Pre-analytical challenges from adsorptive losses associated with thiamine analysis.

Thiamine (vitamin B1) is an essential vitamin serving in its diphosphate form as a cofactor for enzymes in the citric acid cycle and pentose-phosphate pathways. Its concentration reported in the pM and nM range in environmental and clinical analyses prompted our consideration of the components used in pre-analytical processing, including the selection of filters, filter apparatuses, and sample vials. The seemingly innocuous use of glass fiber filters, glass filter flasks, and glass vials, ubiquitous in laboratory analysis of clinical and environmental samples, led to marked thiamine losses. 19.3 nM thiamine was recovered from a 100 nM standard following storage in glass autosampler vials and only 1 nM of thiamine was obtained in the filtrate of a 100 nM thiamine stock passed through a borosilicate glass fiber filter. We further observed a significant shift towards phosphorylated derivatives of thiamine when an equimolar mixture of thiamine, thiamine monophosphate, and thiamine diphosphate was stored in glass (most notably non-silanized glass, where a reduction of 54% of the thiamine peak area was observed) versus polypropylene autosampler vials. The selective losses of thiamine could lead to errors in interpreting the distribution of phosphorylated species in samples. Further, some loss of phosphorylated thiamine derivatives selectively to amber glass vials was observed relative to other glass vials. Our results suggest the use of polymeric filters (including nylon and cellulose acetate) and storage container materials (including polycarbonate and polypropylene) for thiamine handling. Losses to cellulose nitrate and polyethersulfone filters were far less substantial than to glass fiber filters, but were still notable given the low concentrations expected in samples. Thiamine losses were negated when thiamine was stored diluted in trichloroacetic acid or as thiochrome formed in situ, both of which are common practices, but not ubiquitous, in thiamine sample preparation.

Dietary factors potentially impacting thiaminase I-mediated thiamine deficiency.

Fish population declines from thiamine (vitamin B1) deficiency have been widespread in ecologically and economically valuable organisms, ranging from the Great Lakes to the Baltic Sea and, most recently, the California coast. Thiamine deficiencies in predatory fishes are often attributed to a diet of prey fishes with high levels of thiamine-degrading (e.g., thiaminase) enzymes, such as alewives, rainbow smelt, and anchovies. Since their discovery, thiaminase I enzymes have been recognized for breaking down thiamine into its pyrimidine and thiazole moieties using various nucleophilic co-substrates to afford cleavage, but these studies have not thoroughly considered other factors that could modify enzyme activity. We found the thiaminase I enzyme from Clostridium botulinum efficiently degrades thiamine in the presence of pyridoxine (vitamin B6) as a co-substrate but has relatively limited activity in the presence of nicotinic acid (vitamin B3). Using fluorescence measurements, thiamine degradation in an over-the-counter complete multivitamin formulation was inhibited, and a B-complex formulation required co-substrate supplementation for maximal thiamine depletion. These studies prompted the evaluation of specific constituents contributing to thiaminase I inhibition by both chromatography and fluorescence assays: Cu2+ potently and irreversibly inhibited thiamine degradation; ascorbic acid was a strong but reversible inhibitor; Fe2+, Mn2+ and Fe3+ modulated thiamine degradation to a lesser degree. The enhancement by pyridoxine and inhibition by Cu2+ extended to thiaminase-mediated degradation from Burkholderia thailandensis, Paenibacillus thiaminolyticus, and Paenibacillus apiarius in tryptic soy broth supernatants. These co-substrate limitations and the common presence of inhibitory dietary factors complement recent studies reporting that the intended function of thiaminase enzymes is to recycle thiamine breakdown products for thiamine synthesis, not thiamine degradation.

Retraction notice to "Comparison of thiaminase activity in fish using the radiometric and 4-nitrothiophenol colorimetric methods" [J. Great Lakes Res. 36 (2010) 641-645].

[This retracts the article PMC6042866.].

Temporal trends in fish mercury concentrations in an Adirondack Lake managed with a continual predator removal program.

