Freshwater mussels (Unionida) are suffering mass mortality events worldwide, but the causes remain enigmatic. Here, we describe an analysis of bacterial loads, community structure, and inferred metabolic pathways in the hemolymph of pheasantshells (Actinonaias pectorosa) from the Clinch River, USA, during a multi-year mass mortality event. Bacterial loads were approximately 2 logs higher in moribund mussels (cases) than in apparently healthy mussels (controls). Bacterial communities also differed between cases and controls, with fewer sequence variants (SVs) and higher relative abundances of the proteobacteria Yokenella regensburgei and Aeromonas salmonicida in cases than in controls. Inferred bacterial metabolic pathways demonstrated a predominance of degradation, utilization, and assimilation pathways in cases and a predominance of biosynthesis pathways in controls. Only two SVs correlated with Clinch densovirus 1, a virus previously shown to be strongly associated with mortality in this system: Deinococcota and Actinobacteriota, which were associated with densovirus-positive and densovirus-negative mussels, respectively. Overall, our results suggest that bacterial invasion and shifts in the bacterial microbiome during unionid mass mortality events may result from primary insults such as viral infection or environmental stressors. If so, bacterial communities in mussel hemolymph may be sensitive, if generalized, indicators of declining mussel health.

Bat flies (Hippoboscoidea: Nycteribiidae and Streblidae) are obligate hematophagous ectoparasites of bats. We collected streblid bat flies from the New World (México) and the Old World (Uganda), and used metagenomics to identify their viruses. In México, we found méjal virus (Rhabdoviridae; Vesiculovirus), Amate virus (Reoviridae: Orbivirus), and two unclassified viruses of invertebrates. Méjal virus is related to emerging zoonotic encephalitis viruses and to the agriculturally important vesicular stomatitis viruses (VSV). Amate virus and its sister taxon from a bat are most closely related to mosquito- and tick-borne orbiviruses, suggesting a previously unrecognized orbivirus transmission cycle involving bats and bat flies. In Uganda, we found mamucuso virus (Peribunyaviridae: Orthobunyavirus) and two unclassified viruses (a rhabdovirus and an invertebrate virus). Mamucuso virus is related to encephalitic viruses of mammals and to viruses from nycteribiid bat flies and louse flies, suggesting a previously unrecognized orthobunyavirus transmission cycle involving hippoboscoid insects. Bat fly virus transmission may be neither strictly vector-borne nor strictly vertical, with opportunistic feeding by bat flies occasionally leading to zoonotic transmission. Many “bat-associated” viruses, which are ecologically and epidemiologically associated with bats but rarely or never found in bats themselves, may actually be viruses of bat flies or other bat ectoparasites.

Freshwater mussels (Unionida) are globally imperiled, in part due to largely unexplained mass mortality events (MMEs). While recent studies have begun to investigate the possibility that mussel MMEs in the Eastern USA may be caused by infectious diseases, mussels in the Western USA have received relatively little attention in this regard. We conducted a two-year epidemiologic investigation of the role of viruses in ongoing MMEs of the Western pearlshell (Margaritifera falcata) and the Western ridged mussel (Gonidea angulata) in the Chehalis River and Columbia River watersheds in the Western USA. We characterized viromes of mussel hemolymph from 5 locations in 2018 and 2020 using metagenomic methods and identified 557 viruses based on assembled contiguous sequences, most of which are novel. We also characterized the distribution and diversity of a previously identified mussel Gammarhabdovirus related to pathogenic finfish viruses. Overall, we found few consistent associations between viruses and mussel health status. Variation in mussel viromes was most strongly driven by location, with little influence from date, species, or health status, though these variables together only explained ~1/3 of variation in virome composition. Our results demonstrate that Western freshwater mussels host remarkably diverse viromes, but no single virus or combination of viruses appears to be associated with morbidity or mortality during MMEs. Our findings have implications for the conservation of imperiled freshwater mussels, including efforts to enhance natural populations through captive propagation.

