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Balancing selection is important for the maintenance of polymorphism as it can prevent either fixation of one morph through directional selection or genetic drift, or speciation by disruptive selection. Polychromatism, the presence of multiple genetically determined colour phenotypes, can be maintained if the fitness of alternative morphs depends on the relative frequency in a population. In aggressive species, negative frequency-dependent antagonism can prevent an increase in the frequency of rare morphs as they would only benefit from increased fitness while they are rare. Heterospecific aggression is common in nature and has the potential to contribute to rare morph advantage. Here we carry out field observations and laboratory aggression experiments with mbuna cichlids from Lake Malawi, to investigate the role of con- and heterospecific aggression in the maintenance of polychromatism and identify benefits to rare morphs which are likely to result from reduced aggression. We hypothesize that rare morph individuals receive less aggression than common morph individuals and therefore have an ecological advantage. Within species we found that males and females bias aggression towards their own morph, adding to the evidence that inherent own-morph aggression biases can contribute to balancing selection. Over-representation of rare morph territory owners may be influenced by two factors; higher tolerance of different morph individuals as neighbours, and the ability of rare morphs to spend more time feeding. Reduced aggression to rare morph individuals by heterospecifics may also contribute to rare morph advantage., (© 2021 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

Amongst cephalopods microplastics have been reported only in jumbo squid gut. We investigated microplastics in the digestive system of wild cuttlefish (Sepia officinalis) as they are predators and prey and compared the stomach, caecum/intestine and digestive gland (DG) of wild and cultured animals, exposed to seawater from a comparable source. Fibers were the most common type (≈90% of total count) but were ≈2× higher in relation to body weight in wild vs. cultured animals. Fibers were transported to the DG where the count was ≈2× higher /g in wild (median 1.85 fibers/g) vs. cultured. In wild-caught animals the DG was the predominant location but in cultured animals the fibers were more evenly distributed in the digestive tract. The potential impact of microplastics on health of cuttlefish is discussed. Cuttlefish represent a previously unrecognized source of microplastic trophic transfer to fish and finding fibers in cultured animals has implications for aquaculture., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In representative species of all vertebrate classes, the oral ejection of upper digestive tract contents by vomiting or regurgitation is used to void food contaminated with toxins or containing indigestible material not voidable in the feces. Vomiting or regurgitation has been reported in a number of invertebrate marine species ( Exaiptasia diaphana , Cancer productus , and Pleurobranchaea californica ), prompting consideration of whether cephalopods have this capability. This "hypothesis and theory" paper reviews four lines of supporting evidence: (1) the mollusk P. californica sharing some digestive tract morphological and innervation similarities with Octopus vulgaris is able to vomit or regurgitate with the mechanisms well characterized, providing an example of motor program switching; (2) a rationale for vomiting or regurgitation in cephalopods based upon the potential requirement to void indigestible material, which may cause damage and ejection of toxin contaminated food; (3) anecdotal reports (including from the literature) of vomiting- or regurgitation-like behavior in several species of cephalopod ( Sepia officinalis , Sepioteuthis sepioidea , O. vulgaris , and Enteroctopus dofleini ); and (4) anatomical and physiological studies indicating that ejection of gastric/crop contents via the buccal cavity is a theoretical possibility by retroperistalsis in the upper digestive tract (esophagus, crop, and stomach). We have not identified any publications refuting our hypothesis, so a balanced review is not possible. Overall, the evidence presented is circumstantial, so experiments adapting current methodology (e.g., research community survey, in vitro studies of motility, and analysis of indigestible gut contents and feces) are described to obtain additional evidence to either support or refute our hypothesis. We recognize the possibility that further research may not support the hypothesis; therefore, we consider how cephalopods may protect themselves against ingestion of toxic food by external chemodetection prior to ingestion and digestive gland detoxification post-ingestion. Reviewing the evidence for the hypothesis has identified a number of gaps in knowledge of the anatomy (e.g., the presence of sphincters) and physiology (e.g., the fate of indigestible food residues, pH of digestive secretions, sensory innervation, and digestive gland detoxification mechanisms) of the digestive tract as well as a paucity of recent studies on the role of epithelial chemoreceptors in prey identification and food intake., (Copyright © 2020 Sykes, Almansa, Ponte, Cooke and Andrews.)

