a view of a laptop computer connected to 2 monitors and a microplate reader. The monitors display data generated from an assay including a standard curve.
Functional and Molecular Bioassay Core Technology Team Active
Primary leukocytes have been isolated from experimental fish
USGS scientist preparing plates for yeast bioassay screening
Primary hepatocytes isolated from experimental smallmouth bass
nCounter SPRINT† system simultaneously analyzes transcript abundance
Image-based flow cytometer for cell population analysis
About the Research
The Functional and Molecular Bioassay Core Technology Team (CTT) as part of the Environmental Health Program utilizes reporter assays, quantitative gene expression analyses, and high-throughput sequencing methods to produce functional endpoints across a broad scope of environmental topics and sample matrices.
The Functional and Molecular Bioassay CTT applies and modifies microbial and eukaryotic reporter bioassays to detect the presence of bioactive chemicals in environmental water samples.
These analyses compliment analytical chemistry and biological datasets. Analyses include functional endpoints and quantitative gene expression analysis, utilizing both designed codesets and/or custom oligos. In the instance of insufficient genetic data for the non-model species of interest, the laboratory establishes in-house de novo sequence databases via high throughput sequencing methods. Bioassay capabilities were recently expanded by incorporating high content screening using a ImageExpress Pico. Methods adoption and development on this instrument include: functional immune assays previously run using microplates or flow cytometry, genetically engineered translocation-based assay to evaluate nuclear receptor binding, and cytotoxicity-based endpoints.
Key Analytical Capabilities
- Bioassay screening for genotoxic, mutagenic and endocrine active compounds
- In vitro screening and molecular/ functional response profiling of cultured cells
- Automated cell imaging and High Content Screening
- Functional genomics, quantitative gene expression analysis (quantitative polymerase chain reaction [qPCR] or digital, absolute copy measurements)
- Identification of biomarker genes in non-model organisms
- Virus discovery
Key Instrumentation
- Multimode plate reader for bioassay end-point measurements and ELISA
- nCounter Max (Nanostring) for multiplex, digital, gene expression analysis
- QX200 Droplet Digital PCR System
- QuantStudio 5 Real-Time PCR System
- High throughput sequencing (MiSeq)
- Image analysis-based flow cytometric analysis for functional cell assays
- ImageXpress Pico
- Extraction equipment for solid phase extraction of water samples
Environmental Health Integrated Science Team Collaborators
-
Drinking Water and Wastewater Infrastructure Science Team
The team studies toxicants and pathogens in water resources from their sources, through watersheds, aquifers, and infrastructure to human and wildlife exposures. That information is used to develop decision tools that protect human and wildlife health.Immunomodulation Science Team
The Immunomodulation Integrated Science Team focuses on contaminant and pathogen exposures in the environment that might influence the immune systems of wildlife and the connection to their shared environment with humans. In collaboration with public-health officials, the Team also addresses potential human-health risks stemming from similar exposures. If actual risks are identified, this Team...Energy Integrated Science Team
The Energy Lifecycle Integrated Science Team focuses on the potential for contaminant exposures in the environment that might originate from energy resource activities including, extraction, production, transportation, storage, extraction, waste management and restoration. Perceived health risks to humans and other organisms will be distinguished from actual risks, if any. If actual risks are...Food Resources Lifecycle Integrated Science Team
The team studies the movement of toxicants and pathogens that could originate from the growing, raising, and processing/manufacturing of plant and animal products through the environment where exposure can occur. This information is used to understand if there are adverse effects upon exposure and to develop decision tools to protect health.Fishing and Hunting Integrated Science Team
The Fishing and Hunting integrated science team focuses on contaminant and pathogen exposures in the environment that could impact the presence and vitality of fish and wildlife populations that drive commercial, recreational and subsistence activities such as hunting and fishing. If actual risks are identified this project will inform how to economically and effectively minimize risk by providing...
Science activities related to the Functional and Molecular Bioassay Core Technology Team can be found below.
