Co-Exposure of Phenanthrene and the cyp-Inducer 3-Methylchrysene Leads to Altered Biotransformation and Increased Toxicity in Fish Egg and Larvae.

Polycyclic aromatic hydrocarbons (PAHs) have frequently been suspected of governing crude oil toxicity because of similar morphological defects in fish. However, PAH concentrations are often not high enough to explain the observed crude oil toxicity. We hypothesize that one PAH can enhance the metabolism and toxicity of another PAH when administered as a mixture. Early life stage Atlantic haddock (Melanogrammus aeglefinus) were in this study exposed to phenanthrene in the presence and absence of 3-methylchrysene that is known to induce the metabolic enzyme cytochrome P450 1A via cyp1a gene expression. Uptake, metabolism, and multiple toxicity endpoints were then measured in a time-course study up to 3 days post-hatching. Passive dosing provided aqueous concentrations ≈180 µg/L for phenanthrene and ≈0.6 µg/L for 3-methylchrysene, which resulted in tissue concentrations ≈60 µg/g ww for phenanthrene and ≈0.15 µg/g ww for 3-methylchrysene. The low concentration of 3-methylchrysene led to the elevated expression of cyp1a but no toxicity. Levels of phenanthrene metabolites were 5-fold higher, and morphological defects and cardiotoxicity were consistently greater when co-exposed to both compounds relative to phenanthrene alone. This work highlights the metabolic activation of PAH toxicity by a co-occurring PAH, which can lead to excess toxicity, synergistic effects, and the overproportional contribution of PAHs to crude oil toxicity.

Assessing hydroxylated polycyclic aromatic hydrocarbon (OHPAH) metabolites in bile of English sole (Parophrys vetulus) from Puget Sound, WA, USA by liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are widely monitored in marine biota from urbanized areas, due to their toxicity to aquatic organisms. Teleost fish can quickly metabolize PAHs into hydroxylated forms (OHPAHs) that, in some cases, are more toxic than the parent (unmetabolized) PAHs. But due to this fast metabolism, monitoring traditional parent PAHs in fish can cause underestimation on assessing PAH exposure. In addition, environmental levels of individual OHPAH metabolites are lacking in the literature worldwide. Therefore, we developed a rapid and accurate analytical method in which a number of individual OHPAHs metabolites are measured simultaneously in fish bile, via liquid chromatography coupled with tandem mass spectrometry, including low and high molecular weight mono- and diol-OHPAHs. We analyzed bile samples of 119 English sole (Parophrys vetulus) collected from 14 Puget Sound, WA, USA, sites, which has multiple sources of PAHs, including urban stormwater runoff, wastewater effluents, as well as an inactive creosote facility. The mean (± SD) biliary summed OHPAH (∑OHPAH) concentrations determined in English sole from urban, near-urban, and non-urban sites were 790 ± 1400 (n = 46), 310 ± 330 (n = 44) and 130 ± 200 (n = 29) ng/mL, respectively, with a maximum reaching 9400 ng/mL in a sample from an urban site. We compared these novel biliary OHPAH metabolite data with parent PAHs measured in stomach content of the same individual sole. Biliary ∑OHPAH concentrations were significantly correlated with the levels of ∑PAH in stomach content, however, with major differences in their distribution. We also demonstrated that biliary OHPAH metabolite data in English sole can potentially be used to distinguish different sampling sites due to a specific variety and intensity of PAH sources in the aquatic environment, which makes this a very important analytical approach for assessing PAH exposure in the environment.

Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart.