Cephalopods are the first invertebrate class regulated by the European Union (EU) under Directive 2010/63/EU on the protection of animals used for scientific purposes, which requires prospective assessment of severity of procedures. To assist the scientific community in establishing severity classification for cephalopods, we undertook a web-based survey of the EU cephalopod research community as represented by the participants in the European COoperation on Science and Technology (COST) Action FA1301, Cephs In Action'. The survey consisted of 50 scenarios covering a range of procedures involving several cephalopod species at different life stages. Respondents (59 people from 15 countries) either allocated a severity classification to each scenario or indicated that they were unable to decide (UTD). Analyses evaluated score distributions and clustering. Overall, the UTD scores were low (7.0 ± 0.6%) and did not affect the severity classification. Procedures involving paralarvae and killing methods (not specified in Annexe IV) had the highest UTD scores. Consensus on non-recovery procedures was reached consistently, although occasionally non-recovery appeared to be confused with killing methods. Scenarios describing procedures above the lower threshold for regulation, including those describing behavioural studies, were also identified and allocated throughout the full range of severity classifications. Severity classification for scenarios based on different species (e.g. cuttlefish vs. octopus) was consistent, comparable and dependent on potentially more harmful interventions. We found no marked or statistically significant differences in the overall scoring of scenarios between the demographic subgroups (age, sex, PhD and cephalopod experience). The COST Action FA1301 survey data provide a basis for a prospective severity classification for cephalopods to serve as guide for researchers, project assessors and regulators.

Previous research into public perceptions of live prey feeding has been focused on terrestrial animals. The reasons for this likely relate to the difficulty humans have in being compassionate to animals who are phylogenetically distantly related. In order to test these assumptions, the general public (two groups; one who had just visited an aquarium; and one group who had just visited a zoo), aquarium professionals in the UK/US and terrestrial zoo animal professionals (UK) were investigated to see how they would differ in their responses when asked about feeding various live aquatic animals to one another. Likert based surveys were used to obtain data face to face and via online social media. Demographics in previous research identified a lower acceptance of live prey feeding by females, however in aquatic animals this was not reflected. Instead, separations in perception were seen to exist between participants dependent on whether they had just visited a zoo or aquarium, or worked with animals., Competing Interests: The authors have declared that no competing interests exist.

Xenopus tropicalis is an increasingly important animal model in a variety of biological research fields. In many countries legislation exists to promote and increase welfare wherever possible, including the ability to view animals during daily husbandry with minimal stress to the animal. X. tropicalis ( n = 16) refuge use was investigated; it was found that the animals significantly preferred black opaque overhead cover to open-ended pipes or closed-off ceramic plants pots in refuge choice experiments. This experiment was repeated by replacing the opaque black overhead cover with red filters. A significant preference for overhead cover was seen for the red translucent cover compared with other available refuges, suggesting that X. tropicalis may adopt translucent refuges due to their visual inabilities. The inability of frogs to see certain wavelengths of light may allow staff to view them whilst simultaneously providing the refuge of choice.

Limited dispersal is commonly used to explain differences in diversification rates. An obvious but unexplored factor affecting dispersal is the mode of locomotion used by animals. Whether individuals walk, swim or fly can dictate the type and severity of geographical barriers to dispersal, and determine the general range over which genetic differentiation might occur. We collated information on locomotion mode and genetic differentiation (F ST ) among vertebrate populations from over 400 published articles. Our results showed that vertebrate species that walk tend to have higher genetic differentiation among populations than species that swim or fly. Within species that swim, vertebrates in freshwater systems have higher genetic differentiation than those in marine systems, which is consistent with the higher number of species in freshwater environments. These results show that locomotion mode can impact gene flow among populations, supporting at a broad-scale what has previously been proposed at smaller taxonomical scales., (© 2018 John Wiley & Sons Ltd/CNRS.)

Maintenance of health and welfare of a cephalopod is essential whether it is in a research, aquaculture or public display. The inclusion of cephalopods in the European Union legislation (Directive 2010/63/EU) regulating the use of animals for scientific purposes has prompted detailed consideration and review of all aspects of the care and welfare of cephalopods in the laboratory but the information generated will be of utility in other settings. We overview a wide range of topics of relevance to cephalopod digestive tract physiology and their relationship to the health and welfare of these animals. Major topics reviewed include: (i) Feeding cephalopods in captivity which deals with live food and prepared diets, feeding frequency ( ad libitum vs. intermittent) and the amount of food provided; (ii) The particular challenges in feeding hatchlings and paralarvae, as feeding and survival of paralarvae remain major bottlenecks for aquaculture e.g., Octopus vulgaris ; (iii) Digestive tract parasites and ingested toxins are discussed not only from the perspective of the impact on digestive function and welfare but also as potential confounding factors in research studies; (iv) Food deprivation is sometimes necessary (e.g., prior to anesthesia and surgery, to investigate metabolic control) but what is the impact on a cephalopod, how can it be assessed and how does the duration relate to regulatory threshold and severity assessment? Reduced food intake is also reviewed in the context of setting humane end-points in experimental procedures; (v) A range of experimental procedures are reviewed for their potential impact on digestive tract function and welfare including anesthesia and surgery, pain and stress, drug administration and induced developmental abnormalities. The review concludes by making some specific recommendations regarding reporting of feeding data and identifies a number of areas for further investigation. The answer to many of the questions raised here will rely on studies of the physiology of the digestive tract.