Agricultural best management practices can improve water quality and conditions for fisheries in the Chesapeake Bay Watershed
Fish and Aquatic Animal Health Publications, 2021 – 2022
Investigating blotchy bass syndrome in black basses
Human and Ecological Health Impacts Associated with Water Reuse and Conservation Practices
Endocrine Disrupting Chemicals and Prevalence of Intersex in Fish Populations in New Jersey
Prevalence of novel, emerging hepatitis viruses in wild white suckers and their association with liver tumors in the Great Lakes region
Endocrine Disrupting Chemicals and Prevalence of Intersex in Fish Populations in New Jersey
Data related to the Functional and Molecular Bioassay Core Technology Team can be found below.
Water quality and contaminants in stream surface waters collected in the Shenandoah Valley, 2021
Results from U.S. Geological Survey Environmental Health Food Resources Lifecycle Integrated Science Team, Graton Pesticides (GRAPE) Study, in Graton, California, April–May 2021
Physiological and molecular endpoints observed in juvenile largemouth bass in response to an estrogen (17α-ethinylestradiol) and subsequently a bacterial challenge (Edwardsiella piscicida) exposure under laboratory conditions.
Water-quality results from a wastewater reuse study: Inorganic and organic compositions of wastewater effluent and select urban and agricultural water types during rain-induced runoff, Chickasha, Oklahoma, 2018-2019
SARS-CoV-2 Data from National Wastewater Surveillance System Surge Capacity Sampling, September 2021
Estrogen equivalents of surface water in Pennsylvania, 2012-2017
Water-quality data for a pharmaceutical study at Muddy Creek in North Liberty and Coralville, Iowa, 2017-2018
Multimedia items related to the Functional and Molecular Bioassay Core Technology Team can be found below.
a view of a laptop computer connected to 2 monitors and a microplate reader. The monitors display data generated from an assay including a standard curve.
Overhead view of a microplate with a dilution gradient. The targeted bioassay is designed to identify bioreactivity at varying concentrations.
Overhead view of a microplate with a dilution gradient. The targeted bioassay is designed to identify bioreactivity at varying concentrations.
image of the automated prep station for the Nanostring nCounter Max. The device prepares cartridges for CodeSet Gene Expression Analysis
image of the automated prep station for the Nanostring nCounter Max. The device prepares cartridges for CodeSet Gene Expression Analysis
Clay Raines, a USGS scientist, performing a dermal swab on a Largemouth Bass at a Bass Pro Shops retail store with a presumptive case of blotchy bass syndrome. The collected swabs can be used to isolate DNA and RNA which can be used to identify pathogens including those associated with hyperpigmentation.
Clay Raines, a USGS scientist, performing a dermal swab on a Largemouth Bass at a Bass Pro Shops retail store with a presumptive case of blotchy bass syndrome. The collected swabs can be used to isolate DNA and RNA which can be used to identify pathogens including those associated with hyperpigmentation.
Photo of a technician working at a biological safety cabinet.
Photo of a technician working at a biological safety cabinet.
Functional and Molecular Bioassay Laboratory — Kearneysville, West Virginia. Multiwell cell culture array
Functional and Molecular Bioassay Laboratory — Kearneysville, West Virginia. Multiwell cell culture array
Scientific publications related to the Functional and Molecular Bioassay Core Technology Team can be found below.
Transcriptome signatures of wastewater effluent exposure in larval zebrafish vary with seasonal mixture composition in an effluent-dominated stream
Using biological responses to monitor freshwater post-spill conditions over 3 years in Blacktail Creek, North Dakota, USA
Site- and individual-level contaminations affect infection prevalence of an emerging infectious disease of amphibians
Tandem field and laboratory approaches to quantify attenuation mechanisms of pharmaceutical and pharmaceutical transformation products in a wastewater effluent-dominated stream
Emerging investigator series: Municipal wastewater as a year-round point source of neonicotinoid insecticides that persist in an effluent-dominated stream
Hyperpigmented melanistic skin lesions of smallmouth bass Micropterus dolomieu from the Chesapeake Bay watershed
Reconnaissance of surface water estrogenicity and the prevalence of intersex in smallmouth bass (Micropterus dolomieu) inhabiting New Jersey
External partners that the Functional and Molecular Bioassay Core Technology Team collaborates with are linked below.
