Aerobic anoxygenic phototrophs play important roles in nutrient cycling within cyanobacterial Microcystis bloom microbiomes.

BACKGROUND: During the bloom season, the colonial cyanobacterium Microcystis forms complex aggregates which include a diverse microbiome within an exopolymer matrix. Early research postulated a simple mutualism existing with bacteria benefitting from the rich source of fixed carbon and Microcystis receiving recycled nutrients. Researchers have since hypothesized that Microcystis aggregates represent a community of synergistic and interacting species, an interactome, each with unique metabolic capabilities that are critical to the growth, maintenance, and demise of Microcystis blooms. Research has also shown that aggregate-associated bacteria are taxonomically different from free-living bacteria in the surrounding water. Moreover, research has identified little overlap in functional potential between Microcystis and members of its microbiome, further supporting the interactome concept. However, we still lack verification of general interaction and know little about the taxa and metabolic pathways supporting nutrient and metabolite cycling within Microcystis aggregates. RESULTS: During a 7-month study of bacterial communities comparing free-living and aggregate-associated bacteria in Lake Taihu, China, we found that aerobic anoxygenic phototrophic (AAP) bacteria were significantly more abundant within Microcystis aggregates than in free-living samples, suggesting a possible functional role for AAP bacteria in overall aggregate community function. We then analyzed gene composition in 102 high-quality metagenome-assembled genomes (MAGs) of bloom-microbiome bacteria from 10 lakes spanning four continents, compared with 12 complete Microcystis genomes which revealed that microbiome bacteria and Microcystis possessed complementary biochemical pathways that could serve in C, N, S, and P cycling. Mapping published transcripts from Microcystis blooms onto a comprehensive AAP and non-AAP bacteria MAG database (226 MAGs) indicated that observed high levels of expression of genes involved in nutrient cycling pathways were in AAP bacteria. CONCLUSIONS: Our results provide strong corroboration of the hypothesized Microcystis interactome and the first evidence that AAP bacteria may play an important role in nutrient cycling within Microcystis aggregate microbiomes. Video Abstract.

Microcystis pangenome reveals cryptic diversity within and across morphospecies.

Microcystis, a common harmful algal bloom (HAB) taxon, threatens water supplies and human health, yet species delimitation is contentious in this taxon, leading to challenges in research and management of this threat. Historical and common morphology-based classifications recognize multiple morphospecies, most with variable and diverse ecologies, while DNA sequence-based classifications indicate a single species with multiple ecotypes. To better delimit Microcystis species, we conducted a pangenome analysis of 122 genomes. Core- and non-core gene phylogenetic analyses placed 113 genomes into 23 monophyletic clusters containing at least two genomes. Overall, genome-related indices revealed that Microcystis contains at least 16 putative genospecies. Fifteen genospecies included at least one Microcystis aeruginosa morphospecies, and 10 genospecies included two or more morphospecies. This classification system will enable consistent taxonomic identification of Microcystis and thereby aid in resolving some of the complexities and controversies that have long characterized eco-evolutionary research and management of this important HAB taxon.

Potential biomarkers of endocrine and habitat disruption identified via RNA-Seq in Salvelinus fontinalis with proximity to fracking operations in Pennsylvania headwater stream ecosystems.

Unconventional natural gas development (fracking) has been a rapidly expanding technique used for the extraction of natural gas from the Marcellus Shale formation in Pennsylvania. There remains a knowledge gap regarding the ecological impacts of fracking, especially regarding the long-term health of native Brook trout (Salvelinus fontinalis) populations. During the summer of 2015, Brook trout were sampled from twelve streams located in forested, northwestern Pennsylvania in order to evaluate the impacts of fracking on Brook trout. Four stream sites were undisturbed (no fracking activity), three had a developed well pad without fracking activity, and five had active fracking with natural gas production. Liver tissue was isolated from two to five fish per stream and underwent RNA-Seq analysis to identify differentially expressed genes between ecosystems with differing fracking status. Data were analyzed individually and with samples pooled within-stream to account for hierarchical data structure and variation in sample coverage within streams. Differentially expressed and differentially alternatively spliced genes had functions related to lipid and steroid metabolism, mRNA processing, RNA polymerase and protein regulation. Unique to our study, genes related to xenobiotic and stress responses were found as well as potential markers for endocrine disruption and saline adaptation that were identified in watersheds with active fracking activity. These results support the utility of RNA-Seq to assess trout health and suggest detrimental impacts of fracking on sensitive trout populations.