Ensuring the health and welfare of animals in research is paramount, and the normal functioning of the digestive tract is essential for both. Here we critically assess non- or minimally-invasive techniques which may be used to assess a cephalopod's digestive tract functionality to inform health monitoring. We focus on: (i) predatory response as an indication of appetitive drive; (ii) body weight assessment and interpretation of deviations (e.g., digestive gland weight loss is disproportionate to body weight loss in starvation); (iii) oro-anal transit time requiring novel, standardized techniques to facilitate comparative studies of species and diets; (iv) defecation frequency and analysis of fecal color (diet dependent) and composition (parasites, biomarkers, and cytology); (v) digestive tract endoscopy, but passage of the esophagus through the brain is a technical challenge; (vi) high resolution ultrasound that offers the possibility of imaging the morphology of the digestive tract (e.g., food distribution, indigestible residues, obstruction) and recording contractile activity; (vii) needle biopsy (with ultrasound guidance) as a technique for investigating digestive gland biochemistry and pathology without the death of the animal. These techniques will inform the development of physiologically based assessments of health and the impact of experimental procedures. Although intended for use in the laboratory they are equally applicable to cephalopods in public display and aquaculture.

We know little about on how frequently transitions into new habitats occur, especially the colonization of novel environments that are the most likely to instigate adaptive evolution. One of the most extreme ecological transitions has been the shift in habitat associated with the move from water to land by amphibious fish. We provide the first phylogenetic investigation of these transitions for living fish. Thirty-three families have species reported to be amphibious and these are likely independent evolutionary origins of fish emerging onto land. Phylogenetic reconstructions of closely related taxa within one of these families, the Blenniidae, inferred as many as seven convergences on a highly amphibious lifestyle. Taken together, there appear to be few constraints on fish emerging onto land given amphibious behavior has evolved repeatedly many times across ecologically diverse families. The colonization of novel habitats by other taxa resulting in less dramatic changes in environment should be equally, if not, more frequent in nature, providing an important prerequisite for subsequent adaptive differentiation., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

Quantifying the spatial scale of population connectivity is important for understanding the evolutionary potential of ecologically divergent populations and for designing conservation strategies to preserve those populations. For marine organisms like fish, the spatial scale of connectivity is generally set by a pelagic larval phase. This has complicated past estimates of connectivity because detailed information on larval movements are difficult to obtain. Genetic approaches provide a tractable alternative and have the added benefit of estimating directly the reproductive isolation of populations. In this study, we leveraged empirical estimates of genetic differentiation among populations with simulations and a meta-analysis to provide a general estimate of the spatial scale of genetic connectivity in marine environments. We used neutral genetic markers to first quantify the genetic differentiation of ecologically-isolated adult populations of a land dwelling fish, the Pacific leaping blenny (Alticus arnoldorum), where marine larval dispersal is the only probable means of connectivity among populations. We then compared these estimates to simulations of a range of marine dispersal scenarios and to collated FST and distance data from the literature for marine fish across diverse spatial scales. We found genetic connectivity at sea was extensive among marine populations and in the case of A. arnoldorum, apparently little affected by the presence of ecological barriers. We estimated that ~5000 km (with broad confidence intervals ranging from 810-11,692 km) was the spatial scale at which evolutionarily meaningful barriers to gene flow start to occur at sea, although substantially shorter distances are also possible for some taxa. In general, however, such a large estimate of connectivity has important implications for the evolutionary and conservation potential of many marine fish communities.