Resistance or susceptibility to T. cruzi infection is dependent on the host immunological profile. Innate immune receptors, such as Toll-like receptors (TLRs/TLR2, TLR4, TLR7, and TLR9) and Nod-like receptors (NLRs/NOD1 and NLRP3 inflammasome) are involved with the resistance against acute experimental T. cruzi infection. Here, we evaluated the impact of T. cruzi virulence on the expression of innate immune receptors and its products in mice. For that, we used six T. cruzi strains/isolates that showed low (AM64/TcIV and 3253/Tc-V), medium (PL1.10.14/TcIII and CL/TcVI), or high (Colombian/Tc-I and Y/TcII) virulence and pathogenicity to the vertebrate host and belonging to the six discrete typing units (DTUs)-TcI to TcVI. Parasitemia, mortality, and myocarditis were evaluated and correlated to the expression of TLRs, NLRs, adapter molecules, cytokines, and iNOS in myocardium by real time PCR. Cytokines (IL-1ß, IL-12, TNF-α, and IFN-γ) were quantified in sera 15 days after infection. Our data indicate that high virulent strains of T. cruzi, which generate high parasitemia, severe myocarditis, and 100% mortality in infected mice, inhibit the expression of TLR2, TLR4, TLR9, TRIF, and Myd88 transcripts, leading to a low IL-12 production, when compared to medium and low virulent T. cruzi strains. On the other hand, the high virulent T. cruzi strains induce the upregulation of NLRP3, caspase-1, IL-1ß, TNF-α, and iNOS mRNA in heart muscle, compared to low and medium virulent strains, which may contribute to myocarditis and death. Moreover, high virulent strains induce higher levels of IL-1ß and TNF-α in sera compared to less virulent parasites. Altogether the data indicate that differential TLR and NLR expression in heart muscle is correlated with virulence and pathogenicity of T cruzi strains. A better knowledge of the immunological mechanisms involved in resistance to T. cruzi infection is important to understand the natural history of Chagas disease, can lead to identification of immunological markers and/or to serve as a basis for alternative therapies.

Untangling mechanisms of crude oil toxicity: Linking gene expression, morphology and PAHs at two developmental stages in a cold-water fish.

Early life stages of fish are highly sensitive to crude oil exposure and thus, short term exposures during critical developmental periods could have detrimental consequences for juvenile survival. Here we administered crude oil to Atlantic haddock (Melanogrammus aeglefinus) in short term (3-day) exposures at two developmental time periods: before first heartbeat, from gastrulation to cardiac cone stage (early), and from first heartbeat to one day before hatching (late). A frequent sampling regime enabled us to determine immediate PAH uptake, metabolite formation and gene expression changes. In general, the embryotoxic consequences of an oil exposure were more severe in the early exposure animals. Oil droplets on the eggshell resulted in severe cardiac and craniofacial abnormalities in the highest treatments. Gene expression changes of Cytochrome 1 a, b, c and d (cyp1a, b, c, d), Bone morphogenetic protein 10 (bmp10), ABC transporter b1 (abcb1) and Rh-associated G-protein (rhag) were linked to PAH uptake, occurrence of metabolites of phenanthrene and developmental and functional abnormalities. We detected circulation-independent, oil-induced gene expression changes and separated phenotypes linked to proliferation, growth and disruption of formation events at early and late developmental stages. Changes in bmp10 expression suggest a direct oil-induced effect on calcium homeostasis. Localized expression of rhag propose an impact on osmoregulation. Severe eye abnormalities were linked to possible inappropriate overexpression of cyp1b in the eyes. This study gives an increased knowledge about developmentally dependent effects of crude oil toxicity. Thus, our findings provide more knowledge and detail to new and several existing adverse outcome pathways of crude oil toxicity.

Plant DNA Barcoding Principles and Limits: A Case Study in the Genus Vanilla.

Powerful DNA barcodes have been much more difficult to define in plants than in animals. In 2009, the international Consortium for the Barcoding Of Life (CBOL) chose the combination of the chloroplast genes (rbcL + matK) as the proposed official barcode for plants. However, this system has got important limits. First, any barcode system will only be useful if there is a clear barcode gap and if species are monophyletic. Second, chloroplast and mitochondrial (COI gene used for animals) barcodes will not be usable for discriminating hybrid species. Moreover, it was also shown that, using chloroplast regions, maximum species discrimination would be around 70% and very variable among plant groups. This is why many authors have more recently advocated for the addition of the nuclear ITS region to this barcode because it reveals more variations and allows the resolution of hybrid or closely related species. We tested different chloroplast genes (rbcL, matK, psaB, psbC) and the nuclear ITS region in the genus Vanilla, a taxonomically complex group and therefore a good model to test for the efficiency of different barcode systems. We found that the CBOL official barcode system performed relatively poorly in Vanilla (76% species discrimination), and we demonstrate that adding ITS to this barcode system allows to increase resolution (for closely related species and to the subspecies level) and to identify hybrid species. The best species discrimination attained was 96.2% because of one paraphyletic species that could not be resolved.