Early-life formula feeding is associated with infant gut microbiota alterations and an increased antibiotic resistance load.

BACKGROUND: Infants are at a high risk of acquiring fatal infections, and their treatment relies on functioning antibiotics. Antibiotic resistance genes (ARGs) are present in high numbers in antibiotic-naive infants' gut microbiomes, and infant mortality caused by resistant infections is high. The role of antibiotics in shaping the infant resistome has been studied, but there is limited knowledge on other factors that affect the antibiotic resistance burden of the infant gut. OBJECTIVES: Our objectives were to determine the impact of early exposure to formula on the ARG load in neonates and infants born either preterm or full term. Our hypotheses were that diet causes a selective pressure that influences the microbial community of the infant gut, and formula exposure would increase the abundance of taxa that carry ARGs. METHODS: Cross-sectionally sampled gut metagenomes of 46 neonates were used to build a generalized linear model to determine the impact of diet on ARG loads in neonates. The model was cross-validated using neonate metagenomes gathered from public databases using our custom statistical pipeline for cross-validation. RESULTS: Formula-fed neonates had higher relative abundances of opportunistic pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Klebsiella oxytoca, and Clostridioides difficile. The relative abundance of ARGs carried by gut bacteria was 69% higher in the formula-receiving group (fold change, 1.69; 95% CI: 1.12-2.55; P = 0.013; n = 180) compared to exclusively human milk-fed infants. The formula-fed infants also had significantly less typical infant bacteria, such as Bifidobacteria, that have potential health benefits. CONCLUSIONS: The novel finding that formula exposure is correlated with a higher neonatal ARG burden lays the foundation that clinicians should consider feeding mode in addition to antibiotic use during the first months of life to minimize the proliferation of antibiotic-resistant gut bacteria in infants.

Comparative meta-omics for identifying pathogens associated with prosthetic joint infection.

Prosthetic joint infections (PJI) are economically and personally costly, and their incidence has been increasing in the United States. Herein, we compared 16S rRNA amplicon sequencing (16S), shotgun metagenomics (MG) and metatranscriptomics (MT) in identifying pathogens causing PJI. Samples were collected from 30 patients, including 10 patients undergoing revision arthroplasty for infection, 10 patients receiving revision for aseptic failure, and 10 patients undergoing primary total joint arthroplasty. Synovial fluid and peripheral blood samples from the patients were obtained at time of surgery. Analysis revealed distinct microbial communities between primary, aseptic, and infected samples using MG, MT, (PERMANOVA p = 0.001), and 16S sequencing (PERMANOVA p < 0.01). MG and MT had higher concordance with culture (83%) compared to 0% concordance of 16S results. Supervised learning methods revealed MT datasets most clearly differentiated infected, primary, and aseptic sample groups. MT data also revealed more antibiotic resistance genes, with improved concordance results compared to MG. These data suggest that a differential and underlying microbial ecology exists within uninfected and infected joints. This study represents the first application of RNA-based sequencing (MT). Further work on larger cohorts will provide opportunities to employ deep learning approaches to improve accuracy, predictive power, and clinical utility.

A Metatranscriptomics Survey of Microbial Diversity on Surfaces Post-Intervention of cleanSURFACES® Technology in an Intensive Care Unit.

Hospital-acquired infections (HAIs) pose a serious threat to patients, and hospitals spend billions of dollars each year to reduce and treat these infections. Many HAIs are due to contamination from workers' hands and contact with high-touch surfaces. Therefore, we set out to test the efficacy of a new preventative technology, AIONX® Antimicrobial Technologies, Inc's cleanSURFACES®, which is designed to complement daily chemical cleaning events by continuously preventing re-colonization of surfaces. To that end, we swabbed surfaces before (Baseline) and after (Post) application of the cleanSURFACES® at various time points (Day 1, Day 7, Day 14, and Day 28). To circumvent limitations associated with culture-based and 16S rRNA gene amplicon sequencing methodologies, these surface swabs were processed using metatranscriptomic (RNA) analysis to allow for comprehensive taxonomic resolution and the detection of active microorganisms. Overall, there was a significant (P < 0.05) global reduction of microbial diversity in Post-intervention samples. Additionally, Post sample microbial communities clustered together much more closely than Baseline samples based on pairwise distances calculated with the weighted Jaccard distance metric, suggesting a defined shift after product application. This shift was characterized by a general depletion of several microbes among Post samples, with multiple phyla also being reduced over the duration of the study. Notably, specific clinically relevant microbes, including Staphylococcus aureus, Clostridioides difficile and Streptococcus spp., were depleted Post-intervention. Taken together, these findings suggest that chemical cleaning events used jointly with cleanSURFACES® have the potential to reduce colonization of surfaces by a wide variety of microbes, including many clinically relevant pathogens.