Mercury is a neurotoxic pollutant and contamination in remote ecosystems due to atmospheric mercury deposition coupled with watershed characteristics that influence mercury bioavailability. Biological interactions that affect mercury bioaccumulation are especially relevant as fish assemblages change in response to species introductions and lake management practices. We studied the influence of shifting food web dynamics on mercury in fisheries of Little Moose Lake in the southwestern Adirondack Mountains of New York, USA. Annual removal of non-native Smallmouth Bass (Micropterus dolomieu) has been used as a management strategy since 2000 to restore the native fish assemblage and food web in favor of Lake Trout (Salvelinus namaycush). Changes in total mercury, stable carbon (13C/12C) and nitrogen (15N/14N) isotopes, and growth were evaluated for Lake Trout and Smallmouth Bass. Growth rates increased for both predators and trophic position increased for Lake Trout post-removal. Mercury concentrations in Lake Trout increased over the 16-year study period influenced by a diet shift from invertebrates to higher trophic level prey fish, regardless of increased growth. Smallmouth Bass mercury concentrations decreased with compensatory growth from a reduced population size. These contrasting trends indicate that changes in mercury deposition were not the primary driver for mercury bioaccumulation responses in Little Moose Lake. Stable isotope values changed for both predators and for several lower trophic level organisms, likely reflecting changes in nutrient cycling and/or inputs. Our findings emphasize the potential role of fisheries management on whole-lake and predatory fish responses to mercury contamination in temperate lakes.

Periplasmic binding protein-based magnetic isolation and detection of thiamine in complex biological matrices.

Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine periplasmic binding protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5 nM ranging up to 240 nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2 nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0 nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.

Thiaminase I Provides a Growth Advantage by Salvaging Precursors from Environmental Thiamine and Its Analogs in Burkholderia thailandensis.

Thiamine is essential to life, as it serves as a cofactor for enzymes involved in critical carbon transformations. Many bacteria can synthesize thiamine, while thiamine auxotrophs must obtain it or its precursors from the environment. Thiaminases degrade thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, and thiaminase I specifically has been implicated in thiamine deficiency syndromes in animals. The biological role of this secreted enzyme has been a long-standing mystery. We used the thiaminase I-producing soil bacterium Burkholderia thailandensis as a model to ascertain its function. First, we generated thiamine auxotrophs, which are still able to use exogenous precursors (thiazole and hydroxymethyl pyrimidine), to synthesize thiamine. We found that thiaminase I extended the survival of these strains, when grown in defined media where thiamine was serially diluted out, compared to isogenic strains that could not produce thiaminase I. Thiamine auxotrophs grew better on thiamine precursors than thiamine itself, suggesting thiaminase I functions to convert thiamine to useful precursors. Furthermore, our findings demonstrate that thiaminase I cleaves phosphorylated thiamine and toxic analogs, which releases precursors that can then be used for thiamine synthesis. This study establishes a biological role for this perplexing enzyme and provides additional insight into the complicated nature of thiamine metabolism and how individual bacteria may manipulate the availability of a vital nutrient in the environment.IMPORTANCE The function of thiaminase I has remained a long-standing, unsolved mystery. The enzyme is only known to be produced by a small subset of microorganisms, although thiaminase I activity has been associated with numerous plants and animals, and is implicated in thiamine deficiencies brought on by consumption of organisms containing this enzyme. Genomic and biochemical analyses have shed light on potential roles for the enzyme. Using the genetically amenable thiaminase I-producing soil bacterium Burkholderia thailandensis, we were able to demonstrate that thiaminase I helps salvage precursors from thiamine derivatives in the environment and degrades thiamine to its precursors, which are preferentially used by B. thailandensis auxotrophs. Our study establishes a biological role for this perplexing enzyme and provides insight into the complicated nature of thiamine metabolism. It also establishes B. thailandensis as a robust model system for studying thiamine metabolism.

Thiamine Assays-Advances, Challenges, and Caveats.