Genetic structuring among colonies of a pantropical seabird: Implication for subspecies validation and conservation.

Investigations of the genetic structure of populations over the entire range of a species yield valuable information about connectivity among populations. Seabirds are an intriguing taxon in this regard because they move extensively when not breeding, facilitating intermixing of populations, but breed consistently on the same isolated islands, restricting gene flow among populations. The degree of genetic structuring of populations varies extensively among seabird species but they have been understudied in their tropical ranges. Here, we address this across a broad spatial scale by using microsatellite and mitochondrial data to explore the population connectivity of 13 breeding populations representing the six subspecies of the white-tailed tropicbird (Phaethon lepturus) in the Atlantic, Indian, and Pacific Oceans. Our primary aim was to identify appropriate conservation units for this little known species. Three morphometric characters were also examined in the subspecies. We found a clear pattern of population structuring with four genetic groups. The most ancient and the most isolated group was in the northwestern Atlantic Ocean. The South Atlantic populations and South Mozambique Channel population on Europa were genetically isolated and may have had a common ancestor. Birds from the Indo-Pacific region showed unclear and weak genetic differentiation. This structuring was most well defined from nuclear and mtDNA markers but was less well resolved by morphological data. The validity of classifying white-tailed tropicbirds into six distinct subspecies is discussed in light of our new findings. From a conservation standpoint our results highlight that the three most threatened conservation units for this species are the two subspecies of the tropical North and South Atlantic Oceans and that of Europa Island in the Indian Ocean.

NOD2 receptor is crucial for protecting against the digestive form of Chagas disease.

Digestive and cardiodigestive forms of Chagas' disease are observed in 2% to 27% of the patients, depending on their geographic location, Trypanosoma cruzi strain and immunopathological responses. The aim of this work was to evaluate the role of NOD2 innate immune receptor in the pathogenesis of the digestive system in Chagas' disease. Patients with digestive form of the disease showed lower mRNA expression of NOD2, higher expression of RIP2 and α-defensin 6, compared to indeterminate form, detected by Real-time PCR in peripheral blood mononuclear cells. In addition, there was a negative correlation between the expression of NOD2 and the degree of dilation of the esophagus, sigmoid and rectum in those patients. The infection of NOD2-/- mice with T. cruzi strain isolated from the digestive patient induced a decrease in intestinal motility. Histopathological analysis of the colon and jejunum of NOD2-/- and wild type C57BL/6 animals revealed discrete inflammatory foci during the acute phase of infection. Interestingly, during the chronic phase of the infection there was inflammation and hypertrophy of the longitudinal and circular muscular layer more pronounced in the colon and jejunum from NOD2-/- animals, when compared to wild type C57BL/6 mice. Together, our results suggest that NOD2 plays a protective role against the development of digestive form of Chagas' disease.

Temporal trends in aluminum smelter-derived polycyclic aromatic hydrocarbons in outmigrant juvenile Chinook salmon from Kitimat, British Columbia, Canada.

Aluminum smelter-derived polycyclic aromatic hydrocarbons (PAHs) in outmigrant juvenile Chinook salmon were evaluated in Kitimat Arm, British Columbia, Canada from 2000 to 2004, and in 2015. Decades of continual smelter operations by Rio Tinto resulted in PAH contamination of marine sediments at levels associated with adverse effects in juvenile salmon. Recently, smelter operations have undergone process changes to reduce PAH input to the environment. The PAH concentrations in juvenile Chinook salmon observed in 2000 to 2004, at sites nearest the smelter were comparable to salmon in other urban areas where reduced disease resistance was observed; the levels were lower in 2015 than 2000-2004 suggesting that the recent process changes were effective. Further, these data establish a benchmark for assessing any future changes affecting PAH input and the potential risks to the receiving environment.

