Assessing the effects of exposure timing on biomarker expression using β-estradiol

Temporal and spatial variability in estrogenicity has been documented formanytreated wastewater effluents with the consequences of this variability on the expression of biomarkers of endocrine disruption being largely unknown. Laboratory exposure studies usually utilize constant exposure concentrations which may produce biological effects that differ from those observed in organisms exposed in natural environments. In this study, we investigated the effects of differential timing of exposures with 17β - estradiol (E2) on a range of fathead minnow biomarkers to simulate diverse environmentally relevant exposure profiles. Two 21-day, replicate experiments were performed exposing mature male fathead minnows to E2 at time-weighted mean concentrations (similar average exposure to the contaminant during the 21-day exposure period; 17 ng E2/L experiment 1; 12 ng E2/L experiment 2) comparable to E2 equivalency values (EEQ) reported for several anthropogenically altered environments. A comparable time-weighted mean concentration of E2 was applied to five treatments which varied in the daily application schema: E2 was either applied at a steady rate (ST), in a gradual decreasing concentration (HI), a gradual increasing concentration (LO), applied intermittently (IN), or at a randomly varying concentration (VA). We assessed a range of widely used physiological (vitellogenin mRNA induction and plasma concentrations), anatomical (body and organ indices, secondary sex characteristics, and histopathology), and behavioral (nest holding) biomarkers reported to change following exposure to endocrine active compounds (EACs). All treatments responded with a rise in plasma vitellogenin concentration when compared with the ethanol carrier control. Predicatively, vitellogenin mRNA induction, which tracked closely with plasma vitellogenin concentrations in most treatments was not elevated in the HI treatment, presumably due to the lack of E2 exposure immediately prior to analysis. The ability of treatment male fish to hold nest sites in direct competition with control males was sensitive to E2 exposure and did yield statistically significant differences between treatments and carrier control. Other biological endpoints assessed in this study (organosomatic indices, secondary sex characteristics) varied little between treatments and controls. This study indicates that a broad suite of endpoints is necessary to fully assess the biological consequences of fish exposure to estrogens and that for at least field studies, a combination of vitellogenin mRNA and plasma vitellogenin analysis are most promising in deciphering exposure histories of wild-caught and caged fishes

Effects of Triclosan and Triclocarban, Two Ubiquitous Environmental Contaminants, on Anatomy, Physiology, and Behavior of The Fathead Minnow (Pimephales Promelas)

Triclosan (TCS) and triclocarban (TCC) are two common antimicrobial agents found in many personal care products and subsequently are detected ubiquitously in wastewater effluent and receiving waters. Both compounds are of recent regulatory interest due to their omnipresence in the environment, including in humans. Although TCS and TCC have been suggested to be endocrine active, little information exists about their effects on organismal end points in development (growth, escape performance), anatomy (morphological indices, histology), physiology (vitellogenin), and behavior of exposed aquatic organisms. In this study, newly hatched fathead minnows were exposed for 12 days, and mature male and female fathead minnows (Pimephales promelas) were exposed for 21 days to environmentally realistic concentrations (nanograms per liter) of these two compounds singularly and in mixtures. At the end of the exposure, larvae were assessed for growth and predator-avoidance performance, and a subset of mature fish was assessed for plasma vitellogenin induction, expression of secondary sexual characteristics, relative size of liver and gonads, and histopathological changes to both organs. The remaining exposed mature fish were placed in breeding pairs of one male and one female minnow from the same treatment to assess their ability to defend a nest site and reproduce. Exposure to either antimicrobial compound, alone or as a mixture, caused no changes to larval fish, gonad size, or vitellogenin concentrations in mature fathead minnows. In contrast, decreased aggression was seen in adult male fathead minnows exposed to TCC (1.6 μg/l) or a mixture (560 ng/l TCS + 179 ng/l TCC and 1.6 μg/l TCS + 450 ng/l TCC). Decreased aggression would likely decrease their ability to defend and hold a nest site needed for spawning and reproduction. Substantial variability was found in the severity of observed effects within treatments, suggesting that environmentally realistic concentrations of these compounds may only affect particularly sensitive individuals. © Springer Science+Business Media, LLC 2012

