Evolutionary and ecological correlates of thiaminase in fishes.

Thiamine (vitamin B1) is required by all living organisms in multiple metabolic pathways. It is scarce in natural systems, and deficiency can lead to reproductive failure, neurological issues, and death. One major cause of thiamine deficiency is an overreliance on diet items containing the enzyme thiaminase. Thiaminase activity has been noted in many prey fishes and linked to cohort failure in salmonid predators that eat prey fish with thiaminase activity, yet it is generally unknown whether evolutionary history, fish traits, and/or environmental conditions lead to production of thiaminase. We conducted literature and GenBank BLAST sequence searches to collect thiaminase activity data and sequence homology data in expressed protein sequences for 300 freshwater and marine fishes. We then tested whether presence or absence of thiaminase could be predicted by evolutionary relationships, trophic level, omega-3 fatty acid concentrations, habitat, climate, invasive potential, and body size. There was no evolutionary relationship with thiaminase activity. It first appears in Class Actinoptergyii (bony ray-finned fishes) and is present across the entire Actinoptergyii phylogeny in both primitive and derived fish orders. Instead, ecological factors explained the most variation in thiaminase: fishes were more likely to express thiaminase if they fed closer to the base of the food web, were high in polyunsaturated fatty acids, lived in freshwater, and were from tropical climates. These data provide a foundation for understanding sources of thiaminase leading to thiamine deficiency in fisheries and other organisms, including humans that eat uncooked fish.

Toward invasive mussel genetic biocontrol: Approaches, challenges, and perspectives.

Invasive freshwater mussels, such as the zebra (Dreissena polymorpha), quagga (Dreissena rostriformis bugensis), and golden (Limnoperna fortunei) mussel have spread outside their native ranges throughout many regions of the North American, South American, and European continents in recent decades, damaging infrastructure and the environment. This review describes ongoing efforts by multiple groups to develop genetic biocontrol methods for invasive mussels. First, we provide an overview of genetic biocontrol strategies that have been applied in other invasive or pest species. Next, we summarize physical and chemical methods that are currently in use for invasive mussel control. We then describe the multidisciplinary approaches our groups are employing to develop genetic biocontrol tools for invasive mussels. Finally, we discuss the challenges and limitations of applying genetic biocontrol tools to invasive mussels. Collectively, we aim to openly share information and combine expertise to develop practical tools to enable the management of invasive freshwater mussels.

When are environmental DNA early detections of invasive species actionable?

Environmental DNA (eDNA) sampling provides sensitive early detection capabilities for recently introduced taxa. However, natural resource managers struggle with how to integrate eDNA results into an early detection rapid response program because positive eDNA detections are not always indicative of an eventual infestation. We used a structured decision making (SDM) framework to evaluate appropriate response actions to hypothetical eDNA early detections of an introduced aquatic plant in Sebago Lake (Maine, USA). The results were juxtaposed to a recent study that used a similar SDM approach to evaluate response actions to hypothetical eDNA early detections of introduced mussels in Jordanelle Reservoir (Utah, USA). We found that eDNA early detections were not actionable in Sebago Lake because the plant's invasion potential was spatially constrained and the current management activities provided acceptable levels of mitigation. In Jordanelle Reservoir, eDNA detections were actionable due to high invasion potential and analyses supported management actions to contain the invasion. The divergent outcomes of the two case studies are related to the unique attributes of the habitats and species, highlighting the utility of the SDM approach when considering an eDNA monitoring program. We use these two case studies to present a general SDM framework and a set of heuristics that can be efficiently applied to eDNA early detection rapid response scenarios and other instances associated with indeterminant eDNA detections, especially when there is an imperative to make decisions as quickly as possible.

Genetic basis of thiaminase I activity in a vertebrate, zebrafish Danio rerio.

Thiamine (vitamin B1) metabolism is an important driver of human and animal health and ecological functioning. Some organisms, including species of ferns, mollusks, and fish, contain thiamine-degrading enzymes known as thiaminases, and consumption of these organisms can lead to thiamine deficiency in the consumer. Consumption of fish containing thiaminase has led to elevated mortality and recruitment failure in farmed animals and wild salmonine populations around the world. In the North American Great Lakes, consumption of the non-native prey fish alewife (Alosa pseudoharengus) by native lake trout (Salvelinus namaycush) led to thiamine deficiency in the trout, contributed to elevated fry mortality, and impeded natural population recruitment. Several thiaminases have been genetically characterized in bacteria and unicellular eukaryotes, and the source of thiaminase in multicellular organisms has been hypothesized to be gut microflora. In an unexpected discovery, we identified thiaminase I genes in zebrafish (Danio rerio) with homology to bacterial tenA thiaminase II. The biochemical activity of zebrafish thiaminase I (GenBank NP_001314821.1) was confirmed in a recombinant system. Genes homologous to the zebrafish tenA-like thiaminase I were identified in many animals, including common carp (Cyprinus carpio), zebra mussel (Dreissena polymorpha) and alewife. Thus, the source of thiaminase I in alewife impacting lake trout populations is likely to be de novo synthesis.

