Influence of Environmental Stressors on the Microbiota of Zebra Mussels (Dreissena polymorpha).

Host-associated microbiota play a critical role in host fitness by providing nutrition, enhancing digestion capabilities, and by providing protection from pathogens. Here, we investigated the effects of two environmental stressors, temperature, and salinity, on the microbiota associated with zebra mussels (ZMs), a highly invasive bivalve in North America. To examine this in detail, lake-collected ZMs were acclimated to laboratory conditions, and subjected to temperature and salinity stress conditions. The impact of these stressors on the diversity, composition, and dynamics of ZM-associated microbiota were assessed by using amplicon- and shotgun-based sequencing, and qPCR-based approaches. Elevated temperature was found to be the primary driver of ZM mortality, although salinity alone also increased its likelihood. Stressor-induced ZM mortality, which ranged between 53 and 100%, was concomitant with significant increases in the relative abundance of several genera of putative opportunistic pathogens including Aeromonas. These genera were only present in low relative abundance in ZMs obtained from the control tank with 0% mortality. Shotgun sequencing and qPCR analyses indicated that the relative and absolute abundances of pathogenic Aeromonas species (particularly A. veronii) were significantly greater in temperature-induced dead ZMs. Taken together, our results show that environmental stress, especially elevated temperature (> 25 °C), is associated with the rapid mortality of ZMs as well as the proliferation of putative opportunistic bacterial pathogens.

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States.

Koi herpesvirus (KHV; cyprinid herpesvirus-3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real-time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.

Genomic characterization of a novel calicivirus, FHMCV-2012, from baitfish in the USA.

During regulatory sampling of fathead minnows (Pimephales promelas), a novel calicivirus was isolated from homogenates of kidney and spleen inoculated into bluegill fry (BF-2) cells. Infected cell cultures exhibiting cytopathic effects were screened by PCR-based methods for selected fish viral pathogens. Illumina HiSeq next generation sequencing of the total RNA revealed a novel calicivirus genome that showed limited protein sequence similarity to known homologs in a BLASTp search. The complete genome of this fathead minnow calicivirus (FHMCV) is 6564 nt long, encoding a polyprotein of 2114 aa in length. The complete polyprotein shared only 21% identity with Atlantic salmon calicivirus,followed by 11% to 14% identity with mammalian caliciviruses. A molecular detection assay (RT-PCR) was designed from this sequence for screening of field samples for FHMCV in the future. This virus likely represents a prototype species of a novel genus in the family Caliciviridae, tentatively named "Minovirus".

Evolution of a malaria resistance gene in wild primates.

The ecology, behaviour and genetics of our closest living relatives, the nonhuman primates, should help us to understand the evolution of our own lineage. Although a large amount of data has been amassed on primate ecology and behaviour, much less is known about the functional and evolutionary genetic aspects of primate biology, especially in wild primates. As a result, even in well-studied populations in which nongenetic factors that influence adaptively important characteristics have been identified, we have almost no understanding of the underlying genetic basis for such traits. Here, we report on the functional consequences of genetic variation at the malaria-related FY (DARC) gene in a well-studied population of yellow baboons (Papio cynocephalus) living in Amboseli National Park in Kenya. FY codes for a chemokine receptor normally expressed on the erythrocyte surface that is the known entry point for the malarial parasite Plasmodium vivax. We identified variation in the cis-regulatory region of the baboon FY gene that was associated with phenotypic variation in susceptibility to Hepatocystis, a malaria-like pathogen that is common in baboons. Genetic variation in this region also influenced gene expression in vivo in wild individuals, a result we confirmed using in vitro reporter gene assays. The patterns of genetic variation in and around this locus were also suggestive of non-neutral evolution, raising the possibility that the evolution of the FY cis-regulatory region in baboons has exhibited both mechanistic and selective parallels with the homologous region in humans. Together, our results represent the first reported association and functional characterization linking genetic variation and a complex trait in a natural population of nonhuman primates.