Intra- and inter-specific morphological diversity of amphidromous gobies influences waterfall-climbing performance

The morphology of an individual can affect functional performance and, ultimately, survival and fitness. To study these links, a first step is to evaluate the relationship between morphology and performance. Sicydiine fishes are an interesting model for such studies, because successful completion of their life cycle depends on a functionally demanding task: climbing waterfalls when they return to freshwater from the ocean. Previous studies identified two different climbing modes among sicydiines: 'inching' and 'powerburst'. Relationships between morphology and climbing performance have been found for inching and powerburst species present on different islands from distant oceans (Pacific and Caribbean), but species from the same island have not been compared. In this study, we challenged two different sicydiine species from Reunion Island to an artificial climbing ramp: the inching climber Sicyopterus lagocephalus and the powerburst climber Cotylopus acutipinnis. For each species, we evaluated differences in morphology between successful and unsuccessful climbers. We predicted that species with similar climbing styles (S. lagocephalus and S. stimpsoni) would show differences between successful and unsuccessful climbers that more closely resembled each other than differences between successful and unsuccessful climbers across species from the same island (S. lagocephalus and C. acutipinnis). This prediction was only partially met. S. lagocephalus individuals with a streamlined body showed better climbing performance, as observed for S. stimpsoni. However, the size of the pelvic sucker, which fish use to attach to the substrate while resting, appeared less important for S. lagocephalus than for S. stimpsoni and C. acutipinnis. Instead, the size of the mouth, which inching species use to attach to the substrate while moving up, seemed important for S. lagocephalus but not for other species. Thus, differences in the morphology-performance relationship among sicydiines may relate not only to differences in climbing mode, but also to species-specific specializations within each climbing mode

The anatomy of the larynx of the bowhead whale, Balaena mysticetus, and its sound-producing functions

This study describes the morphology of the laryngeal apparatus in bowhead whales (Balaena mysticetus) with respect to respiration, deglutition, and vocalization. We also examined the intrinsic cricoarytenothyroid muscle (Musculus (M.) diverticuli laryngei) which forms the laryngeal diverticulum, to ascertain its interactions with the laryngeal cartilages during respiration and sound production. Five fetal larynges and four from adult whales were studied using noninvasive imaging, as well as macroscopic and microscopic techniques. The larynx extends from the skull base into the thoracic inlet. The dorsally curved laryngeal stalk, supported by epiglottis and the corniculate processes of arytenoid cartilages, is situated within the nasopharynx. The epiglottic cartilage exhibits a prominent medial ridge. The arytenoid cartilages are rod-shaped, and extend through the laryngeal cavity. The thyroid cartilage possesses a prominent caudal horn with a fibrous articulation to the ventrally incomplete cricoid cartilage. The M. thyroepiglotticus forms the connection between epiglottic and thyroid cartilages. The M. cricothyroideus lateralis connects the caudal horn of the thyroid cartilage with the cricoid cartilage and the M. cricothyroideus medialis connects the cricoid and thyroid cartilage. An extensive laryngeal diverticulum (Diverticulum laryngis), formed by the laryngeal mucosa and M. diverticuli laryngei, is positioned caudo-ventral to the laryngeal vestibule. The mucosa thickens into a fold medial to the vocal processes of the arytenoid cartilages. Experiments with airflow combined with histological and anatomical evidence strongly suggest a sound producing function for these (vocal) folds. This analysis provides the first account of sound producing structures and function in bowhead whales

Ontogenetic change in novel functions: waterfall climbing in adult Hawaiian gobiid fishes