Declines in Reproductive Condition of Male Largemouth Bass (Micropterus salmoides) Following Seasonal Exposure to Estrogenic Endocrine-Disrupting Compounds.

Reproductive abnormalities, that could lead to possible effects at the population level, have been observed in wild fish throughout the United States, with high prevalence in largemouth bass (LMB; Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Estrone (E1) and atrazine (ATR) are common environmental contaminants often associated with agricultural land use. 17alpha-ethinylestradiol (EE2) is a contaminant associated with wastewater treatment effluent, and a representative, well-studied estrogen commonly used for fish toxicity testing. Our objective was to assess whether early gonad recrudescence in adult fish was a period of sensitivity for alterations in reproductive condition and function. Adult male LMB were exposed from post-spawning to early gonad recrudescence to either a mixture of E1 (47.9 ng/L) + ATR (5.4 µg/L), or EE2 (2.4 ng/L) in outdoor experimental ponds. Gonad samples were collected from fish just prior to the start of exposure (July), at the end of the exposure period (December), the following spring just prior to spawning (April), and post spawning (May). Gonadosomatic index (GSI) was significantly reduced in E1 + ATR-exposed and EE2-exposed males compared to control at every post-exposure time point. Reduced sperm count and sperm motility were observed in the mixture treatment (E1 + ATR) compared to the control. Sperm motility was also reduced in the EE2 treatment. These data together indicate that estrogenic endocrine-disrupting compounds can lessen the reproductive condition of adult male LMB, and that effects of exposure during early gonad recrudescence can persist at least through the subsequent spawning cycle.

Exposure to 17α-Ethinylestradiol Results in Differential Susceptibility of Largemouth Bass (Micropterus salmoides) to Bacterial Infection.

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass (Micropterus salmoides) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2Low, 0.87 ng/L) or high (EE2High, 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD40 dose of the Gram-negative bacteria Edwardsiella piscicida. Unexpectedly, this prior exposure to EE2High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2Low-exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2High treatment that could underlie the observed protective effect and metabolic shift in EE2High-infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Effects of early life stage exposure of largemouth bass to atrazine or a model estrogen (17α-ethinylestradiol).

Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.

Evaluation of potential mechanisms of atrazine-induced reproductive impairment in fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes).

Atrazine has been implicated in reproductive dysfunction of exposed organisms, and previous studies documented decreased egg production in Japanese medaka (Oryzias latipes) and fathead minnows (Pimephales promelas) during 30-d to 38-d exposures to 0.5 µg/L, 5 µg/L, and 50 µg/L atrazine. The authors evaluated possible mechanisms underlying the reduction in egg production. Gene expression in steroidogenesis pathways and the hypothalamus-pituitary-gonad axis of male and female fish was measured. Atrazine did not significantly induce gonad aromatase (cyp19a1a) expression. An atrazine-induced shift in the number of females in an active reproductive state was observed. Expression of the egg maturation genes vitellogenin 1 (vtg1) and zona pellucida glycoprotein 3.1 (zp3.1) in medaka females was correlated and had a bimodal distribution. In both species, females with low vtg1 or zp3.1 expression also had low expression of steroidogenesis genes in the gonad, estrogen receptor in the liver, and gonadotropins in the brain. In the medaka, the number of females per tank that had high expression of zp3.1 was significantly correlated with egg production per tank. The number of medaka females with low expression of zp3.1 increased significantly with atrazine exposure. Thus, the decline in egg production observed in response to atrazine exposure may be the result of a coordinated downregulation of genes required for reproduction in a subset of females. Environ Toxicol Chem 2016;35:2230-2238. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.

Atrazine reduces reproduction in Japanese medaka (Oryzias latipes).

