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Freshwater mussels are integral components of riverine ecosystems, influencing water quality, nutrient cycling, and habitat characteristics. Enigmatic freshwater mussel declines, often characterized by sudden mass mortality events, pose significant challenges to conservation efforts. The Clinch River, a freshwater biodiversity hotspot in Virginia and Tennessee, USA, has experienced several enigmatic mass mortality events since 2016. Studies have reported bacteria associated with moribund Pheasantshell ( Actinonaias pectorosa ) during mortality events in the Clinch River, specifically Yokenella regensburgei . Despite reports of bacterial infection, little is known about their role as pathogens. Through a multiyear case-control study, combining in-situ experiments, field surveys, histology, bacterial isolation, and high-throughput sequencing, we assessed the role of bacteria in Pheasantshell ( Actinonais pectorosa ) mortality at three sites in the Clinch River. Between May 2021 and December 2023, we collected 21 wild moribund free-living A . pectorosa and 68 hatchery-reared A . pectorosa maintained in silos at the same sites and investigated differences in pathology and microbiologye between groups. No silo mussels presented clinical signs of disease, or gross or microscopic lesions associated with pathological conditions leading to mortality. Our findings reveal a significant association between Yokenella regensburgei and severe multisystemic and multifocal infiltrative hemocytosis with necrosis, consistent with sepsis. Lesions associated with yokenellosis were of sufficient severity and physiological significance to explain mortality in infected hosts. Although our study does not explain the cause of these infections, it confirms that mussels at our study sites are ultimately dying with an infectious disease and that Y . regensburgei can be pathogenic in free-living mussels. Our results underscore the importance of considering bacterial diseases in wild mussel populations and emphasize the need for further research to elucidate the epidemiology and pathogenicity of Y . regensburgei . Overall, our study highlights the importance of integrated approaches combining pathology, microbiology, and epidemiology in freshwater mussel conservation efforts.

Translocations of freshwater species have become a widespread conservation strategy to mitigate the impacts of habitat fragmentation, yet they are not often rigorously monitored using animal movement data to determine their success. We demonstrate the value of monitoring pre- and post-translocation movements and home-range sizes of a fully-aquatic, benthic stream salamander, the eastern hellbender ( Cryptobranchus a . alleganiensis ) to determine translocation success. We studied the home range sizes, movements, and habitat use of individuals (n = 27) in two self-sustaining populations (S1 & S2) for one year, and then subsequently collected similar data from a subset of these individuals (n = 17) that were translocated into two nearby streams (T1 & T2) with dam-isolated, declining populations in the Blue Ridge Ecoregion of Tennessee. We collected 1,571 location data points (869 pre-translocation and 715 post-translocation) from four study sites, and evaluated effects of mass, sex, and pre-translocation home range size/sedentariness, as well as habitat covariates on home range size and movements. Hellbender home range sizes increased from pre-translocation estimates at both sites, but response depended primarily on physical characteristics of release sites. Home range and fine-scale movement metrics indicated that hellbenders translocated from S1 to T1 settled in more quickly, had greater site fidelity, and smaller home ranges than hellbenders translocated from S2 to T2. Hellbender movements were influenced by cover rock size and density rather than individual characteristics. Study-long survival rates of translocated hellbenders increased from S1 to T1 (80% to 100%) and decreased from S2 to T2 (76% to 33%). Monitoring pre- and post-translocation movements was a valuable method for evaluating short-term translocation success in a freshwater environment. For future hellbender translocations, managers should prioritize selecting suitable release sites with contiguous boulder-dense areas (1–2 per m 2 ), adequate prey (crayfish) densities (>1/m 2 ), and habitats with low risk of predation.