Juveniles from three species of Hawaiian gobiid fishes climb waterfalls as part of an amphidromous life cycle, allowing them to re-penetrate adult upstream habitats after being swept out to the ocean upon hatching. The importance of climbing for juvenile stream gobies is well established, but adult fish in upstream island habitats also face potential downstream displacement by periodic disturbances. Thus, retention of climbing ability could be advantageous for adult stream gobies. Climbing performance might be expected to decline among adults, however, due to the tendency for mass-specific muscular power production to decrease with body size, and a lack of positively allometric growth among structures like the pelvic sucker that support body weight against gravity. To evaluate changes in waterfall-climbing ability with body size in Hawaiian stream gobies, we compared climbing performance and kinematics between adults and juveniles from three species: Awaous guamensis, Sicyopterus stimpsoni and Lentipes concolor. For species in which juveniles climbed using ‘powerbursts’ of axial undulation, adult performance and kinematics showed marked changes: adult A. guamensis failed to climb, and adult L. concolor used multiple pectoral fin adductions to crutch up surfaces at slow speeds, rather than rapid powerbursts. Adult S. stimpsoni, like juveniles, still used oral and pelvic suckers to ‘inch’ up surfaces and climbed at speeds comparable to those of juveniles. However, unlike juveniles, adult S. stimpsoni also add pectoral fin crutching to every climbing cycle. Thus, although powerburst species appear to be particularly susceptible to size-related declines in waterfall-climbing performance, the addition of compensatory mechanisms pre- vents the loss of this novel function in some species

Selective Uptake and Biological Consequences of Environmentally Relevant Antidepressant Pharmaceutical Exposures on Male Fathead Minnows

Antidepressant pharmaceuticals have been reported in wastewater effluent at the nanogram to low microgram-per-liter range, and include bupropion (BUP), fluoxetine (FLX), sertraline (SER), and venlafaxine (VEN). To assess the effects of antidepressants on reproductive anatomy, physiology, and behavior, adult male fathead minnows (Pimephales promelas) were exposed for 21 days either to a single concentration of the antidepressants FLX, SER, VEN, or BUP, or to an antidepressant mixture. The data demonstrated that exposure to VEN (305. ng/L and 1104. ng/L) and SER (5.2. ng/L) resulted in mortality. Anatomical alterations were noted within the testes of fish exposed to SER and FLX, both modulators of the neurotransmitter serotonin. Additionally, FLX at 28. ng/L induced vitellogenin in male fish-a common endpoint for estrogenic endocrine disruption. Significant alterations in male secondary sex characteristics were noted with single exposures. Effects of single compound exposures neither carried over, nor became additive in the antidepressant mixtures, and reproductive behavior was not affected. Analysis of brain tissues from the exposed fish suggested increased uptake of FLX, SER and BUP and minimal uptake of VEN when compared to exposure water concentrations. Furthermore, the only metabolite detected consistently in the brain tissues was norfluoxetine. Similar trends of uptake by brain tissue were observed when fish were exposed to antidepressant mixtures. The present study demonstrates that anatomy and physiology, but not reproductive behavior, can be disrupted by exposure to environmental concentrations of some antidepressants. The observation that antidepressant uptake into fish tissues is selective may have consequences on assessing the mode-of-action and effects of these compounds in future studies. © 2011 Elsevier B.V

Environmental Estrogens in an Urban Aquatic Ecosystem: Ii. Biological Effects

Urban aquatic ecosystems are often overlooked in toxicological studies even though they serve many ecosystem functions and sustain fish populations despite large-scale habitat alterations. However, urban fish populations are likely exposed to a broad range of stressors, including environmental estrogens (EEs) that may affect anatomy, physiology and reproduction of exposed fish. Although significant progress has been made in establishing ecological consequences of EE exposure, these studies have focused largely on hydrologically simple systems that lack the complexity of urban aquatic environments. Therefore, the objective of this study was to assess the occurrence and biological effects of EEs across a large urbanized aquatic ecosystem. A multi-pronged study design was employed relying on quantitative determination of select EEs by liquid chromatography tandem mass spectrometry and repeated biological monitoring of wild-caught and caged fish for indications of endocrine disruption. Over three years, EEs were measured in aqueous samples (n = 42 samples) and biological effects assessed in \u3e 1200 male fish across the 2000 km2 aquatic ecosystems of the Greater Metropolitan Area of Chicago, IL. Our study demonstrated that in addition to water reclamation plant (WRP) effluents, non-WRP sources contribute significant EE loads to the aquatic ecosystem. While resident and caged male fish responded with the induction of the egg-yolk protein vitellogenin, an indicator of EE exposure, neither resident nor caged sunfish exhibited prevalent histopathological changes to their reproductive organs (i.e., intersex) that have been reported in other studies. Vitellogenin induction was greater in spring than the fall and was not correlated with body condition factor, gonadosomatic index or hepatosomatic index. Exposure effects were not correlated with sites downstream of treated effluent discharge further affirming the complexity of sources and effects of EEs in urban aquatic ecosystems. © 2013 Elsevier Ltd. All rights reserved