Atrazine is an effective broadleaf herbicide and the second most heavily used herbicide in the United States. Effects along the hypothalamus-pituitary-gonad axis in a number of vertebrate taxa have been demonstrated. Seasonally elevated concentrations of atrazine in surface waters may adversely affect fishes, but only a few studies have examined reproductive effects of this chemical. The present study was designed to evaluate a population endpoint (egg production) in conjunction with histological (reproductive stage, gonad pathology) and biochemical (aromatase activity, sex hormone production) phenotypes associated with atrazine exposure in Japanese medaka. Adult virgin breeding groups of one male and four females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 µg/L (0, 2.3, 23.2, 231 nM) of atrazine in a flow-through diluter for 14 or 38 days. Total egg production was lower (36-42%) in all atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of atrazine-treated fish were significant by exposure day 24. Reductions in total egg production in atrazine treatment groups were most attributable to a reduced number of eggs ovulated by females in atrazine-treated tanks. Additionally, males exposed to atrazine had a greater number of abnormal germ cells. There was no effect of atrazine on gonadosomatic index, aromatase protein, or whole body 17 ß-estradiol or testosterone. Our results suggest that atrazine reduces egg production through alteration of final maturation of oocytes. The reduced egg production observed in this study was very similar to our previously reported results for fathead minnow. This study provides further information with which to evaluate atrazine's risk to fish populations.

Methylmercury-induced changes in gene transcription associated with neuroendocrine disruption in largemouth bass (Micropterus salmoides).

Methyl-mercury (MeHg) is a potent neuroendocrine disruptor that impairs reproductive processes in fish. The objectives of this study were to (1) characterize transcriptomic changes induced by MeHg exposure in the female largemouth bass (LMB) hypothalamus under controlled laboratory conditions, (2) investigate the health and reproductive impacts of MeHg exposure on male and female largemouth bass (LMB) in the natural environment, and (3) identify MeHg-associated gene expression patterns in whole brain of female LMB from MeHg-contaminated habitats. The laboratory experiment was a single injection of 2.5 µg MeHg/g body weight for 96 h exposure. The field survey compared river systems in Florida, USA with comparably lower concentrations of MeHg (Wekiva, Santa Fe, and St. Johns Rivers) in fish and one river system with LMB that contained elevated concentrations of MeHg (St. Marys River). Microarray analysis was used to quantify transcriptomic responses to MeHg exposure. Although fish at the high-MeHg site did not show overt health or reproductive impairment, there were MeHg-responsive genes and pathways identified in the laboratory study that were also altered in fish from the high-MeHg site relative to fish at the low-MeHg sites. Gene network analysis suggested that MeHg regulated the expression targets of neuropeptide receptor and steroid signaling, as well as structural components of the cell. Disease-associated gene networks related to MeHg exposure, based upon expression data, included cerebellum ataxia, movement disorders, and hypercalcemia. Gene responses in the CNS are consistent with the documented neurotoxicological and neuroendocrine disrupting effects of MeHg in vertebrates.

Dose-related estrogen effects on gene expression in fetal mouse prostate mesenchymal cells.

Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were enriched in the glycolytic pathway. At the highest dose (100 nM), E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- ß signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high.

Identification of the thiamin pyrophosphokinase gene in rainbow trout: characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development.

Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

Estrogenic environmental chemicals and drugs: mechanisms for effects on the developing male urogenital system.

Development and differentiation of the prostate from the fetal urogenital sinus (UGS) is dependent on androgen action via androgen receptors (AR) in the UGS mesenchyme. Estrogens are not required for prostate differentiation but do act to modulate androgen action. In mice exposure to exogenous estrogen during development results in permanent effects on adult prostate size and function, which is mediated through mesenchymal estrogen receptor (ER) alpha. For many years estrogens were thought to inhibit prostate growth because estrogenic drugs studied were administered at very high concentrations that interfered with normal prostate development. There is now extensive evidence that exposure to estrogen at very low concentrations during the early stages of prostate differentiation can stimulate fetal/neonatal prostate growth and lead to prostate disease in adulthood. Bisphenol A (BPA) is an environmental endocrine disrupting chemical that binds to both ER receptor subtypes as well as to AR. Interest in BPA has increased because of its prevalence in the environment and its detection in over 90% of people in the USA. In tissue culture of fetal mouse UGS mesenchymal cells, BPA and estradiol stimulated changes in the expression of several genes. We discuss here the potential involvement of estrogen in regulating signaling pathways affecting cellular functions relevant to steroid hormone signaling and metabolism and to inter- and intra-cellular communications that promote cell growth. The findings presented here provide additional evidence that BPA and the estrogenic drug ethinylestradiol disrupt prostate development in male mice at administered doses relevant to human exposures.

Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury.

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5 µg MeHg/g. Gene expression changes in the brain were examined using a 22,000-feature zebrafish microarray. At a significance level of p < 0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.

Atrazine reduces reproduction in fathead minnow (Pimephales promelas).