Thiamine (vitamin B1) is essential to the health of all living organisms and deficiency has long been associated with diseases in animals such as fish, birds, alligators, and domesticated ruminant mammals. Thiamine is also implicated in several human diseases including Alzheimer's, diabetes, dementia, depression and, most notably, Wernicke-Korsakoff syndrome and Beriberi disease. Yet, highly sensitive and specific detection of thiamine remains an analytical challenge, as pM to nm levels of thiamine need to be detected in environmental and human samples, respectively, various phosphorylated variants need to be discriminated, and rapid on-site detection would be highly desirable. Furthermore, appropriate sample preparation is mandatory, owing to the complexity of the relevant sample matrices including fish tissues, ocean water, and body fluids. This Review has two objectives. First, it provides a thorough overview of analytical techniques published for thiamine detection over the last 15 years. Second, it describes the principles of analytical approaches that are based on biorecognition and may open up new avenues for rapid and high-throughput thiamine analysis. Most notably, periplasmic binding proteins, ribozymes, and aptamers are of particular interest, as they function as bioaffinity recognition elements that can fill an important assay technology gap, owing to the unavailability of thiamine-specific commercial antibodies. Finally, the authors provide brief evaluations of key outcomes of the major assay concepts and suggest how innovative techniques could help develop sensitive and specific thiamine analytical test systems.

Nonnative fish stocking alters stream ecosystem nutrient dynamics.

Each year, millions of hatchery-raised fish are stocked into streams and rivers worldwide, yet the effects of hatchery-raised fish on stream nutrient cycles have seldom been examined. We quantified the influence of supplemental nonnative fish stocking, a widespread recreational fishery management practice, on in-stream nutrient storage and cycling. We predicted that supplemental, hatchery-raised brown trout (Salmo trutta) stocking would result in increased N and P supply relative to in-stream biotic demand for those nutrients and that stocked fishes would remineralize and store a significantly greater amount of N and P than the native fish community, due to higher areal biomass. To test these predictions, we measured the biomass, nutrient (NH4+ -N and soluble reactive phosphorus [SRP]) remineralization rates, and body carbon, nitrogen, and phosphorus content of the native fish community and trout stocked into four study streams. We then estimated fish growth rates to determine species-specific nutrient sequestration rates in body tissues for both stocked and native fish and measured ammonium and phosphorus uptake rates to determine the relative influence of net fish nutrient remineralization on stream nutrient cycles. When brown trout were stocked in these systems at density levels that were orders of magnitude higher than ambient native fish density, they provided a sizeable source of NH4+ -N that could account for up to 85% of demand for that nutrient. Stocked trout had minimal effects on in-stream SRP cycles even at high release densities, likely due to low per capita SRP excretion rates. A unique feature of our study was that we evaluated the temporal component of the stocked trout nutrient subsidy by estimating the number of fish removed from the system through natural mortality and angler harvest, which indicated that the stocked trout subsidy lasted approximately 6-8 weeks after stocking. By combining population models with areal nutrient excretion rates and estimates of biotic nutrient uptake, we showed that trout stocking provided a strong pulsed nutrient subsidy.

Competition for vitamin B1 (thiamin) structures numerous ecological interactions.

Thiamin (vitamin B1) is a cofactor required for essential biochemical reactions in all living organisms, yet free thiamin is scarce in the environment. The diversity of biochemical pathways involved in the acquisition, degradation, and synthesis of thiamin indicates that organisms have evolved numerous ecological strategies for meeting this nutritional requirement. In this review we synthesize information from multiple disciplines to show how the complex biochemistry of thiamin influences ecological outcomes of interactions between organisms in environments ranging from the open ocean and the Australian outback to the gastrointestinal tract of animals. We highlight population and ecosystem responses to the availability or absence of thiamin. These include widespread mortality of fishes, birds, and mammals, as well as the thiamin-dependent regulation of ocean productivity. Overall, we portray thiamin biochemistry as the foundation for molecularly mediated ecological interactions that influence survival and abundance of a vast array of organisms.

Acid rain recovery may help to mitigate the impacts of climate change on thermally sensitive fish in lakes across eastern North America.