Freshwater mussels (Unionida) are among the world’s most imperiled taxa, but the relationship between freshwater mussel mortality events and infectious disease is largely unstudied. We surveyed viromes of a widespread and abundant species (mucket, Actinonaias ligamentina; syn: Ortmanniana ligamentina) experiencing a mortality event of unknown etiology in the Huron River, Michigan, in 2019–2020 and compared them to viromes from mucket in a healthy population in the St. Croix River, Wisconsin and a population from the Clinch River, Virginia and Tennessee, where a mortality event was affecting the congeneric pheasantshell (Actinonaias pectorosa; syn: Ortmanniana pectorosa) population. We identified 38 viruses, most of which were associated with mussels collected during the Huron River mortality event. Viral richness and cumulative viral read depths were significantly higher in moribund mussels from the Huron River than in healthy controls from each of the three populations. Our results demonstrate significant increases in the number and intensity of viral infections for freshwater mussels experiencing mortality events, whereas individuals from healthy populations have a substantially reduced virome comprising a limited number of species at low viral read depths.

Infectious disease is a major concern for both wild and captive primate populations. Primate sanctuaries in Africa provide critical protection to thousands of wild‐born, orphan primates confiscated from the bushmeat and pet trades. However, uncertainty about the infectious agents these individuals potentially harbor has important implications for their individual care and long‐term conservation strategies. We used metagenomic next‐generation sequencing to identify viruses in blood samples from chimpanzees (Pan troglodytes) in three sanctuaries in West, Central, and East Africa. Our goal was to evaluate whether viruses of human origin or other “atypical” or unknown viruses might infect these chimpanzees. We identified viruses from eight families: Anelloviridae, Flaviviridae, Genomoviridae, Hepadnaviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, and Rhabdoviridae. The majority (15/26) of viruses identified were members of the family Anelloviridae and represent the genera Alphatorquevirus (torque teno viruses) and Betatorquevirus (torque teno mini viruses), which are common in chimpanzees and apathogenic. Of the remaining 11 viruses, 9 were typical constituents of the chimpanzee virome that have been identified in previous studies and are also thought to be apathogenic. One virus, a novel tibrovirus (Rhabdoviridae: Tibrovirus) is related to Bas‐Congo virus, which was originally thought to be a human pathogen but is currently thought to be apathogenic, incidental, and vector‐borne. The only virus associated with disease was rhinovirus C (Picornaviridae: Enterovirus) infecting one chimpanzee subsequent to an outbreak of respiratory illness at that sanctuary. Our results suggest that the blood‐borne virome of African sanctuary chimpanzees does not differ appreciably from that of their wild counterparts, and that persistent infection with exogenous viruses may be less common than often assumed.

Biologists monitoring freshwater mussel (order Unionida) populations rely on behavioral, often subjective, signs to identify moribund (“sick”) or stressed mussels, such as gaping valves and slow response to probing, and they lack clinical indicators to support a diagnosis. As part of a multi-year study to investigate causes of reoccurring mortality of pheasantshell (Ortmanniana pectorosa; synonym Actinonaias pectorosa) in the Clinch River, Virginia and Tennessee, USA, we analyzed the hemolymph metabolome of a subset of mussels from the 2018 sampling period. Mussels at the mortality sites were diagnosed in the field as affected (case) or unaffected (control) based on behavioral and physical signs. Hemolymph was collected in the field by non-lethal methods from the anterior adductor muscle for analysis. We used ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectroscopy to detect targeted and untargeted metabolites in hemolymph and compared metabolomic profiles by field assessment of clinical status. Targeted biomarker analysis found 13 metabolites associated with field assessments of clinical status. Of these, increased gamma-linolenic acid and N-methyl-l-alanine were most indicative of case mussels, while adenine and inosine were the best indicators of control mussels. Five pathways in the targeted analysis differed by clinical status; two of these, purine metabolism and glycerophospholipid metabolism, were also indicated in the untargeted analysis. In the untargeted nalysis, 22 metabolic pathways were associated with clinical status. Many of the impacted pathways in the case group were catabolic processes, such as degradation of amino acids and fatty acids. Hierarchical clustering analysis matched clinical status in 72% (18 of 25) of mussels, with control mussels more frequently (5 of 16) not matching clinical status. Our study demonstrated that metabolomic analysis of hemolymph is suitable for assessing mussel condition and complements field-based indicators of health.