Antidepressant Pharmaceuticals in Two U.S. Effluent-Impacted Streams: Occurrence and Fate in Water and Sediment and Selective Uptake in Fish Neural Tissue

Antidepressant pharmaceuticals are widely prescribed in the United States; release of municipal wastewater effluent is a primary route introducing them to aquatic environments, where little is known about their distribution and fate. Water, bed sediment, and brain tissue from native white suckers (Catostomus commersoni)were collected upstream and atpoints progressively downstream from outfalls discharging to two effluentimpacted streams, Boulder Creek (Colorado) and Fourmile Creek (Iowa). A liquid chromatography/tandem mass spectrometry method was used to quantify antidepressants, including fluoxetine, norfluoxetine (degradate), sertraline, norsertraline (degradate), paroxetine, Citalopram, fluvoxamine, duloxetine, venlafaxine, and bupropion in all three sample matrices. Antidepressants were not present above the limit of quantitation in water samples upstream from the effluent outfalls but were present at points downstream at ng/L concentrations, even at the farthest downstream sampling site 8.4 km downstream from the outfall. The antidepressants with the highest measured concentrations in both streams were venlafaxine, bupropion, and Citalopram and typically were observed at concentrations of at least an order of magnitude greater than the more commonly investigated antidepressants fluoxetine and sertraline. Concentrations of antidepressants in bed sediment were measured at ng/g levels; venlafaxine and fluoxetine were the predominant chemicals observed. Fluoxetine, sertraline, and their degradates were the principal antidepressants observed in fish brain tissue, typically at low ng/g concentrations. Aqualitatively different antidepressant profile was observed in brain tissue compared to streamwater samples. This study documents that wastewater effluent can be a point source of antidepressants to stream ecosystems and that the qualitative composition of antidepressants in brain tissue from exposed fish differs substantially from the compositions observed in streamwater and sediment, suggesting selective uptake. © 2010 American Chemical Society

Diversité fonctionnelle des innovations évolutives: l’exemple de la cinématique et des performances de grimpe des chutes d’eau des juvéniles de gobies