From the 1970s to 1990s, more stringent air quality regulations were implemented across North America and Europe to reduce chemical emissions that contribute to acid rain. Surface water pH slowly increased during the following decades, but biological recovery lagged behind chemical recovery. Fortunately, this situation is changing. In the past few years, northeastern US fish populations have begun to recover in lakes that were historically incapable of sustaining wild fish due to acidic conditions. As lake ecosystems across the eastern United States recover from acid deposition, the stress to the most susceptible populations of native coldwater fish appears to be shifting from acidification effects to thermal impacts associated with changing climate. Extreme summer temperature events - which are expected to occur with increasing frequency in the coming century - can stress and ultimately kill native coldwater fish in lakes where thermal stratification is absent or highly limited. Based on data from northeastern North America, we argue that recovery from acid deposition has the potential to improve the resilience of coldwater fish populations in some lakes to impacts of climate change. This will occur as the amount of dissolved organic carbon (DOC) in the water increases with increasing lake pH. Increased DOC will reduce water clarity and lead to shallower and more persistent lake thermoclines that can provide larger areas of coldwater thermal refuge habitat. Recovery from acidification will not eliminate the threat of climate change to coldwater fish, but secondary effects of acid recovery may improve the resistance of coldwater fish populations in lakes to the effects of elevated summer temperatures in historically acidified ecosystems. This analysis highlights the importance of considering the legacy of past ecosystem impacts and how recovery or persistence of those effects may interact with climate change impacts on biota in the coming decades.

High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition.

Although antibodies and aptamers are commonly used bioaffinity recognition elements, they are not available for many important analytes. As an alternative, we demonstrate use of a periplasmic binding protein (PBP) to provide high affinity recognition for thiamine (vitamin B1), an analyte of great importance to human and environmental health for which, like so many other small molecules, no suitable biorecognition element is available. We demonstrate that with an appropriate competitive strategy, a highly sensitive (limit of detection of 0.5 nM) and specific bioassay for thiamine and its phosphorylated derivatives can be designed. The high-throughput method relies upon the thiamine periplasmic binding protein (TBP) from Escherichia coli for thiamine biorecognition and dye-encapsulating liposomes for signal-enhancement. A thiamine monosuccinate-PEG-biotin derivative was synthesized to serve as an immobilized competitor that overcame constraints imposed by the deep binding cleft and structural recognition requirements of PBPs. The assay was applied to ambient environmental samples with high reproducibility. These findings demonstrate that PBPs can serve as highly specific and sensitive affinity recognition elements in bioanalytical assay formats, thereby opening up the field of affinity sensors to a new range of analytes.

Chemical and biological recovery from acid deposition within the Honnedaga Lake watershed, New York, USA.

Honnedaga Lake in the Adirondack region of New York has sustained a heritage brook trout population despite decades of atmospheric acid deposition. Detrimental impacts from acid deposition were observed from 1920 to 1960 with the sequential loss of acid-sensitive fishes, leaving only brook trout extant in the lake. Open-lake trap net catches of brook trout declined for two decades into the late 1970s, when brook trout were considered extirpated from the lake but persisted in tributary refuges. Amendments to the Clean Air Act in 1990 mandated reductions in sulfate and nitrogen oxide emissions. By 2000, brook trout had re-colonized the lake coincident with reductions in surface-water sulfate, nitrate, and inorganic monomeric aluminum. No changes have been observed in surface-water acid-neutralizing capacity (ANC) or calcium concentration. Observed increases in chlorophyll a and decreases in water clarity reflect an increase in phytoplankton abundance. The zooplankton community exhibits low species richness, with a scarcity of acid-sensitive Daphnia and dominance by acid-tolerant copepods. Trap net surveys indicate that relative abundance of adult brook trout population has significantly increased since the 1970s. Brook trout are absent in 65 % of tributaries that are chronically acidified with ANC of <0 µeq/L and toxic aluminum levels (>2 µmol/L). Given the current conditions, a slow recovery of chemistry and biota is expected in Honnedaga Lake and its tributaries. We are exploring the potential to accelerate the recovery of brook trout abundance in Honnedaga Lake through lime applications to chronically and episodically acidified tributaries.

A rapid method for assaying thiaminase I activity in diverse biological samples.

Vitamin B1 (thiamine) deficiencies can lead to neurological disorders, reproductive failure and death in wild and domestic animal populations. In some cases, disease is brought about by the consumption of foods high in thiaminase I activity. Levels of thiaminase activity in these foods are highly variable and the factors leading to production of this enzyme are poorly understood. Here we describe improvements in a spectrophotometric thiaminase I activity assay that measures the disappearance of 4-nitrothiophenol, a favored nucleophile co-substrate that replaces the thiazole portion of thiamine during the inactivation of thiamine by the enzyme. Scalable sample processing protocols and a 96-well microtiter plate format are presented that allow the rapid evaluation of multiple, replicated samples in the course of only a few hours. Observed levels of activity in bacterial culture supernatant, fish, ferns and molluscs using this colorimetric assay were similar to previously published reports that employed a radiometric method. Organisms devoid of thiaminase I, based upon previous work, showed no activity with this assay. In addition, activity was found in a variety of fishes and one fern species from which this enzyme had not previously been reported. Overall, we demonstrate the suitability of this technique for measuring thiaminase I activity within small amounts of tissue and environmental samples with replication levels that were heretofore prohibitive. The assay provides a considerable improvement in the ability to examine and understand the properties of an enzyme that has a substantial influence on organism and ecosystem health.