- Overview
About the Research
The Functional and Molecular Bioassay Core Technology Team (CTT) as part of the Environmental Health Program utilizes reporter assays, quantitative gene expression analyses, and high-throughput sequencing methods to produce functional endpoints across a broad scope of environmental topics and sample matrices.
The Functional and Molecular Bioassay CTT applies and modifies microbial and eukaryotic reporter bioassays to detect the presence of bioactive chemicals in environmental water samples.
These analyses compliment analytical chemistry and biological datasets. Analyses include functional endpoints and quantitative gene expression analysis, utilizing both designed codesets and/or custom oligos. In the instance of insufficient genetic data for the non-model species of interest, the laboratory establishes in-house de novo sequence databases via high throughput sequencing methods. Bioassay capabilities were recently expanded by incorporating high content screening using a ImageExpress Pico. Methods adoption and development on this instrument include: functional immune assays previously run using microplates or flow cytometry, genetically engineered translocation-based assay to evaluate nuclear receptor binding, and cytotoxicity-based endpoints.
Key Analytical Capabilities
- Bioassay screening for genotoxic, mutagenic and endocrine active compounds
- In vitro screening and molecular/ functional response profiling of cultured cells
- Automated cell imaging and High Content Screening
- Functional genomics, quantitative gene expression analysis (quantitative polymerase chain reaction [qPCR] or digital, absolute copy measurements)
- Identification of biomarker genes in non-model organisms
- Virus discovery
Key Instrumentation
- Multimode plate reader for bioassay end-point measurements and ELISA
- nCounter Max (Nanostring) for multiplex, digital, gene expression analysis
- QX200 Droplet Digital PCR System
- QuantStudio 5 Real-Time PCR System
- High throughput sequencing (MiSeq)
- Image analysis-based flow cytometric analysis for functional cell assays
- ImageXpress Pico
- Extraction equipment for solid phase extraction of water samples
Environmental Health Integrated Science Team Collaborators-
Drinking Water and Wastewater Infrastructure Science Team
The team studies toxicants and pathogens in water resources from their sources, through watersheds, aquifers, and infrastructure to human and wildlife exposures. That information is used to develop decision tools that protect human and wildlife health.Immunomodulation Science Team
The Immunomodulation Integrated Science Team focuses on contaminant and pathogen exposures in the environment that might influence the immune systems of wildlife and the connection to their shared environment with humans. In collaboration with public-health officials, the Team also addresses potential human-health risks stemming from similar exposures. If actual risks are identified, this Team...Energy Integrated Science Team
The Energy Lifecycle Integrated Science Team focuses on the potential for contaminant exposures in the environment that might originate from energy resource activities including, extraction, production, transportation, storage, extraction, waste management and restoration. Perceived health risks to humans and other organisms will be distinguished from actual risks, if any. If actual risks are...Food Resources Lifecycle Integrated Science Team
The team studies the movement of toxicants and pathogens that could originate from the growing, raising, and processing/manufacturing of plant and animal products through the environment where exposure can occur. This information is used to understand if there are adverse effects upon exposure and to develop decision tools to protect health.Fishing and Hunting Integrated Science Team
The Fishing and Hunting integrated science team focuses on contaminant and pathogen exposures in the environment that could impact the presence and vitality of fish and wildlife populations that drive commercial, recreational and subsistence activities such as hunting and fishing. If actual risks are identified this project will inform how to economically and effectively minimize risk by providing...
- Science
Science activities related to the Functional and Molecular Bioassay Core Technology Team can be found below.