International audienceDie Funktionelle Leistung von Evolutionären Neuheiten: Erkenntnisse durch Kinematische- und Leistungsstudien von Wasserfallklettern an jugendlichen Gobiid-Fischen Zusammenfassung Die Entwicklung neuartiger funktioneller Merkmale kann wesentlich zur Diversifizierung von Abstammungslinien beitragen. Ältere funktionelle Merkmale können aufgrund der im Laufe der Zeit auftretenden evolutionären Veränderungen größere Unterschiede aufweisen als neuere Merkmale. Die funktionale Komplexität und die Eins-zu-Eins-Zuordnung von Struktur-zu-Funktion können solche Erwartungen jedoch erschweren. In diesem Zusammenhang haben wir die Kinematik und Leistung von Jungtieren verschiedener Spezien von Gobiid-Fischen für zwei Arten des Wasserfallkletterns verglichen: das ältere „Powerburst “-Klettern und das in jüngster Zeit entwickelte „Inching“, das nur in einer Gattung von Gobiid- Fischen vorkommt, die in der Gruppe der Powerburst-Kletterer eingeschlossen ist. Ähnliche Netto-Klettergeschwindigkeiten bei “Inching”-Spezien scheinen zunächst darauf hinzudeuten, dass diese neuere Art des Kletterns wenig funktionelle Vielfalt aufweist. Allerdings ist die ähnliche Nettogeschwindigkeit das Resultat von verschiedenen Faktoren: Sicyopterus stimpsoni aus Hawaii bewegt sich langsamer als S. lagocephalus aus La Réunion aber bewegt sich mehr kontinuierlich. Das Ergebnis einer ähnlichen Leistung durch zweierlei Funktionspfade ähnelt dem Phänomen der Eins-zu-Eins Zuordnung von Struktur-zu-Funktion. Eine solche Ähnlichkeit kann die angemessene Interpretation der relativen funktionalen Vielfalt zwischen den Linien erschweren, sofern nicht die der Leistung zugrundeliegenden Mechanismen untersucht werden. Insbesondere zeigt die Ähnlichkeit in der Nettoleistung zwischen “Powerburst” - und “Inching” -Kletterstilen, dass die Entwicklung des neuen Kletterstils wahrscheinlich wenig mit verbesserter Kletterleistung zu tun hat. In diesem Zusammenhang könnten andere Prozesse (z. B. Exaptation) an der Entstehung dieser funktionellen Neuheit beteiligt sein. Translated to German by Heiko Schoenfuss ([email protected])The evolution of novel functional traits can contribute substantially to the diversification of lineages. Older functional traits might show greater variation than more recently evolved novelties, due to the accrual of evolutionary changes through time. However, functional complexity and many-to-one mapping of structure to function could complicate such expectations. In this context, we compared kinematics and performance across juveniles from multiple species for two styles of waterfall-climbing that are novel to gobiid fishes: ancestral “powerburst” climbing, and more recently evolved “inching”, which has been confirmed only among species of a single genus that is nested within the clade of powerburst climbers. Similar net climbing speeds across inching species seem, at first, to indicate that this more recently evolved mode of climbing exhibits less functional diversity. However, these similar net speeds arise through different pathways: Sicyopterus stimpsoni from Hawai’i move more slowly than S. lagocephalus from La Réunion, but may also spend more time moving. The production of similar performance between multiple functional pathways reflects a situation that resembles the phenomenon of many-to-one mapping of structure to function. Such similarity has the potential to mask appropriate interpretations of relative functional diversity between lineages, unless the mechanisms underlying performance are explored. More specifically, similarity in net performance between “powerburst” and “inching” styles indicates that selection on climbing performance was likely a limited factor in promoting the evolution of inching as a new mode of climbing. In this context, other processes (e.g., exaptation) might be implicated in the origin of this functional novelty.Diversité fonctionnelle des innovations évolutives: l’exemple de la cinématique et des performances de grimpe des chutes d’eau des juvéniles de gobies Résumé L’évolution de nouveaux traits fonctionnels peut contribuer significativement à la diversification des lignées. Les traits fonctionnels les plus anciens peuvent montrer plus de variabilité que les plus récents du fait de l’accumulation de changements évolutif au cours du temps. Cependant, ces prédictions peuvent être complexifiées par la diversité des fonctions et par l’implication de plusieurs structures dans une même fonction. Dans ce contexte, nous avons étudié la cinématique et les performances de grimpe des chutes d’eau de plusieurs