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish.

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17ß-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11ß-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20ß,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.

Evaluation of polycyclic aromatic hydrocarbons bioavailability on Santos Bay (Brazil) through levels of biliary metabolites.

This study evaluated the PAH bioavailability from Santos Bay (Brazil) in 4 species of fish, using PAH biliary metabolites. The collection was done monthly, between July and December, in three different regions of Santos Bay. The metabolites were analyzed through a high performance liquid chromatograph with fluorescence detectors. Total metabolites concentrations ranged from 65.5 to 589µgg-1 of bile, evidencing PAH bioavailability on Santos Bay. Levels of phenanthrene and benzo[a]pyrene metabolites were in the classification range of areas moderate contaminated. Those concentrations were lower in Nebris microps and higher in Sphoeroides testudineus (p<0.05). Naphthalene metabolites concentrations did not differ significantly among fish species and were in the classification range of low contaminated areas. There were no significant spatial and temporal differences in levels among sampled areas. These results are environmentally important given the high levels of urbanization and the absence of biomonitoring data in this area.

Raman spectral characteristics of neck and head of femur in low-density lipoprotein receptor gene knockout mice submitted to treadmill aerobic training.

Hypercholesterolemia is associated with deterioration of bone tissue. Trough Raman spectroscopy, the present study aimed to assess the molecular changes in terms of mineral and organic bone-tissue remodeling in low-density lipoprotein receptor gene knockout mice (LDLr-/-) submitted to treadmill aerobic training. In order to evaluate alterations in trabecular bone and cartilage surface, neck and head femoral were assessed into four groups: (CON, n=7), the 1) animals did not perform any physical activity and had hypercholesterolemia (CON, n=7); The trained groups where animals underwent a protocols of low, moderate and high training for 8weeks and 4 times per week on a treadmill with progressive intensities: running speed (8-13m/min) and time of exercise (20-60min). These progressive intensities were needed to adjust the working load for the physical training to 2) 30-49% (LOW, n=7), 3) 50-79% (MOD, n=7), and 4) 80-100% (HIGH, n=7) of the maximum running speed. Each group was examined for molecular changes in both bone phases (mineral and organic) using principal components analysis (PCA). All exercised groups showed a significant increase (p<0.05) of bone remodeling in trabecular area and a higher content of proteins (organic content) in cartilage independently of physical activity intensity. Thus, this work demonstrated that physical training could act as a bone-protector factor in hyperlipidemic animal model as well as Raman technique associated with PCA analysis may be feasible methodology for assessment or monitoring of bone at the molecular level.

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17ß-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish.

Treatment of poultry litter does not improve performance or carcass lesions in broilers / El tratamiento de la cama no afecta el desempeño ni las lesiones en la canal del pollo de engorde / Desempenho produtivo e lesões na carcaça de frangos de corte não são afetados pelo tratamento da cama de frango

Background: broilers spend most of their lives in contact with litter; litter quality can affect their health and performance. Objective: the effects of litter treatment on performance and carcass lesions were evaluated in five consecutive flocks with 640 male broilers each. Methods: a completely randomized model was used comprising eight treatments and four replicates. The treatments included (1) untreated litter, (2) litter subjected to in-house composting, (3) litter treated (LT) with aluminum sulfate, (4) LT with gypsum, (5) LT with quicklime, (6) LT with dolomitic limestone, (7) LT with zeolite, and (8) LT with charcoal. Chopped elephant-grass hay was used as poultry litter in all flocks. Results: none of the litter treatments were found to influence the performance and carcass lesions of the male broilers in all five flocks. Furthermore, poultry litter treatments were not economically viable. Conclusion: poultry litter treatments did not affect the performance and scores of carcass lesions of male broilers, but increased the cost of poultry production.