The bald eagle ( Haliaeetus leucocephalus ) is a culturally and ecologically vital species in North America that embodies conservation success but continues to face threats that include emerging pathogens. The introduction of A/goose/Guangdong/1/1996 lineage highly pathogenic (HP) clade 2.3.4.4b H5N1 influenza A virus (IAV) in North America in late 2021 resulted in high rates of mortality among bald eagles. Here we show an alarming rate of bald eagle nest failure and mortality attributed to HP IAV. We documented fatal, systemic HP IAV infection in breeding adult and nestling bald eagles along the southeastern U.S. coast. Concurrently, annual bald eagle nest surveys in Georgia and Florida revealed a precipitous drop in success in coastal counties compared with previous years, portending negative impacts on population recruitment. As an apex predator and efficient scavenger, it is likely that bald eagles become infected through consumption of infected waterfowl. These results and similar reports of raptor mortality in Europe, Asia, and Africa, indicate a clear threat to raptor health. The possible long-term persistence of HP H5N1 IAV in North America poses an impending threat to bald eagle populations not only related to direct mortality but also decreased recruitment and warrants continued efforts to understand these potential impacts.

Translocations of freshwater species have become a widespread conservation strategy to mitigate the impacts of habitat fragmentation, yet they are not often rigorously monitored using animal movement data to determine their success. We demonstrate the value of monitoring pre- and post-translocation movements and home-range sizes of a fully-aquatic, benthic stream salamander, the eastern hellbender (Cryptobranchus a. alleganiensis) to determine translocation success. We studied the home range sizes, movements, and habitat use of individuals (n = 27) in two self-sustaining populations (S1 & S2) for one year, and then subsequently collected similar data from a subset of these individuals (n = 17) that were translocated into two nearby streams (T1 & T2) with dam-isolated, declining populations in the Blue Ridge Ecoregion of Tennessee. We collected 1,571 location data points (869 pre-translocation and 715 post-translocation) from four study sites, and evaluated effects of mass, sex, and pre-translocation home range size/sedentariness, as well as habitat covariates on home range size and movements. Hellbender home range sizes increased from pre-translocation estimates at both sites, but response depended primarily on physical characteristics of release sites. Home range and fine-scale movement metrics indicated that hellbenders translocated from S1 to T1 settled in more quickly, had greater site fidelity, and smaller home ranges than hellbenders translocated from S2 to T2. Hellbender movements were influenced by cover rock size and density rather than individual characteristics. Study-long survival rates of translocated hellbenders increased from S1 to T1 (80% to 100%) and decreased from S2 to T2 (76% to 33%). Monitoring pre- and post-translocation movements was a valuable method for evaluating short-term translocation success in a freshwater environment. For future hellbender translocations, managers should prioritize selecting suitable release sites with contiguous boulder-dense areas (1-2 per m.sup.2 ), adequate prey (crayfish) densities (>1/m.sup.2 ), and habitats with low risk of predation.

Hellbenders ( Cryptobranchus alleganiensis ) are large, aquatic salamanders from the eastern United States. Both subspecies, eastern and Ozark hellbenders, have experienced declines resulting in federal listing of Ozark hellbenders. The globally distributed chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been detected in both subspecies, and Batrachochytrium salamandrivorans ( Bsal ) poses a new threat if introduced into North America. Ozark hellbenders also suffer a high prevalence of toe lesions of unknown etiology, with changes in host immunocompetence hypothesized to contribute. Antimicrobial peptides (AMPs) secreted from dermal granular glands may play a role in hellbender health. We collected skin secretions from free-ranging hellbenders and enriched them for small cationic peptides used for growth inhibition assays against Bd and Bsal . Generalized linear mixed models revealed the presence of active toe lesions as the strongest and only significant predictor of decreased Bd inhibition by skin peptides. We also found skin secretions were more inhibitory of Bsal than Bd . MALDI-TOF mass spectrometry revealed candidate peptides responsible for anti-chytrid activity. Results support the hypothesis that hellbender skin secretions are important for innate immunity against chytrid pathogens, and decreased production or release of skin peptides may be linked to other sub-lethal effects of disease associated with toe lesions.