espèces de gobies utilisant deux styles de grimpe originaux au sein de cette famille: le mode « powerburst » plus ancestral et le mode « inching » qui a évolué plus récemment. Le mode inching n’a été confirmé que pour deux espèces du même genre incluses au sein du clade des powerburst. Des vitesses de grimpe similaires entre les espèces utilisant le mode inching paraissent indiquer que ce mode de grimpe, qui a évolué plus récemment, présente une diversité fonctionnelle moins élevée. Toutefois, la similarité des vitesses de grimpe entre les deux espèces s’explique par des processus différents: le Sicyopterus stimpsoni d’Haiwaï se déplace plus lentement que le S. lagocephalus de La Réunion mais passe plus de temps en mouvement. La production de performances similaires, résultant de processus différents, reflète un phénomène semblable à celui de l’implication de plusieurs structures dans une même fonction. Si les mécanismes sous-jacents ne sont pas explorés, ces similarités peuvent perturber l’interprétation des différences relatives de diversité fonctionnelle entre les lignées. Par ailleurs, les performances de grimpe similaires entre certaines espèces utilisant le mode inching et d’autres le mode powerburst paraissent indiquer que la force de sélection sur les performances de grimpe est sans doute un facteur réduisant l’avantage évolutif du mode de grimpe inching. Dans ce contexte, d’autres mécanismes (e.g., exaptation) pourraient être à l’origine de cette innovation fonctionnelle. Translated to French by Raphael Lagarde ([email protected])進化的ノベルティの機能的多様性：滝登りハゼの稚魚による岩登り時の運動学とパフォーマンスからの洞察 （Abstract: 梗概） 進化的に新しい機能特性は系統の多様化に大きく貢献することが可能である。より古い機能特性は、時間の経過に伴う進化的変化の蓄積により、より新しく発現した特性よりも大きな偏差を示すと考えられるのであるが、機能の複雑さと、構造と機能における多対一の関連性により、この様な予測はより複雑になる可能性がある。これを踏まえて、我々は祖先的岩登りスタイルの“パワーバースト”タイプとその系統群の中から進化的に新しく発現した単一の属が獲得したとされる“インチング”タイプを示す複数種の滝登りハゼの稚魚による岩登り時の運動学とパフォーマンス比較分析した。“インチング”タイプの実質的なクライミング速度はどれも種間の差異が認められなかった、そのためこのもっとも最近進化的に発現した岩登りのスタイルは、機能的多様性をあまり示さないことを示唆しているよう考えられる。しかし、ハワイ島のSicyopterus stimpsoniは、レユニオン島のS. lagocephalusよりも遅いが、クライミング移動により多くの時間を費やす傾向にあることが分かった。したがって、この種間に差異のない実質的速度は異なる経路を介して発生していると考えられ、複数の機能経路間で同様のパフォーマンスが得られたことは、多対一の関連性があると言える。そしてこれはパフォーマンスの基礎となるメカニズムが明確にされない限り、系統間の相対的な機能的多様性の適切な解釈を複雑にするであろう。より具体的に言うなれば、“パワーバースト”タイプと“インチング”タイプの実質的パフォーマンスの類似性は、クライミングパフォーマンスのへ淘汰が進化的に新しい岩登りスタイルとしての“インチング”タイプを確立する上で制限要因になり得る可能性が高いことを示している。さらには、他のプロセス（例えば、外適応）がこの機能的ノベルティの起源に関係している可能性があると考えられる。Translated to Japanese by Takashi Maie ([email protected])Diversidade funcional de novidades evolucionárias: percepções da cinemática da escalada em cascatas e desempenho de peixes juvenis gobiídeos ResumoA evolução de novos traços funcionais pode contribuir substancialmente para a diversificação de linhagens. Os traços funcionais mais antigos podem mostrar maior variação do que as novidades desenvolvidas mais recentemente, devido ao acúmulo de mudanças evolutivas ao longo do tempo. No entanto, a complexidade funcional e os inúmeros mapeamentos de uma estrutura para uma única função podem complicar essas expectativas. Nesse contexto, comparamos a cinemática e a performance em juvenis de várias espécies para dois estilos de escalada em cascata que são novidades em peixes gobiídeos: a ancestral escalada por “explosão” e o evolutivamente mais recente chamado de “avançamento”, que foi confirmado apenas entre espécies de um único gênero que dentro do clado de escaladores por explosão. Velocidades finais de escalada semelhantes entre espécies usando “avançamento” parecem, inicialmente, indicar que esse modo de escalada evolutivamente mais recente exibe menor diversidade funcional. No entanto, essas velocidades finais similares ocorrem por diferentes formas: Sicyopterus stimpsoni do Havaí se move mais devagar que S. lagocephalus das Ilhas Reunião, mas pode gastar mais tempo se movendo. Desempenhos semelhantes entre várias vias funcionais refletem uma situação que se assemelha ao fenômeno de mapeamento de uma estrutura para um única função. Essa semelhança tem o potencial de ocultar interpretações apropriadas sobre relativa diversidade funcional entre linhagens, a menos que os mecanismos que afetam o desempenho sejam explorados. Mais especificamente, a semelhança no desempenho final entre os estilos “explosão” e “avançamento”indica que a seleção na performance em escalada provavelmente foi um fator limitante na promoção da evolução por “avançamento” como um novo modo de escalada. Nesse contexto, outros processos (e.g., exaptação) podem estar relacionados com a origem dessa novidade funcional.Translated to Portuguese by Diego Vaz ([email protected]