Comparison of thiaminase activity in fish using the radiometric and 4-nitrothiophenol colorimetric methods.

Thiaminase induced thiamine deficiency occurs in fish, humans, livestock and wild animals. A non-radioactive thiaminase assay was described in 2007, but a direct comparison with the radioactive 14C-thiamine method which has been in use for more than 30 years has not been reported. The objective was to measure thiaminase activity in forage fish (alewife Alosa pseudoharengus, rainbow smelt Osmerus mordax, and slimy sculpin Cottus cognatus) consumed by predators that manifest thiamine deficiency using both methods. Modifications were made to the colorimetric assay to improve repeatability. Modification included a change in assay pH, enhanced sample clean-up, constant assay temperature (37 °C), increase in the concentration of 4-nitrothiophenol (4NTP) and use of a spectrophotometer fitted with a 0.2 cm cell. A strong relationship between the two assays was found for 51 alewife (R2=0.85), 36 smelt (R2=0.87) and 20 sculpin (R2=0.82). Thiaminase activity in the colorimetric assay was about 1000 times higher than activity measured by the radioactive method. Application of the assay to fish species from which no thiaminase activity has previously been reported resulted in no 4NTP thiaminase activity being found in bloater Coregonus hoyi, lake trout Salvelinus namaycusch, steelhead trout Oncorhynchus mykiss or Chinook salmon Oncorhynchus tshawytscha. In species previously reported to contain thiaminase, 4NTP thiaminase activity was measured in bacteria Paenibacillus thiaminolyticus, gizzard shad Dorosoma cepedianum, bracken fern Pteridium aquilinum, quagga mussel Dreissena bugensis and zebra mussels D. polymorpha.

When can efforts to control nuisance and invasive species backfire?

Population control through harvest has the potential to reduce the abundance of nuisance and invasive species. However, demographic structure and density-dependent processes can confound removal efforts and lead to undesirable consequences, such as overcompensation (an increase in abundance in response to harvest) and instability (population cycling or chaos). Recent empirical studies have demonstrated the potential for increased mortality (such as that caused by harvest) to lead to overcompensation and instability in plant, insect, and fish populations. We developed a general population model with juvenile and adult stages to help determine the conditions under which control harvest efforts can produce unintended outcomes. Analytical and simulation analyses of the model demonstrated that the potential for overcompensation as a result of harvest was significant for species with high fecundity, even when annual stage-specific survivorship values were fairly low. Population instability as a result of harvest occurred less frequently and was only possible with harvest strategies that targeted adults when both fecundity and adult survivorship were high. We considered these results in conjunction with current literature on nuisance and invasive species to propose general guidelines for assessing the risks associated with control harvest based on life history characteristics of target populations. Our results suggest that species with high per capita fecundity (over discrete breeding periods), short juvenile stages, and fairly constant survivorship rates are most likely to respond undesirably to harvest. It is difficult to determine the extent to which overcompensation and instability could occur during real-world removal efforts, and more empirical removal studies should be undertaken to evaluate population-level responses to control harvests. Nevertheless, our results identify key issues that have been seldom acknowledged and are potentially generic across taxa.

Changes in mercury bioaccumulation in an apex predator in response to removal of an introduced competitor.

We evaluated methylmercury (MeHg) concentrations in native apex predators, lake trout Salvelinus namaycush before and after the large-scale removal of introduced predators, smallmouth bass Micropterus dolomieu in a 270 ha Adirondack lake. Previous studies show that removing competitors can result in increased growth and decreased mercury concentrations in remaining fish. Instead, we observed a significant increase in lake trout MeHg concentrations despite observed increases in lake trout growth. Bioenergetics simulations predicted similar increases in lake trout MeHg concentrations. Higher MeHg in prey fish (post-removal diet) relative to invertebrates (pre-removal diet) was the most important factor increasing lake trout MeHg concentrations. However, this effect was counteracted by increased lake trout growth (i.e., growth dilution) likely due to a combination of decreased foraging costs and an increase in prey energy density. These data provide evidence for a mechanism (diet shift due to reduced competition) by which changes in food web structure can influence MeHg concentrations in top predators.