Walnuts and Vegetable Oils Containing Oleic Acid Differentially Affect the Gut Microbiota and Associations with Cardiovascular Risk Factors: Follow-up of a Randomized, Controlled, Feeding Trial in Adults at Risk for Cardiovascular Disease.

BACKGROUND: It is unclear whether the favorable effects of walnuts on the gut microbiota are attributable to the fatty acids, including α-linolenic acid (ALA), and/or the bioactive compounds and fiber. OBJECTIVE: This study examined between-diet gut bacterial differences in individuals at increased cardiovascular risk following diets that replace SFAs with walnuts or vegetable oils. METHODS: Forty-two adults at cardiovascular risk were included in a randomized, crossover, controlled-feeding trial that provided a 2-wk standard Western diet (SWD) run-in and three 6-wk isocaloric study diets: a diet containing whole walnuts (WD; 57-99 g/d walnuts; 2.7% ALA), a fatty acid-matched diet devoid of walnuts (walnut fatty acid-matched diet; WFMD; 2.6% ALA), and a diet replacing ALA with oleic acid without walnuts (oleic acid replaces ALA diet; ORAD; 0.4% ALA). Fecal samples were collected following the run-in and study diets to assess gut microbiota with 16S rRNA sequencing and Qiime2 for amplicon sequence variant picking. RESULTS: Subjects had elevated BMI (30 ± 1 kg/m2), blood pressure (121 ± 2/77 ± 1 mmHg), and LDL cholesterol (120 ± 5 mg/dL). Following the WD, Roseburia [relative abundance (RA) = 4.2%, linear discriminant analysis (LDA) = 4], Eubacterium eligensgroup (RA = 1.4%, LDA = 4), LachnospiraceaeUCG001 (RA = 1.2%, LDA = 3.2), Lachnospiraceae UCG004 (RA = 1.0%, LDA = 3), and Leuconostocaceae (RA = 0.03%, LDA = 2.8) were most abundant relative to taxa in the SWD (P ≤ 0.05 for all). The WD was also enriched in Gordonibacter relative to the WFMD. Roseburia (3.6%, LDA = 4) and Eubacterium eligensgroup (RA = 1.5%, LDA = 3.4) were abundant following the WFMD, and Clostridialesvadin BB60group (RA = 0.3%, LDA = 2) and gutmetagenome (RA = 0.2%, LDA = 2) were most abundant following the ORAD relative to the SWD (P ≤ 0.05 for all). Lachnospiraceae were inversely correlated with blood pressure and lipid/lipoprotein measurements following the WD. CONCLUSIONS: The results indicate similar enrichment of Roseburia following the WD and WFMD, which could be explained by the fatty acid composition. Gordonibacter enrichment and the inverse association between Lachnospiraceae and cardiovascular risk factors following the WD suggest that the gut microbiota may contribute to the health benefits of walnut consumption in adults at cardiovascular risk. This trial was registered at clinicaltrials.gov as NCT02210767.

Microbial Communities Associated With Passive Acidic Abandoned Coal Mine Remediation.