Antecedentes: los pollos de engorde pasan la mayoría de su vida en contacto con la cama; la calidad de la cama puede afectar la salud y desempeño del ave. Objetivo: fueron evaluados los efectos de diferentes tratamientos de la cama sobre el desempeño y lesiones en la canal de pollos de engorde durante cinco lotes consecutivos con 640 aves cada uno. Métodos: se empleó un modelo completamente aleatorizado con ocho tratamientos y cuatro repeticiones. Los tratamientos incluyeron (1) cama no tratada, (2) cama sometida a compostaje en el galpón, (3) cama tratada (CT) con sulfato de aluminio, (4) CT con yeso agrícola, (5) CT con cal, (6) CT con calcáreo dolomítico, (7) CT con zeolita y (8) CT con carbón vegetal. Heno de pasto elefante picado fue usado como cama en todos los lotes. Resultado: los tratamientos no influenciaron el desempeño ni las lesiones en la canal de los pollos en ningún lote. Además, ninguno de los tratamientos de las camas fue económicamente viables. Conclusión: el tratamiento de la cama de pollo no solo no afecta el desempeño ni las lesiones en la canal de los pollos sino que eleva los costos de producción de las aves.

Antecedentes: os frangos de corte passam a maioria de suas vidas em contato com a cama e a qualidade desta pode afetar a saúde e o desempenho produtivo da ave. Objetivo: Avaliou-se os efeitos dos tratamentos da cama de frango sobre o desempenho no crescimento e lesões na carcaça de frangos de corte. Métodos: avaliaram-se 640 aves por lote, durante cinco lotes consecutivos, analisaram-se empregando um modelo completamente casualizado com oito tratamentos e quatro repetições. Os tratamentos consistiram de (1) cama não tratada, (2) cama submetida a compostagem no galpão, (3) cama tratada (CT) com sulfato de alumínio, (4) CT com gesso agrícola, (5) CT com cal virgem, (6) CT com calcário dolomítico, (7) CT com zeolita e (8) CT com carvão vegetal. Feno de capim elefante picado foi usado como cama em todos os lotes. Resultado: os diferentes tratamentos não influenciaram o desempenho na produção e as lesões na carcaça dos frangos em nenhum lote, entretanto, os tratamentos da cama foram economicamente inviáveis. Conclusão: o tratamento da cama de frango não afeta o desempenho produtivo e os escores de lesões na carcaça dos frangos de corte, além, eleva os custos da produção avícola.

Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria).

Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17α-hydroxyprogesterone, androstenedione (A4), T and 17ß-estradiol (E2) production. Treatment with T increased production of A4, 11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, E2, 11-ketotestosterone, and 5α-dihydrotestosterone (DHT). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and DHT over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and DHT, a steroid hormone whose role in reproduction of fishes remains unknown.

Diarrhetic shellfish toxins and other lipophilic toxins of human health concern in Washington State.

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 µg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

Endocrine disrupting chemicals in fish bile: a rapid method of analysis using English sole (Parophrys vetulus) from Puget Sound, WA, USA.

This study describes a recently developed and rapid method to measure bisphenol A (BPA), 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2) in bile of fish using enzymatic hydrolysis of samples followed by solid-phase extraction and ultra-performance liquid chromatography with tandem mass spectrometry. The limits of quantitation (LOQ) for BPA, EE2 and E2 were 6.3ngmL(-1), 12.5ngmL(-1) and 6.3ngmL(-1), respectively. These compounds were analyzed in bile of male English sole (Parophrys vetulus) collected from urban and non-urban sites in Puget Sound, WA, USA. The BPA and E2 concentrations (and occurrence) ranged from

Molecular biology, phytochemistry and bioactivity of three endemic Aloe species from Mauritius and Réunion Islands.