High numbers of black vultures ( Coragyps atratus ) have died from clade 2.3.4.4b highly pathogenic (HP) avian influenza A(H5N1) virus (IAV) since its initial detection in North America in November 2021. Year-round mortality in 2022 may have been maintained by conspecific scavenging. HP IAV was detected in samples from 113/134 (84.3%) dead vultures collected from seven southeastern states in 2022–23. Results indicated that 53/73 (72.6%) of those with gross evaluation in 2022–23 consistently had grossly enlarged and mottled spleen and liver. Histologically, spleens and livers of 14/14 black vultures had widespread, severe necrosis concurrent with immunohistochemical IAV labeling. Additional lesions included segmental digestive tract ulceration, necrosis, and hemorrhage from the oropharynx to the large intestine. Influenza immunolabeling was common in gonad, adrenal gland, kidney, and digestive tract. This pattern, concurrent with field observations, substantiates the digestive tract as the initial site of viral entry during ingestion of high viral doses with strong epitheliotropism. Viral spread outside of seasonal migration is concerning but likely self-limiting in black vulture populations. This efficient, self-perpetuating transmission system may warrant conservation consideration in vultures and other near-obligate scavengers. Continued monitoring of HP IAV in black vultures is needed to evaluate population trends and ecological impacts.

Freshwater mussels are integral components of riverine ecosystems, influencing water quality, nutrient cycling, and habitat characteristics. Enigmatic freshwater mussel declines, often characterized by sudden mass mortality events, pose significant challenges to conservation efforts. The Clinch River, a freshwater biodiversity hotspot in Virginia and Tennessee, USA, has experienced several enigmatic mass mortality events since 2016. Studies have reported bacteria associated with moribund Pheasantshell (Actinonaias pectorosa) during mortality events in the Clinch River, specifically Yokenella regensburgei. Despite reports of bacterial infection, little is known about their role as pathogens. Through a multiyear case-control study, combining in-situ experiments, field surveys, histology, bacterial isolation, and high-throughput sequencing, we assessed the role of bacteria in Pheasantshell (Actinonais pectorosa) mortality at three sites in the Clinch River. Between May 2021 and December 2023, we collected 21 wild moribund free-living A. pectorosa and 68 hatchery-reared A. pectorosa maintained in silos at the same sites and investigated differences in pathology and microbiologye between groups. No silo mussels presented clinical signs of disease, or gross or microscopic lesions associated with pathological conditions leading to mortality. Our findings reveal a significant association between Yokenella regensburgei and severe multisystemic and multifocal infiltrative hemocytosis with necrosis, consistent with sepsis. Lesions associated with yokenellosis were of sufficient severity and physiological significance to explain mortality in infected hosts. Although our study does not explain the cause of these infections, it confirms that mussels at our study sites are ultimately dying with an infectious disease and that Y. regensburgei can be pathogenic in free-living mussels. Our results underscore the importance of considering bacterial diseases in wild mussel populations and emphasize the need for further research to elucidate the epidemiology and pathogenicity of Y. regensburgei. Overall, our study highlights the importance of integrated approaches combining pathology, microbiology, and epidemiology in freshwater mussel conservation efforts.

Background Angiostrongylus cantonensis , the rat lungworm, is a metastrongyloid parasite that uses rodents as definitive hosts, mollusks as intermediate hosts, and a wide range of invertebrate and vertebrate species as paratenic hosts. Although this parasite poses a significant public health concern in many regions of the world, it can also cause disease in numerous domestic and wildlife aberrant host species. When parasite larvae are ingested by one of these aberrant hosts, larval migration in the central nervous system causes extensive damage, resulting in spinal cord and/or brain damage and inflammation, leading to potentially fatal neurological disease. We describe A. cantonensis infection in a novel host, the Florida burrowing owl ( Athene cunicularia floridana ), on Marco Island, Collier County, Florida, USA. The Florida burrowing owl is a state-listed species that has experienced steep population declines across its range, primarily due to habitat loss and fragmentation. Many populations are now restricted to urban environments, which pose novel threats to the owls, such as exposure to anticoagulant rodenticides and novel pathogens, increased risk of predation, vehicular strike, and increased disturbance at nest sites. Methods Through diagnostic evaluation of carcasses and select tissues submitted to the Southeastern Cooperative Wildlife Disease Study from 2019 to 2023, we diagnosed nine confirmed or suspected cases of angiostrongylosis on Marco Island. Results Microscopic examination and polymerase chain reaction (PCR) testing confirmed parasite identification. In addition, ancillary testing ruled out other potential causes of neurological disease, such as rodenticides, West Nile virus, and highly pathogenic avian influenza virus. Conclusions This study underscores the importance of surveillance and monitoring efforts for A. cantonensis , particularly in regions where novel hosts may serve as indicators of public health risk. In addition, as urbanization and habitat fragmentation continue encroaching upon wildlife habitats, understanding the dynamics of host–parasite interactions becomes crucial for mitigating the spread of zoonotic diseases. Graphical Abstract