Acid mine drainage (AMD) is an environmental issue that can be characterized by either acidic or circumneutral pH and high dissolved metal content in contaminated waters. It is estimated to affect roughly 3000 miles of waterways within the state of Pennsylvania, with half being acidic and half being circumneutral. To negate the harmful effects of AMD, ∼300 passive remediation systems have been constructed within the state of Pennsylvania. In this study, we evaluated the microbial community structure and functional capability associated with Middle Branch passive remediation system in central PA. Sediment and water samples were collected from each area within the passive remediation system and its receiving stream. Environmental parameters associated with the remediation system were found to explain a significant amount of variation in microbial community structure. This study revealed shifts in microbial community structure from acidophilic bacteria in raw AMD discharge to a more metabolically diverse set of taxa (i.e., Acidimicrobiales, Rhizobiales, Chthoniobacteraceae) toward the end of the system. Vertical flow ponds and the aerobic wetland showed strong metabolic capability for sulfur redox environments. These findings are integral to the understanding of designing effective passive remediation systems because it provides insight as to how certain bacteria [sulfate reducing bacteria (SRBs) and sulfur oxidizing bacteria (SOBs)] are potentially contributing to a microbially mediated AMD remediation process. This study further supports previous investigations that demonstrated the effectiveness of SRBs in the process of removing sulfate and heavy metals from contaminated water.

Integrated Meta-omics Reveals a Fungus-Associated Bacteriome and Distinct Functional Pathways in Clostridioides difficile Infection.

There has been no prior application of matched metagenomics and metatranscriptomics in Clostridioides difficile infection (CDI) evaluating the role of fungi in CDI or identifying community functions that contribute to the development of this disease. We collected diarrheal stools from 49 inpatients (18 of whom tested positive for CDI) under stringent inclusion criteria. We utilized a tiered sequencing approach to identify enriched bacterial and fungal taxa, using 16S and internal transcribed spacer (ITS) rRNA gene amplicon sequencing, with matched metagenomics and metatranscriptomics performed on a subset of the population. Distinct bacterial and fungal compositions distinguished CDI-positive and -negative patients, with the greatest differentiation between the cohorts observed based on bacterial metatranscriptomics. Bipartite network analyses demonstrated that Aspergillus and Penicillium taxa shared a strong positive relationship in CDI patients and together formed negative cooccurring relationships with several bacterial taxa, including the Oscillospira, Comamonadaceae, Microbacteriaceae, and Cytophagaceae Metatranscriptomics revealed enriched pathways in CDI patients associated with biofilm production primarily driven by Escherichia coli and Pseudomonas, quorum-sensing proteins, and two-component systems related to functions such as osmotic regulation, linoleic acid metabolism, and flagellar assembly. Differential expression of functional pathways unveiled a mechanism by which the causal dysbiosis of CDI may self-perpetuate, potentially contributing to treatment failures. We propose that CDI has a distinct fungus-associated bacteriome, and this first description of metatranscriptomics in human subjects with CDI demonstrates that inflammation, osmotic changes, and biofilm production are key elements of CDI pathophysiology.IMPORTANCE Our data suggest a potential role for fungi in the most common nosocomial bacterial infection in the United States, introducing the concept of a transkingdom interaction between bacteria and fungi in this disease. We also provide the first direct measure of microbial community function in Clostridioides difficile infection using patient-derived tissue samples, revealing antibiotic-independent mechanisms by which C. difficile infection may resist a return to a healthy gut microbiome.

Using Bacterial Transcriptomics to Investigate Targets of Host-Bacterial Interactions in Caenorhabditis elegans.

The interactions between a host and its resident microbes form complicated networks that can affect host physiology. Disentangling these host-microbe interactions can help us better understand mechanisms by which bacteria affect hosts, while also defining the integral commensal protection that host-associated microbiota offer to promote health. Here we utilize a tractable genetic model organism, Caenorhabditis elegans, to study the effects of host environments on bacterial gene expression and metabolic pathways. First, we compared the transcriptomic profiles of E. coli OP50 in vitro (on agar plates) versus in vivo (fed to C. elegans host). Our data revealed that 110 biosynthetic genes were enriched in host-associated E. coli. Several of these expressed genes code for the precursors and products needed for the synthesis of lipopolysaccharides (LPS), which are important for innate immune and stress responses, as well as pathogenicity. Secondly, we compared the transcriptomic profiles of E. coli fed to hosts with different genetic backgrounds, including the long-lived daf-2/insulin like growth factor (IGF) receptor and short lived daf-16/FOXO transcription factor mutants. We find that hosts genetics also alters bacterial metabolic pathways. Given that bacteria influence host health, this transcriptomics approach can elucidate genes mediating host aging.

Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania.