Evaluating the effect of stressors on thiaminase activity in alewife.

No consistent explanation has been found for the variability in the thiaminase activity of alewives Alosa pseudoharengus despite the role of alewife thiaminase in large-scale salmonine mortality in the Laurentian Great Lakes. We conducted experiments to evaluate the effect of two stressors, reduced salt content in the water and food limitation, on alewife thiaminase activity. Alewives were subjected to treatments in replicated tanks in which conductivity was lowered (< 100 microS/cm) for 8 d and feeding was limited for 39 d. Circulating white blood cells, plasma cortisol, plasma glucose, and whole-body thiaminase were measured in individual alewives to assess their response to these experimental treatments. Alewives from the controls had significantly larger numbers of circulating white blood cells than those in the salt-reduced and food-limited treatments (24,000 and 19,000 cells/microL and 11,000 and 9,000 cells/microL for alewives from the two control and salt-reduced treatment tanks, respectively, and 34,000 and 30,000 cells/microL and 21,000 and 16,000 cells/microL for alewives from the two control and food-limited treatment tanks). No significant differences in alewife thiaminase activity were found between treatment fish and their controls. The mean thiaminase activity in the alewives studied increased from 6,900 to 16,000 pmol x g(-1) x min(-1) from the time of their collection in Cayuga Lake to the start of laboratory experiments 1.5-2.5 years later; the latter value was more than twice that of previously reported levels of thiaminase activity from alewives collected in the wild. These data suggest that the variability in alewife thiaminase is not related to stress from salt reduction or food limitation, but laboratory holding conditions significantly increased thiaminase through a mechanism not evaluated by our experimental treatments.

An assay for thiaminase I in complex biological samples.

An alternative method for measuring thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, resulting in a large decrease in absorbance at 411nm. This new assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high-throughput analysis in a 96-well format with complex biological samples.

Mature and old-growth riparian forests: structure, dynamics, and effects on Adirondack stream habitats.

Riparian forests regulate linkages between terrestrial and aquatic ecosystems, yet relationships among riparian forest development, stand structure, and stream habitats are poorly understood in many temperate deciduous forest systems. Our research has (1) described structural attributes associated with old-growth riparian forests and (2) assessed linkages between these characteristics and in-stream habitat structure. The 19 study sites were located along predominantly first- and second-order streams in northern hardwood-conifer forests in the Adirondack Mountains of New York (U.S.A.). Sites were classified as mature forest (6 sites), mature with remnant old-growth trees (3 sites), and old-growth (10 sites). Forest-structure attributes were measured over stream channels and at varying distances from each bank. In-stream habitat features such as large woody debris (LWD), pools, and boulders were measured in each stream reach. Forest structure was examined in relation to stand age using multivariate techniques, ANOVA, and linear regression. We investigated linkages between forest structure and stream characteristics using similar methods, preceded by information-theoretic modeling (AIC). Old-growth riparian forest structure is more complex than that found in mature forests and exhibits significantly greater accumulations of aboveground tree biomass, both living and dead. In-stream LWD volumes were significantly (alpha = 0.05) greater at old-growth sites (200 m3/ha) compared to mature sites (34 m3/ha) and were strongly related to the basal area of adjacent forests. In-stream large-log densities correlated strongly with debris-dam densities. AIC models that included large-log density, debris-dam density, boulder density, and bankfull width had the most support for predicting pool density. There were higher proportions of LWD-formed pools relative to boulder-formed pools at old-growth sites as compared to mature sites. Old-growth riparian forests provide in-stream habitat features that have not been widely recognized in eastern North America, representing a potential benefit from late-successional riparian forest management and conservation. Riparian management practices (including buffer delineation and restorative silvicultural approaches) that emphasize development and maintenance of late-successional characteristics are recommended where the associated in-stream effects are desired.