Atrazine, the widely used herbicide, has shown to affect the hypothalamus-pituitary-gonad axis in certain vertebrate species, but few studies have examined reproductive effects of this chemical on fish. Our study was designed to evaluate a population endpoint (egg production) in conjunction with histological (e.g., gonad development) and biochemical (e.g., hormone production) phenotypes associated with atrazine exposure in fathead minnows. Adult virgin breeding groups of 1 male and 2 females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 microg/L of atrazine in a flow-through diluter for 14 or 30 days. Total egg production was lower (19-39%) in all atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of atrazine treated fish were significant by 17-20 days of exposure. Reductions in egg production in atrazine treatment groups were most attributable to reduced numbers of spawning events with increased atrazine exposure concentrations. Gonad abnormalities were observed in both male and female fish of atrazine-exposed fish. Our results also indicate that atrazine reduces egg production through alteration of final maturation of oocytes. The reproductive effects observed in this study warrant further investigation and evaluation of the potential risks posed by atrazine, particularly feral populations of fish from streams in agricultural areas with high use of this herbicide.

The genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute exposure to the androgen, 17beta-trenbolone.

We investigated the genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute (4 days) exposure to 0.1 or 1.0microg/L of 17beta-trenbolone (TB), the active metabolite of an anabolic androgenic steroid used as a growth promoter in cattle and a contaminant of concern in aquatic systems. Our objectives were to investigate the gene expression profile induced by TB, define biomarkers of exposure to TB, and increase our understanding of the mechanisms of adverse effects of TB on fish reproduction. In female gonad tissue, microarray analysis using a 22K oligonucleotide microarray (EcoArray Inc., Gainesville, FL) showed 99 significantly upregulated genes and 741 significantly downregulated genes in response to 1microg TB/L. In particular, hydroxysteroid (17beta) dehydrogenase 12a (hsd17b12a), zona pellucida glycoprotein 2.2 (zp2.2), and protein inhibitor of activated STAT, 2 (pias2) were all downregulated in gonad. Q-PCR measurements in a larger sample set were consistent with the microarray results in the direction and magnitude of these changes in gene expression. However, several novel potential biomarkers were verified by Q-PCR in the same samples, but could not be validated in independent samples. In liver, Q-PCR measurements showed a significant decrease in vitellogenin 1 (vtg1) mRNA expression. In brain, cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b, previously known as aromatase B) transcript levels were significantly reduced following TB exposure. Our study provides a candidate gene involved in mediating the action of TB, hsd17b12a, and two potential biomarkers sensitive to acute TB exposure, hepatic vtg1 and brain cyp19a1b.

Quantitative polymerase chain reaction (PCR) assays for a bacterial thiaminase I gene and the thiaminase-producing bacterium Paenibacillus thiaminolyticus.

The thiaminase I enzyme produced by the gram-positive bacterium Paenibacillus thiaminolyticus isolated from the viscera of Lake Michigan alewives Alosa pseudoharengus is currently the only defined source of the thiaminase activity linked to thiamine (vitamin B1) deficiency in early mortality syndrome (EMS) in the larvae of Great Lakes salmonines. Diets of alewife or isolated strains of P. thiaminolyticus mixed in a semipurified diet and fed to lake trout Salvelinus namaycush have been shown to produce EMS in fry. We utilized quantitative polymerase chain reaction (Q-PCR) to aid in studies of the sources of P. thiaminolyticus and thiaminase I. Quantitative PCR assays were established to detect the thiaminase I gene of P. thiaminolyticus, the 16S rRNA gene from most species of bacteria, and the 16S rRNA gene specifically from P. thiaminolyticus and a few closely related taxa. The Q-PCR assays are linear over at least six orders of magnitude and can detect the thiaminase I gene of P. thiaminolyticus from as few as 1,000 P. thiaminolyticus cells/g of sample or the Paenibacillus 16S rRNA gene from as few as 100 P. thiaminolyticus cells/g of sample. The initial results from alewife viscera samples with high thiaminase activity yielded unexpectedly low densities of P. thiaminolyticus cells; Paenibacillus thiaminolyticus was detectable in 2 of 6 alewife viscera tested at densities on the order of 100 cells/g out of 100,000,000 total bacterial cells/g. The low numbers of P. thiaminolyticus detected suggest that alewives contain additional non-P. thiaminolyticus sources of thiaminase activity.

In vivo effects of bisphenol A in laboratory rodent studies.

Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/(kg day), in laboratory exposures with mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol and ethinylestradiol. For most effects, the potency of BPA is approximately 10-1000-fold less than that of diethylstilbestrol or ethinylestradiol. Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long lasting, organizational effects in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely, but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that developmental effects occur in the female reproductive system.