Unconventional oil and gas (UOG) extraction, also known as hydraulic fracturing, is becoming more prevalent with the increasing use and demand for natural gas; however, the full extent of its environmental impacts is still unknown. Here we measured physicochemical properties and bacterial community composition of sediment samples taken from twenty-eight streams within the Marcellus shale formation in northeastern Pennsylvania differentially impacted by hydraulic fracturing activities. Fourteen of the streams were classified as UOG+, and thirteen were classified as UOG- based on the presence of UOG extraction in their respective watersheds. One stream was located in a watershed that previously had UOG extraction activities but was recently abandoned. We utilized high-throughput sequencing of the 16S rRNA gene to infer differences in sediment aquatic bacterial community structure between UOG+ and UOG- streams, as well as correlate bacterial community structure to physicochemical water parameters. Although overall alpha and beta diversity differences were not observed, there were a plethora of significantly enriched operational taxonomic units (OTUs) within UOG+ and UOG- samples. Our biomarker analysis revealed many of the bacterial taxa enriched in UOG+ streams can live in saline conditions, such as Rubrobacteraceae. In addition, several bacterial taxa capable of hydrocarbon degradation were also enriched in UOG+ samples, including Oceanospirillaceae. Methanotrophic taxa, such as Methylococcales, were significantly enriched as well. Several taxa that were identified as enriched in these samples were enriched in samples taken from different streams in 2014; moreover, partial least squares discriminant analysis (PLS-DA) revealed clustering between streams from the different studies based on the presence of hydraulic fracturing along the second axis. This study revealed significant differences between bacterial assemblages within stream sediments of UOG+ and UOG- streams and identified several potential biomarkers for evaluating and monitoring the response of autochthonous bacterial communities to potential hydraulic fracturing impacts.

Response of Aquatic Bacterial Communities to Hydraulic Fracturing in Northwestern Pennsylvania: A Five-Year Study.

Horizontal drilling and hydraulic fracturing extraction procedures have become increasingly present in Pennsylvania where the Marcellus Shale play is largely located. The potential for long-term environmental impacts to nearby headwater stream ecosystems and aquatic bacterial assemblages is still incompletely understood. Here, we perform high-throughput sequencing of the 16 S rRNA gene to characterize the bacterial community structure of water, sediment, and other environmental samples (n = 189) from 31 headwater stream sites exhibiting different histories of fracking activity in northwestern Pennsylvania over five years (2012-2016). Stream pH was identified as a main driver of bacterial changes within the streams and fracking activity acted as an environmental selector for certain members at lower taxonomic levels within stream sediment. Methanotrophic and methanogenic bacteria (i.e. Methylocystaceae, Beijerinckiaceae, and Methanobacterium) were significantly enriched in sites exhibiting Marcellus shale activity (MSA+) compared to MSA- streams. This study highlighted potential sentinel taxa associated with nascent Marcellus shale activity and some of these taxa remained as stable biomarkers across this five-year study. Identifying the presence and functionality of specific microbial consortia within fracking-impacted streams will provide a clearer understanding of the natural microbial community's response to fracking and inform in situ remediation strategies.

Antibiotic Treatments for Clostridium difficile Infection Are Associated with Distinct Bacterial and Fungal Community Structures.

