analysis of the Hanford Site. Final Report 1994-1999 (Soll et al., 1999). Ongoing U.S. Department of Energy (DOE), Richland Operations Office (RL) acoustic surveys, including HNF-53759, Summer Bat Monitoring Report for Calendar Year 2012, have also confirmed the presence of the same nine species documented by Soll et al. (1999). An additional species, the spotted bat (Euderma maculatum), was observed roosting on the exterior of a facility by Hanford Site personnel in 2021; the species was confirmed by Hanford Site biologists via photograph (Table 1-1).

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2012

Black-Tailed Jackrabbit Monitoring Report for Fiscal Year 2012

Anecdotal evidence suggests both black-tailed jackrabbits (Lepus californicus) and white-tailed jackrabbits (L. townsendii) were historically abundant; however, the Washington State Department of Fish and Wildlife (WDFW) currently list both the black-tailed and white-tailed jackrabbits as 'candidate' species of concern (WDFW 2012). Threats to jackrabbits include habitat loss and fragmentation. Lack of recent monitoring or sightings on the Hanford Site has led some to believe the population is declining, while others believe that jackrabbits may follow a cyclical pattern of population density. Black-tailed jackrabbits play an important role in the ecosystem, serving as a food source for large raptor and mammal predators including the coyote, golden eagle, and the State Threatened ferruginous hawk. Jackrabbits do not migrate long distances or go into a hibernation or estivation period, and rarely use underground burrows or dens (Best 1996). This highly localized and active ecology provides surveying opportunities through most of the year.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2012

Ground Squirrel Monitoring Report for Calendar Year 2012

The Washington Department of Fish and Wildlife (WDFW) lists the Townsend’s ground squirrel (Urocitellus townsendii) as a “State Candidate” (WDFW 2012). These animals are important to the shrub-steppe ecosystem for many reasons. They serve as a food source for many mammals such as badgers and coyotes and fall prey to predatory birds such as hawks, falcons, and owls. The ground squirrel diet consists of a variety of foods including seeds, which contributes to native plant seed dispersal. The process of digging burrows helps aerate the soil and provides burrows for other species including burrowing owls (Sato 2012). Their decline is due to the loss of suitable habitat and isolation of their communities through fragmentation. Townsend’s ground squirrels can be difficult to monitor due to their concealed underground lifestyle and short seasons of activity.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Towards Commercialization of Hydrophone Flow Shields to Improve Passive Acoustic Data Quality (CRADA 678) (Final Report)

This project performed testing, market research, and manufacturing refinement to ready patent-pending hydrophone flow shields developed at PNNL for commercialization. The flow shields reduce the impact of flow noise on underwater sound recordings in areas of strong flow, like tidal channels or rivers. Flow noise is a challenge in many applications where it is necessary to measure underwater sound, including measuring the sound produced by marine energy converters and monitoring marine mammals. This project addressed several key questions about flow shield performance that are high-priority for potential customers and refined existing manufacturing processes to be scalable for market-level production. Specifically, this project conducted both laboratory and field evaluations of the flow shields to comprehensively evaluate their performance. Overall, the results indicate that the flow shields reduce flow noise by over 20 dB at frequencies below 50 Hz at flow speeds greater than 0.5 m/s and are proven to survive deployments more than 6 months long.

42 ENGINEERING↗

DOE OSTI2022

U.S. Pacific Coast Workshop Report on Preconstruction Research Recommendations (U.S. Offshore Wind Synthesis of Environmental Effects Research (SEER) Project)

In May 2022, the U.S. Offshore Wind Synthesis of Environmental Effects Research (SEER) project team hosted a stakeholder workshop focused on preconstruction (baseline) research needs for potential floating offshore wind (OSW) energy development on the U.S. Pacific Coast, including California, Oregon, and Washington. Prior to the workshop, the SEER team developed a set of initial synthesized research recommendations that were identified based on a review of relevant, publicly available resources and with advisory group input. The workshop covered three marine life breakout groups on subsequent days to discuss research recommendations related to 1) marine mammals and sea turtles, 2) fish and invertebrates, and 3) birds and bats. As part of the workshop, over a hundred participants from the public and private sectors provided feedback on various aspects of the initial research recommendations, including associated data and knowledge gaps, benefits/limitations of available methods and technologies, and technological advancements or infrastructure needed to address the recommendation. Approximately 1,000 total comments were received on the workshop MURAL boards and were synthesized in this report. Based on workshop feedback, SEER developed a final database of over 500 specific research recommendations based on more than 40 resources. In Fall 2022, the full database and a tool with updated synthesized research recommendations were disseminated on Tethys (https://tethys.pnnl.gov) to assist with informing future funding opportunities and research programming. There is a continued need to improve awareness of the potential environmental effects, monitoring technologies, and management strategies for floating OSW energy development on the U.S. Pacific Coast. Coordination of these activities will require the sustained involvement of multiple stakeholders from across sectors. Beyond the baseline considerations discussed in this workshop, future state-of-the-science activities should be planned to consider research needs across wind energy life cycle phases for all relevant wildlife taxa and associated habitat and ecosystem processes.