INTRODUCTION: Aloe tormentorii, A. purpurea and A. macra are used as multipurpose folk medicines in Réunion and Mauritius Islands and are mistaken for the introduced Aloe vera. OBJECTIVE: To compare the phytochemical, antimicrobial and DNA profiles of Aloe endemic to Mauritius and Réunion with the profiles of A. vera. Methodology - Leaf extracts of these Aloe species were analysed using standard phytochemical screening techniques, TLC and by HPLC. These extracts were also assayed for antimicrobial activity using microdilution techniques. Genetic diversity was studied using RAPD markers. RESULTS: Phytochemical and antimicrobial assays and RAPD analysis showed that Mascarene Aloe species were very different from A. vera. CONCLUSION: This study is the first report highlighting the differences between Aloe sp.p from Mascarene and Aloe vera at the metabolic and genomic level.

Distribution of sewage input in marine sediments around a maritime Antarctic research station indicated by molecular geochemical indicators.

Sediments from Admiralty Bay, Antarctica were collected during the austral summers of 2002/2003 and 2003/2004 in order to assess the distribution and concentration of sewage indicators originating from Comandante Ferraz Brazilian Antarctic Station. Fecal sterols (coprostanol+epicoprostanol) and linear alkylbenzenes (LABs) ranged from <0.01 to 0.95 microg g(-1) and <1.0 to 23 ng g(-1) dry weight, respectively. In general, the higher concentrations were found only locally in the vicinity of Ferraz station at Martel Inlet. Baseline values for fecal sterols and coprostanone were calculated as 0.19 and 0.40 microg g(-1), respectively. According to fecal sterols concentrations, sewage contribution to Martel Inlet has increased more than twice since 1997, as result of the increase in the number of researchers at the station especially during the last decade. A low correlation was found between total LABs and fecal steroids, which could be attributed to the contribution of the natural sources of steroids.

Polycyclic aromatic hydrocarbons and petroleum biomarkers in São Sebastião Channel, Brazil: assessment of petroleum contamination.

Polycyclic aromatic hydrocarbons (PAHs) and non-aromatic hydrocarbons (NAHs), including n-alkanes, isoprenoids and petroleum biomarkers (terpanes, hopanes, steranes and diasteranes), were quantified by gas chromatography with flame ionization and mass spectrometer detectors in sediment samples collected from the São Sebastião Channel (SSC), Brazil, where the largest Brazilian maritime petroleum terminal is located. The concentrations of total PAHs, total n-alkanes and petroleum biomarkers ranged from below the detection limits to 370ngg(-1), 28microgg(-1), 2200ngg(-1) (dry weight), respectively. The analysis of PAH distribution suggested combustion sources of PAHs as the main input for these compounds with smaller amount from petroleum contamination. The distribution of petroleum biomarkers undoubtedly demonstrated petroleum as a source of anthropogenic contamination throughout the region. The assessment of petrogenic sources of contamination in marine sediment is more challenging if only PAH analysis were carried out, which demonstrates that more stable hydrocarbons such as petroleum biomarkers are useful for investigating potential presence of petroleum.

Variation in intron length in caffeic acid O-methyltransferase (COMT) in Vanilla species (Orchidaceae).

Variation in intron length in caffeic acid O-methyltransferase (COMT) in Vanilla was studied and demonstrated that COMT genes in Vanilla are organized with four exons and three introns. At least two to four different versions (either allelic or paralogous) of the COMT multigenic family in the genus Vanilla (in terms of intron sizes) were detected. The three introns were differentially variable, with intron-1 being the most length-polymorphic. Patterns of variations were in accordance with known phylogenetic relationships in the genus obtained with neutral markers. In particular, the genus displayed a strong Old World versus New World differentiation with American fragrant species being characterized by a specific 99bp intron-1 size-variant and a unique 226bp intron-3 variant. Conversely, leafless species of the genus displayed unexpected variations in intron lengths. Due to their role in primary (lignin) and secondary (phenolics, e.g., vanillin, alkaloids) metabolisms, COMT genes might not be neutral markers, and represent candidate functional markers for resistance, aromatic or medicinal properties of Vanilla species. Investigating the orthologous/paralogous status of the different genes revealed (in terms of intron size) will allow the evolution of the COMT genes to be studied.