Background Eastern hellbenders ( Cryptobranchus alleganiensis alleganiensis ) have undergone substantial population declines throughout their range, leading them to become the focus of increased conservation efforts, including care in zoo and university settings. However, effective implementation of such conservation strategies often relies on a comprehensive understanding of host health, which can be directly influenced by the gut microbiome, yet characterization of gut microbiota often remains overlooked in ex situ conservation facilities. Additionally, effects on the gut microbiome associated with releasing zoo-reared animals into the wild are poorly understood. Therefore, these circumstances make hellbenders an ideal species to examine the relationship between zoo management strategies and gut microbial dynamics. Methods 16S rRNA sequencing was used to investigate dissimilarities between the gut microbiome of hellbenders in zoo and wild settings and to evaluate the impact of implementing a wild diet in zoo-reared hellbenders. Additionally, the bacterial composition of zoo-released individuals and wild resident hellbenders was compared to examine the response of the gut microbiome upon release into natural habitat. Selected samples were also chosen for ITS1 rDNA sequencing as a preliminary investigation of the hellbender gut mycobiome. Results Human rearing strongly affected the gut microbiome, leading to reduced bacterial richness as well as differing community structure than wild hellbenders. However, implementation of a wild diet in a zoo setting modulated the microbiome and appeared to be mainly driven by bacterial turnover. Additionally, both bacterial and fungal gut assemblages demonstrated the capacity for restructuring upon release into native habitat to become more reflective of a wild-type microbiome. Conclusions We completed the first study elucidating the gut microbial composition patterns of hellbenders, across both zoo and wild settings. These results provide an understanding of the potential impacts of conservation populations in zoos on gut microbial communities and also inform headstart programs of the transition of the gut microbiome post-reintroduction to the wild.

Cryptic species present particular challenges to biodiversity conservation, as true species diversity and distributional boundaries remain obscured. However, modern molecular tools have afforded unparalleled opportunities to elucidate cryptic species, define their distributions, and, ultimately, develop conservation interventions to extend their evolutionary trajectories into the future. The Green Salamander ( Aneides aeneus ) complex provides an evolutionary focal point and the Appalachian Highlands an ecological context for the exploration of cryptic speciation in an imperiled taxon. A recent study uncovered significant levels of genetic and genomic variation geographically structured across the Appalachian Highlands, including up to four lineages, one of which ( A. caryaensis ) was described therein. Here we extend the genetic and genomic examination of the Castaneides species complex by intensive sampling of additional populations along Cumberland Plateau and Appalachian Valley and Ridge of Alabama and Tennessee, employing both mtDNA and RADseq species delimitation approaches to delineate cryptic diversity and boundaries in this region. Analyses of two mitochondrial loci ( nd4 and cytb ) identified two reciprocally monophyletic lineages, which are also supported by population clustering and phylogenetic analyses of SNPs, that identified two population clusters with no evidence of gene flow. Our genetic and genomic results support the recognition of two additional cryptic lineages in the Castaneides species complex. Ultimately, this information is critical in developing successful adaptive management strategies for this important and endemic component of Appalachian Highland biodiversity.