Identification of Francisella noatunensis in novel host species French grunt (Haemulon flavolineatum) and Caesar grunt (Haemulon carbonarium).

Francisella noatunensis is an emerging pathogen of fish that has been isolated from several cultured species worldwide. Here presented is a case involving several hundred marine grunts that were caught near the Florida Keys for display in public aquaria. These fish were maintained in a recirculating system where they began to experience mortalities approximately two weeks post-stocking. Postmortem examination revealed disseminated systemic granulomatous disease most severely affecting spleen and kidney. Splenic and renal tissue homogenates inoculated in modified Thayer Martin agar media yielded colonies consistent with F. noatunensis 4 days post inoculation. Bacterial colonies and tissues were confirmed positive after real-time PCR amplification of the intracellular growth loci gene (iglC) specific for F. noatunensis subspecies orientalis. Consequently, multiple novel host species for this pathogen were identified, including the French grunt (Haemulon flavolineatum) and the Caesar grunt (Haemulon carbonarium).

Health of wild fish exposed to contaminants of emerging concern in freshwater ecosystems utilized by a Minnesota Tribal community.

Fish serve as indicators of exposure to contaminants of emerging concern (CECs)-chemicals such as pharmaceuticals, hormones, and personal care products-which are often designed to impact vertebrates. To investigate fish health and CECs in situ, we evaluated the health of wild fish exposed to CECs in waterbodies across northeastern Minnesota with varying anthropogenic pressures and CEC exposures: waterbodies with no human development along their shorelines, those with development, and those directly receiving treated wastewater effluent. Then, we compared three approaches to evaluate the health of fish exposed to CECs in their natural environment: a refined fish health assessment index, a histopathological index, and high-throughput (ToxCast) in vitro assays. Lastly, we mapped adverse outcome pathways (AOPs) associated with identified ToxCast assays to determine potential impacts across levels of biological organization within the aquatic system. These approaches were applied to subsistence fish collected from the Grand Portage Indian Reservation and 1854 Ceded Territory in 2017 and 2019. Overall, 24 CECs were detected in fish tissues, with all but one of the sites having at least one detection. The combined implementation of these tools revealed that subsistence fish exposed to CECs had histological and macroscopic tissue and organ abnormalities, although a direct causal link could not be established. The health of fish in undeveloped sites was as poor, or sometimes poorer, than fish in developed and wastewater effluent-impacted sites based on gross and histologic tissue lesions. Adverse outcome pathways revealed potential hazardous pathways of individual CECs to fish. A better understanding of how the health of wild fish harvested for consumption is affected by CECs may help prioritize risk management research efforts and can ultimately be used to guide fishery management and public health decisions. Integr Environ Assess Manag 2024;20:846-863. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

A chemical prioritization process: Applications to contaminants of emerging concern in freshwater ecosystems (Phase I).

Contaminants of emerging concern (CECs), such as pharmaceuticals, personal care products, and hormones, are frequently found in aquatic ecosystems around the world. Information on sublethal effects from exposure to commonly detected concentrations of CECs is lacking and the limited availability of toxicity data makes it difficult to interpret the biological significance of occurrence data. However, the ability to evaluate the effects of CECs on aquatic ecosystems is growing in importance, as detection frequency increases. The goal of this study was to prioritize the chemical hazards of 117 CECs detected in subsistence species and freshwater ecosystems on the Grand Portage Indian Reservation and adjacent 1854 Ceded Territory in Minnesota, USA. To prioritize CECs for management actions, we adapted Minnesota Pollution Control Agency's Aquatic Toxicity Profiles framework, a tool for the rapid assessment of contaminants to cause adverse effects on aquatic life by incorporating chemical-specific information. This study aimed to 1) perform a rapid-screening assessment and prioritization of detected CECs based on their potential environmental hazard; 2) identify waterbodies in the study region that contain high priority CECs; and 3) inform future monitoring, assessment, and potential remediation in the study region. In water samples alone, 50 CECs were deemed high priority. Twenty-one CECs were high priority among sediment samples and seven CECs were high priority in fish samples. Azithromycin, DEET, diphenhydramine, fluoxetine, miconazole, and verapamil were high priority in all three media. Due to the presence of high priority CECs throughout the study region, we recommend future monitoring of particular CECs based on the prioritization method used here. We present an application of a chemical hazard prioritization process and identify areas where the framework may be adapted to meet the objectives of other management-related assessments.