Agricultural best management practices can improve water quality and conditions for fisheries in the Chesapeake Bay Watershed
Issue Partners in the Chesapeake Bay Program (CBP) are implementing best management practices (BMPs) to prevent nutrient and sediment from entering waterways across the Chesapeake watershed and reduce loads to the Bay. In addition to reducing nutrients, CBP partners want to better understand how BMPs can provide additional benefits for addressing toxic contaminants, such as pesticides, hormones...Fish and Aquatic Animal Health Publications, 2021 – 2022
Below are journal articles about fish and aquatic animal health from the Eastern Ecological Science Center published in 2021 and 2022.Investigating blotchy bass syndrome in black basses
Black basses (Micropterus spp) are enigmatic North American sportfishes that support the most economically valuable freshwater sport fishery in the United States and serve as keystone predators within aquatic ecosystems. Hyperpigmented melanistic lesions on the surface of black basses have been observed in a number of waterbodies across the country in increasing frequency. We have recently...Human and Ecological Health Impacts Associated with Water Reuse and Conservation Practices
Human and Ecological Health Impacts Associated with Water Reuse and Conservation PracticesEndocrine Disrupting Chemicals and Prevalence of Intersex in Fish Populations in New Jersey
Endocrine disrupting chemicals (EDCs) are compounds with the potential to interfere with normal endocrine communication in organisms.Prevalence of novel, emerging hepatitis viruses in wild white suckers and their association with liver tumors in the Great Lakes region
Prevalence of novel, emerging hepatitis viruses in wild white suckers and their association with liver tumors in the Great Lakes regionEndocrine Disrupting Chemicals and Prevalence of Intersex in Fish Populations in New Jersey
NJ WSC and Leetown Science Center scientists in cooperation with the New Jersey Department of Environmental Protection were tasked with characterizing endocrine disruption in smallmouth bass from New Jersey. - Data
Data related to the Functional and Molecular Bioassay Core Technology Team can be found below.
Water quality and contaminants in stream surface waters collected in the Shenandoah Valley, 2021
The data presented in this data release includes 11 field/water quality parameters, concentrations of 16 nutrients/anions, dissolved organic carbon, 14 organic contaminants, net estrogenicity concentrations, and 51 inorganic constituents in surface water collected twice from 28 stream sites and collected once from 2 stream sites in the Shenandoah Valley (Virginia and West Virginia, USA), in the suResults from U.S. Geological Survey Environmental Health Food Resources Lifecycle Integrated Science Team, Graton Pesticides (GRAPE) Study, in Graton, California, April–May 2021
In the spring of 2021, six households with private wells were selected throughout Graton, California, based on proximity to agriculture (within one mile of agriculture), well depth (i.e., less than 150 feet in depth), and well type (i.e., dug versus drilled). Silicone bands were deployed for 30 days at outdoor locations at each household to passively sample pesticides in ambient air. On May 3, 202Physiological and molecular endpoints observed in juvenile largemouth bass in response to an estrogen (17α-ethinylestradiol) and subsequently a bacterial challenge (Edwardsiella piscicida) exposure under laboratory conditions.
Physiological and molecular endpoints observed in juvenile largemouth bass in response to an estrogen (17α-ethinylestradiol) and subsequently a bacterial challenge (Edwardsiella piscicida) exposure under laboratory conditions. Also included are water quality and chemical concentration data.Water-quality results from a wastewater reuse study: Inorganic and organic compositions of wastewater effluent and select urban and agricultural water types during rain-induced runoff, Chickasha, Oklahoma, 2018-2019
Oklahoma State University South-Central Research Station (SCRS) was used to conduct research to understand the chemical composition of various water types and their potential environmental and human health effects. The study area provided the opportunity to study five water types: (1) receiving surface water (Washita River), (2) urban stormwater, (3) wastewater treatment plant effluent used for irSARS-CoV-2 Data from National Wastewater Surveillance System Surge Capacity Sampling, September 2021
Wastewater-based epidemiology (WBE) is used to determine the consumption of, or exposure to, chemicals or pathogens in human populations, and is conducted by collecting representative samples of untreated wastewater (influent) to quantify pathogens shed in the population served by the sampled wastewater system. The Centers for Disease Control and Prevention (CDC) and the US Department of Health anEstrogen equivalents of surface water in Pennsylvania, 2012-2017
The data were gathered as part of a long-term, seasonal assessment of estrogenicity in selected locations within Pennsylvania from 2012-2017. Data were collected over this time frame to determine seasonal and annual changes in surface water estrogenicity.Estrogen is reported as estrogen equivalents (EEQ) as determined by a bioluminescent yeast estrogen screen. A total of 193 grab water samples werWater-quality data for a pharmaceutical study at Muddy Creek in North Liberty and Coralville, Iowa, 2017-2018
Samples were collected, processed, and analyzed for organics, estrogen equivalents, and fecal indicator bacteria. Filtered organic samples were sent to the National Water Quality Laboratory in Denver, Colorado. Unfiltered estrogen equivalent samples were sent to the Organic Geochemistry Research Lab in Lawrence, Kansas, for extraction, after which they were sent to the National Fish Health Resear - Multimedia
Multimedia items related to the Functional and Molecular Bioassay Core Technology Team can be found below.