Clostridium difficile infection (CDI) is the most common nosocomial infection in the United States, being associated with high recurrence and persistence rates. Though the relationship between intestinal dysbiosis and CDI is well known, it is unclear whether different forms of dysbiosis may potentially affect the course of CDI. How this is further influenced by C. difficile-directed antibiotics is virtually uninvestigated. In this study, diarrheal stool samples were collected from 20 hospitalized patients, half of whom were confirmed to have CDI. Analyzing tissue ex vivo and in duplicate, CDI and non-CDI fecal samples (n = 176) were either not antibiotic treated or treated with metronidazole, vancomycin, or fidaxomicin, the three most common CDI therapies. The microbial community composition, interactions, and predicted metabolic functions were assessed by 16S rRNA gene and internal transcribed spacer sequencing, bipartite network analysis, and phylogenetic investigation of communities by reconstruction of unobserved states. Our results demonstrate that while all C. difficile-directed antibiotics were associated with similar reductions in alpha diversity, beta diversity significantly differed on the basis of the particular antibiotic, with differentiating relative abundances of bacterial and fungal assemblages. With the exception of fidaxomicin, each antibiotic was associated with the emergence of potentially pathogenic fungal operational taxonomic units, with predicted bacterial functions enriched for xenobiotic metabolism that could perpetuate the dysbiosis driving CDI. Toxin-independent mechanisms of colitis related to the relative abundance of pathogenic bacteria and fungi were also noted. This study suggests that a transkingdom interaction between fungi and bacteria may be important in CDI pathophysiology, including being a factor in the historically high persistence and recurrence rates associated with this disease. IMPORTANCE Using human fecal samples and including sequencing for both bacterial and fungal taxa, this study compared the conventional antibiotics used to treat C. difficile infection (CDI) from the perspective of the microbiome, which is particularly relevant, given the relationship between dysbiotic states and the development of CDI. Sequencing and imputed functional analyses suggest that C. difficile-directed antibiotics are associated with distinct forms of dysbiosis that may be influential in the course of CDI. Further, a role for fungal organisms in the perpetuation of the causal dysbiosis of CDI is discussed, suggesting a previously unappreciated, clinically relevant transkingdom interaction that warrants further study.

Bacterial Community Dynamics in Dichloromethane-Contaminated Groundwater Undergoing Natural Attenuation.

The uncontrolled release of the industrial solvent methylene chloride, also known as dichloromethane (DCM), has resulted in widespread groundwater contamination in the United States. Here we investigate the role of groundwater bacterial communities in the natural attenuation of DCM at an undisclosed manufacturing site in New Jersey. This study investigates the bacterial community structure of groundwater samples differentially contaminated with DCM to better understand the biodegradation potential of these autochthonous bacterial communities. Bacterial community analysis was completed using high-throughput sequencing of the 16S rRNA gene of groundwater samples (n = 26) with DCM contamination ranging from 0.89 to 9,800,000 µg/L. Significant DCM concentration-driven shifts in overall bacterial community structure were identified between samples, including an increase in the abundance of Firmicutes within the most contaminated samples. Across all samples, a total of 6,134 unique operational taxonomic units (OTUs) were identified, with 16 taxa having strong correlations with increased DCM concentration. Putative DCM degraders such as Pseudomonas, Dehalobacterium and Desulfovibrio were present within groundwater across all levels of DCM contamination. Interestingly, each of these taxa dominated specific DCM contamination ranges respectively. Potential DCM degrading lineages yet to be cited specifically as a DCM degrading organisms, such as the Desulfosporosinus, thrived within the most heavily contaminated groundwater samples. Co-occurrence network analysis revealed aerobic and anaerobic bacterial taxa with DCM-degrading potential were present at the study site. Our 16S rRNA gene survey serves as the first in situ bacterial community assessment of contaminated groundwater harboring DCM concentrations ranging over seven orders of magnitude. Diversity analyses revealed known as well as potentially novel DCM degrading taxa within defined DCM concentration ranges, indicating niche-specific responses of these autochthonous populations. Altogether, our findings suggest that monitored natural attenuation is an appropriate remediation strategy for DCM contamination, and that high-throughput sequencing technologies are a robust method for assessing the potential role of biodegrading bacterial assemblages in the apparent reduction of DCM concentrations in environmental scenarios.

Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169.

We report here the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, although Fl-0169T possesses unique virulence genes.

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations.

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal behavior questions. The protocol described here provides a simple, reproducible, and controlled method to manipulate DO to study behavioral response in aquatic organisms resulting from hypoxic and anoxic conditions. While performing degasification of water with nitrogen is commonly used in laboratory settings, no explicit method for ecological (aquatic) application exists in the literature, and this protocol is the first to describe a protocol to degasify water to observe organismal response. This technique and protocol were developed for direct application for aquatic macroinvertebrates; however, small fish, amphibians, and other aquatic vertebrates could be easily substituted. It allows for easy manipulation of DO levels ranging from 2 mg/L to 11 mg/L with stability for up to a 5 min animal-observation period. Beyond a 5 min observation period water temperatures began to rise, and at 10 min DO levels became too unstable to maintain. The protocol is scalable to the study organism, reproducible, and reliable, allowing for rapid implementation into introductory teaching labs and high-level research applications. The expected results of this technique should relate dissolved oxygen changes to behavioral responses of organisms.