Anthropogenic factors associated with contaminants of emerging concern detected in inland Minnesota lakes (Phase II).

Contaminants of emerging concern (CECs) include a variety of pharmaceuticals, personal care products, and hormones commonly detected in surface waters. Human activities, such as wastewater treatment and discharge, contribute to the distribution of CECs in water, but other sources and pathways are less frequently examined. This study aimed to identify anthropogenic activities and environmental characteristics associated with the presence of CECs, previously determined to be of high priority for further research and mitigation, in rural inland lakes in northeastern Minnesota, United States. The setting for this study consisted of 21 lakes located within both the Grand Portage Indian Reservation and the 1854 Ceded Territory, where subsistence hunting and fishing are important to the cultural heritage of the indigenous community. We used data pertaining to numbers of buildings, healthcare facilities, wastewater treatment plants, impervious surfaces, and wetlands within defined areas surrounding the lakes as potential predictors of the detection of high priority CECs in water, sediment, and fish. Separate models were run for each contaminant detected in each sample media. We used least absolute shrinkage and selection operator (LASSO) models to account for both predictor selection and parameter estimation for CEC detection. Across contaminants and sample media, the percentage of impervious surface was consistently positively associated with CEC detection. Number of buildings in the surrounding area was often negatively associated with CEC detection, though nonsignificant. Surrounding population, presence of wastewater treatment facilities, and percentage of wetlands in surrounding areas were positively, but inconsistently, associated with CECs, while catchment area and healthcare centers were generally not associated. The results of this study highlight human activities and environmental characteristics associated with CEC presence in a rural area, informing future work regarding specific sources and transport pathways. We also demonstrate the utility of LASSO modeling in the identification of these important relationships.

Isolation and molecular characterization of Newcastle disease viruses from raptors.

The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry.

Comparison of cloacal and oropharyngeal samples for the detection of avian influenza virus in wild birds.

This study was conducted to compare oropharyngeal (OP) and cloacal samples of wild birds (n = 137) for the detection and isolation of avian influenza virus (AIV). A total of 39 (28.5%) cloacal and 85 (62.0%) OP samples were positive for AIV by real-time reverse transcription-PCR (RRT-PCR). The AIV nucleic acid was detected in both cloacal and OP samples from 27 (19.7%) birds, in cloacal samples only from 12 (8.8%) birds, and in OP samples only from 58 (42.3%) birds. Thus, a total of 97 (70.8%) birds were AIV positive by RRT-PCR. The cycle threshold values for the cloacal samples ranged from 16.6 to 36.9 (mean 31.5), and those for OP samples ranged from 18 to 38.9 (mean 34.9). Of the cloacal samples, 12 were positive for H5 subtype influenza virus by RRT-PCR, with one being low pathogenic H5N1. In contrast, five of the OP samples were H5 positive, but none was H5N1. None of the cloacal or OP samples was H7 positive. Eight cloacal samples yielded AIV on inoculation in embryonated chicken eggs, while only one isolate was obtained from OP samples. Thus, from testing of 137 birds, only nine (6.6%) AIV isolates were obtained. The isolates from cloacal samples were subtyped as H6N1 (n = 5), H3N8 (n = 2), and H4N8 (n = 1), and the isolate from OP sample was subtyped as H6N1. No virus was isolated from the corresponding cloacal sample of the bird whose OP sample yielded AIV on virus isolation. These results suggest that surveillance programs for detection of AIV by RRT-PCR may include both sample types (cloacal and OP) to obtain a better picture of AIV prevalence, and OP samples may yield additional isolates of AIV when tested in conjunction with cloacal samples.