EESC Microplate Readera view of a laptop computer connected to 2 monitors and a microplate reader. The monitors display data generated from an assay including a standard curve.
a view of a laptop computer connected to 2 monitors and a microplate reader. The monitors display data generated from an assay including a standard curve.
Bioassay Dilution GradientOverhead view of a microplate with a dilution gradient. The targeted bioassay is designed to identify bioreactivity at varying concentrations.
Overhead view of a microplate with a dilution gradient. The targeted bioassay is designed to identify bioreactivity at varying concentrations.
nCounterMax Automated Prep Stationimage of the automated prep station for the Nanostring nCounter Max. The device prepares cartridges for CodeSet Gene Expression Analysis
image of the automated prep station for the Nanostring nCounter Max. The device prepares cartridges for CodeSet Gene Expression Analysis
C. Raines Blotchy Bass SwabClay Raines, a USGS scientist, performing a dermal swab on a Largemouth Bass at a Bass Pro Shops retail store with a presumptive case of blotchy bass syndrome. The collected swabs can be used to isolate DNA and RNA which can be used to identify pathogens including those associated with hyperpigmentation.
Clay Raines, a USGS scientist, performing a dermal swab on a Largemouth Bass at a Bass Pro Shops retail store with a presumptive case of blotchy bass syndrome. The collected swabs can be used to isolate DNA and RNA which can be used to identify pathogens including those associated with hyperpigmentation.
Technician at BSCPhoto of a technician working at a biological safety cabinet.
Photo of a technician working at a biological safety cabinet.
Primary leukocytes have been isolated from experimental fishPrimary leukocytes have been isolated from experimental fishFunctional and Molecular Bioassay Laboratory — Kearneysville, West Virginia. Multiwell cell culture array
Functional and Molecular Bioassay Laboratory — Kearneysville, West Virginia. Multiwell cell culture array
- Publications
Scientific publications related to the Functional and Molecular Bioassay Core Technology Team can be found below.
Transcriptome signatures of wastewater effluent exposure in larval zebrafish vary with seasonal mixture composition in an effluent-dominated stream
Wastewater treatment plant (WWTP) effluent-dominated streams provide critical habitat for aquatic and terrestrial organisms but also continually expose them to complex mixtures of pharmaceuticals that can potentially impair growth, behavior, and reproduction. Currently, few biomarkers are available that relate to pharmaceutical-specific mechanisms of action. In the experiment reported in this papeAuthorsEmma B. Meade, Luke R. Iwanowicz, Nicklaus Neureuther, Gregory H. LeFevre, Dana W. Kolpin, Hui Zhi, Shannon M. Meppelink, Rachael F. Lane, Angela Schmoldt, Aurash Mohaimani, Olaf Mueller, Rebecca D. KlaperUsing biological responses to monitor freshwater post-spill conditions over 3 years in Blacktail Creek, North Dakota, USA
A pipeline carrying unconventional oil and gas (OG) wastewater spilled approximately 11 million liters of wastewater into Blacktail Creek, North Dakota, USA. Flow of the mix of stream water and wastewater down the channel resulted in storage of contaminants in the hyporheic zone and along the banks, providing a long-term source of wastewater constituents to the stream. A multi-level investigationAuthorsAida Farag, David Harper, Isabelle M. Cozzarelli, Douglas B. Kent, Adam Mumford, Denise M. Akob, Travis W. Schaeffer, Luke R. IwanowiczByEcosystems Mission Area, Contaminant Biology, Environmental Health Program, Toxic Substances Hydrology, Columbia Environmental Research Center, Earth Resources Observation and Science (EROS) Center , Eastern Ecological Science Center, Geology, Energy & Minerals Science Center, Reston Microbiology LaboratorySite- and individual-level contaminations affect infection prevalence of an emerging infectious disease of amphibians