17 WIND ENERGY↗

DOE OSTI2024

N α -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N α -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5 , N 5 -di-methyl N α -acetyl-L-ornithine (k cat /K m = 7.32 ± 0.94 × 10 4 M -1 s -1 ). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC 50 = 58.7 μM; K i = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC 50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril K i = 35.9 ± 5.1 μM). The active site structure of di-Zn Ec ArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2019

The Importance of Shifting Disturbance Regimes in Monarch Butterfly Decline and Recovery

The Eastern migratory monarch butterfly has declined in recent decades, partly because widespread adoption of herbicide-resistant corn and soybean has nearly eliminated common milkweed from crop fields in the US Midwest. We argue that in addition to milkweed loss, monarch declines were likely exacerbated by shifting disturbance regimes within their summer breeding range. Monarchs prefer to lay eggs on younger, vegetative milkweed stems. They also benefit from enemy-free space, as most eggs and early-instar larvae succumb to predators. Historically, ecological disturbances during the growing season could have provided these conditions. During most of the 19th and 20th centuries, milkweed was abundant in crop fields where manual weeding and mechanical cultivation set milkweed back, but rather than killing it would often stimulate regrowth later in the summer. Before European settlement, large mammals and fires (natural and anthropogenic) perturbed grasslands during the summer and could have had similar effects. However, presently most remaining milkweed stems in the Midwest are in perennial grasslands like roadsides, old-fields, parks, and conservation reserves, which often lack growing season disturbance. As a result, monarchs may be left with limited options for oviposition as the summer progresses and could have lower survival in grasslands where predation pressure is high. Our recent work has shown that targeted disturbance during the growing season produces milkweed stems that are attractive to ovipositing monarchs and harbor fewer arthropod predators. Targeted disturbance in perennial grasslands could improve habitat heterogeneity and phenologic diversity of milkweeds, and should be explored as a monarch conservation strategy.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Fencing affects movement patterns of two large carnivores in Southern Africa

The erection of peripheral fences around protected areas has been shown to adversely affect the movement behavior, genetic connectivity and long-term viability of animal populations. However, fencing is also used extensively to reduce human encroachment, limit poaching activities, and mitigate human–wildlife conflicts. Limited information on the movement behavior of carnivores is available along the periphery of protected areas, particularly in semi-arid environments. To quantify the potential effects of the 824 km perimeter fence surrounding the Etosha National Park (Etosha, Namibia) on the movement and space use of carnivores, we collected GPS movement data from 36 lions (Panthera leo) and seven spotted hyenas (Crocuta crocuta) over a 14-year period. For these two apex carnivores, we measured the impact of the Etosha fence on their movements (step lengths and path straightness), calculated the likelihood of fence crossings, and evaluated the spatial–temporal characteristics of these crossings. Our results indicated that lions and hyenas moved faster (longer step lengths) when closer to the fence. Lions showed no variation in path straightness with respect to the fence but hyenas had straighter movement paths when closer to the fence. When moving within the vicinity of the fence, lions had a 9% likelihood of crossing the fence, while hyenas were much more likely (18%) to do so, which suggests that the Etosha fence was more permeable for hyenas than for lions. Fence crossings predominantly occurred at night for both species. Lions were more likely to cross the fence during the cold dry season (May–August) whereas hyenas crossed more often during the warm wet season (December–April). Female and adult lions had a higher probability of crossing the fence than males and subadults. These findings could be useful in the development of appropriate strategies to mitigate human–carnivore conflicts and thus promote the conservation of carnivores in multiple-use areas along the periphery of the park. Further research is needed to better elucidate the impact of perimeter fences on a broader suite of carnivores and other large mammals, including species-specific variation in the response to fences, as well as the economic benefits of fencing provided to local communities.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

The Structure and Immune Regulatory Implications of the Ubiquitin-Like Tandem Domain Within an Avian 2’-5’ Oligoadenylate Synthetase-Like Protein

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host’s ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2’-5’ oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored.

59 BASIC BIOLOGICAL SCIENCES↗