Occurrence of contaminants of emerging concern in aquatic ecosystems utilized by Minnesota tribal communities.

Pharmaceuticals, personal care products, hormones, and other chemicals lacking water quality standards are frequently found in surface water. While evidence is growing that these contaminants of emerging concern (CECs) - those previously unknown, unrecognized, or unregulated - can affect the behavior and reproduction of fish and wildlife, little is known about the distribution of these chemicals in rural, tribal areas. Therefore, we surveyed the presence of CECs in water, sediment, and subsistence fish species across various waterbodies, categorized as undeveloped (i.e., no human development along shorelines), developed (i.e., human development along shorelines), and wastewater effluent-impacted (i.e., contain effluence from wastewater treatment plants), within the Grand Portage Indian Reservation and 1854 Ceded Territory in northeastern Minnesota, U.S.A. Overall, in 28 sites across three years (2016-2018), 117 of the 158 compounds tested were detected in at least one form of medium (i.e., water, sediment, or fish). CECs were detected most frequently at wastewater effluent-impacted sites, with up to 83 chemicals detected in one such lake, while as many as 17 were detected in an undeveloped lake. Although there was no statistically significant difference between the number of CECs present in developed versus undeveloped lakes, a range of 3-17 CECs were detected across these locations. Twenty-two CECs were detected in developed and undeveloped sites that were not detected in wastewater effluent-impacted sites. The detection of CECs in remote, undeveloped locations, where subsistence fish are harvested, raises scientific questions about the safety and security of subsistence foods for indigenous communities. Further investigation is warranted so that science-based solutions to reduce chemical risks to aquatic life and people can be developed locally and be informative for indigenous communities elsewhere.

Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated "Brachyspira hampsonii".

Since 2007, outbreaks of severe bloody diarrhea and hemorrhagic colitis have been reported in the United States and Canada. Though the primary causative agent of swine dysentery is Brachyspira hyodysenteriae, which is strongly hemolytic, the current report describes the isolation of a novel strongly hemolytic Brachyspira sp. This novel Brachyspira sp. was identified from clinical submissions at the Minnesota Veterinary Diagnostic Laboratory, and 40 of such isolates were obtained from 22 clinical submissions representing 5 states. Isolates were confirmed to be different from any known Brachyspira sp. on the basis of phylogenetic analysis of nucleotide sequences of nox and 16S ribosomal RNA (rRNA) genes. Phylogenetic analyses grouped all isolates into 2 clades (clades I and II), and grouping patterns were similar for both nox and 16S rRNA gene sequence analyses. Phenotypically, all isolates were indole and hippurate negative, and enzymatic profiling indicated 2 types of profiles, irrespective of the phylogenetic grouping, differing only in the production of ß-glucosidase. The results suggest that a potentially virulent new species of Brachyspira sp., provisionally named "Brachyspira hampsonii ", is circulating among swine herds in the United States.

Regional specification in the early embryo of the brittle star Ophiopholis aculeata.

Early embryogenesis has been examined experimentally in several echinoderm and hemichordate classes. Although these studies suggest that the mechanisms which underlie regional specification have been highly conserved within the echinoderm + hemichordate clade, nothing is known about these mechanisms in several other echinoderm classes, including the Ophiuroidea. In this study, early embryogenesis was examined in a very little studied animal, the ophiuroid Ophiopholis aculeata. In O. aculeata, the first two cleavage planes do not coincide with the animal-vegetal axis but rather form approximately 45 degrees off this axis. A fate map of the early embryo was constructed using microinjected lineage tracers. Most significantly, this fate map indicates that there is a major segregation of ectodermal from endomesodermal fates at first cleavage. The distribution of developmental potential in the early embryo was also examined by isolating different regions of the early embryo and following these isolates though larval development. These analyses indicate that endomesodermal developmental potential segregates unequally at first, second, and third cleavage in O. aculeata. These results provide insight into the mechanisms of regional specification in O. aculeata and yield new material for the study of the evolution of echinoderm development.