Emerging infectious disease outbreaks are one of multiple stressors responsible for amphibian declines globally. In the northeastern United States, ranaviral diseases are prevalent in amphibians and other ectothermic species, but there is still uncertainty as to whether their presence is leading to population level effects. Further, there is also uncertainty surrounding the potential interactionsAuthorsKelly L. Smalling, Brittany A. Mosher, Luke R. Iwanowicz, Keith Loftin, Adam Boehlke, Michelle Hladik, Carly R. Muletz-Wolz, Nandadevi Córtes-Rodríguez, Robin Femmer, Evan H. Campbell GrantByEcosystems Mission Area, Water Resources Mission Area, Contaminant Biology, Environmental Health Program, Toxic Substances Hydrology, Eastern Ecological Science Center, California Water Science Center, Geology, Geophysics, and Geochemistry Science Center, Kansas Water Science Center, New Jersey Water Science CenterTandem field and laboratory approaches to quantify attenuation mechanisms of pharmaceutical and pharmaceutical transformation products in a wastewater effluent-dominated stream
Evolving complex mixtures of pharmaceuticals and transformation products in effluent-dominated streams pose potential impacts to aquatic species; thus, understanding the attenuation dynamics in the field and characterizing the prominent attenuation mechanisms of pharmaceuticals and their transformation products (TPs) is critical for hazard assessments. Herein, we determined the attenuation dynamicAuthorsHui Zhi, Alyssa L Mianecki, Dana W. Kolpin, Rebecca D. Klaper, Luke R. Iwanowicz, Gregory H. LeFevreEmerging investigator series: Municipal wastewater as a year-round point source of neonicotinoid insecticides that persist in an effluent-dominated stream
Neonicotinoids in aquatic systems have been predominantly associated with agriculture, but some are increasingly being linked to municipal wastewater. Thus, the aim of this work was to understand the municipal wastewater contribution to neonicotinoids in a representative, characterized effluent-dominated temperate-region stream. Our approach was to quantify the spatiotemporal concentrations of imiAuthorsDanielle T. Webb, Hui Zhi, Dana W. Kolpin, Rebecca D. Klaper, Luke R. Iwanowicz, Gregory H. LeFevreHyperpigmented melanistic skin lesions of smallmouth bass Micropterus dolomieu from the Chesapeake Bay watershed
Hyperpigmented melanistic skin lesions (HPMLs) of smallmouth bass Micropterus dolomieu are observed in the Potomac and Susquehanna rivers, Chesapeake Bay watershed, USA. Routine, nonlethal population surveys were conducted at 8 sites on the mainstem Susquehanna River and 9 on the Juniata River, a tributary of the Susquehanna River, between 2012 and 2018, and the prevalence of HPMLs was documented.AuthorsVicki S. Blazer, Kelsey T. Young, Geoffrey D. Smith, Adam Sperry, Luke R. IwanowiczReconnaissance of surface water estrogenicity and the prevalence of intersex in smallmouth bass (Micropterus dolomieu) inhabiting New Jersey
The observation of testicular oocytes in male fishes has been utilized as a biomarker of estrogenic endocrine disruption. A reconnaissance project led in the Northeastern United States (US) during the period of 2008–2010 identified a high prevalence of intersex smallmouth bass on or near US Fish & Wildlife Service National Wildlife Refuges that included the observation of 100% prevalence in smallmAuthorsLuke Iwanowicz, Kelly L. Smalling, Vicki S. Blazer, Ryan P. Braham, Lakyn R. Sanders, Anna Boetsma, Nick Procopio, Sandra Goodrow, Gary Buchanan, Daniel Millemann, Bruce Ruppel, John Vile, Brian Henning, John Abatemarco - Partners
External partners that the Functional and Molecular Bioassay Core Technology Team collaborates with are linked below.