3-Methylfuran is produced in foods during food processing and preservation techniques that involve heat treatment such as cooking, jarring, canning, and pasteurization. Currently, there are no studies available on the toxicity of 3-methylfuran. We conducted a 28-day gavage toxicity study (7 days per week) using doses of 0.0, 0.1, 0.3, 1.5, 3.0, 6.0, 12.0, and 25.0 mg/kg bw/day in order to determine the dose range needed to establish a no observed adverse effect level and to better characterize nonneoplastic effects including those affecting hematology, clinical biochemistry, gross morphology, and histopathology. Histological changes of the liver were noted in all treated animals and gross changes were noted beginning at 3.0 mg/kg bw/kg. Alterations in the activity of serum enzymes indicative of effects on the liver were observed, including increases in levels of alanine transaminase and alkaline phosphatase at the highest dose. There was a significant increase in serum thyroxine (T4) and triiodothyronine (T3), which was not accompanied by histological changes in the thyroid. For the most part, statistically significant changes were seen only at the highest dose for hematology and at the 2 highest doses for clinical chemistry parameters. In contrast, mild histological lesions in the liver were observed even at the lowest dose of 0.1 mg/kg bw/day., (© 2014 by The Author(s).)

Evolution creates and sustains biodiversity via adaptive changes in ecologically relevant traits. Ecologically mediated selection contributes to genetic divergence both in the presence or absence of geographic isolation between populations, and is considered an important driver of speciation. Indeed, the genetics of ecological speciation is becoming increasingly studied across a variety of taxa and environments. In this paper we review the literature of ecological speciation in the tropics. We report on low research productivity in tropical ecosystems and discuss reasons accounting for the rarity of studies. We argue for research programs that simultaneously address biogeographical and taxonomic questions in the tropics, while effectively assessing relationships between reproductive isolation and ecological divergence. To contribute toward this goal, we propose a new framework for ecological speciation that integrates information from phylogenetics, phylogeography, population genomics, and simulations in evolutionary landscape genetics (ELG). We introduce components of the framework, describe ELG simulations (a largely unexplored approach in ecological speciation), and discuss design and experimental feasibility within the context of tropical research. We then use published genetic datasets from populations of five codistributed Amazonian fish species to assess the performance of the framework in studies of tropical speciation. We suggest that these approaches can assist in distinguishing the relative contribution of natural selection from biogeographic history in the origin of biodiversity, even in complex ecosystems such as Amazonia. We also discuss on how to assess ecological speciation using ELG simulations that include selection. These integrative frameworks have considerable potential to enhance conservation management in biodiversity rich ecosystems and to complement historical biogeographic and evolutionary studies of tropical biotas.

This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleotide polymorphisms, indels and sequence repeats and are subjected to diversification analyses. The sequence elements that compose He185/333 are different to those identified for Sp185/333. Differences between Sp185/333 and He185/333 genes are also evident in the complexity of the sequences of the introns. He185/333 proteins show a diverse range of molecular weights on Western blots. The observed sizes and pIs of the proteins differ from predicted values, suggesting post-translational modifications and oligomerization. Immunofluorescence microscopy shows that He185/333 proteins are mainly located on the surface of coelomocyte subpopulations. Our data demonstrate that He185/333 bears the same substantial characteristics as their S. purpuratus homologues. However, we also identify several unique characteristics of He185/333 (such as novel element patterns, sequence repeats, distribution of positively-selected codons and introns), suggesting species-specific adaptations. All sequences in this publication have been submitted to Genbank (accession numbers JQ780171-JQ780321) and are listed in table S1.

Furan is produced in foods during processing and preservation techniques that involve heat treatment. Previously, we reported that furan-exposed rats exhibited dose-dependent gross and histological changes in liver which correlated with changes in liver serum enzymes ALT, AST and ALP. Here we report the effects of furan on the male reproductive system. There were no histological or weight changes in the reproductive organs. Serum testosterone levels were increased in a dose-dependent manner whereas serum LH was decreased. There were no changes in 17-OHase, 3β-HSD and 17β-HSD activities or serum FSH. Furan did not alter mRNA expression levels for the LH receptor or Tspo but in contrast, mRNA levels of StAR were increased in all doses of furan. The mRNA for the cholesterol side-chain cleavage enzyme (Cyp11a1) was increased by furan at the high dose, as was the level of intratesticular testosterone. We conclude that subchronic furan exposure affects testicular steroidogenesis., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Ecological speciation involves the evolution of reproductive isolation and niche divergence in the absence of a physical barrier to gene flow. The process is one of the most controversial topics of the speciation debate, particularly in tropical regions. Here, we investigate ecologically based divergence across an Amazonian ecotone in the electric fish, Steatogenys elegans. We combine phylogenetics, genome scans, and population genetics with a recently developed individual-based evolutionary landscape genetics approach that incorporates selection. This framework is used to assess the relative contributions of geography and divergent natural selection between environments as biodiversity drivers. We report on two closely related and sympatric lineages that exemplify how divergent selection across a major Amazonian aquatic ecotone (i.e., between rivers with markedly different hydrochemical properties) may result in replicated ecologically mediated speciation. The results link selection across an ecological gradient with reproductive isolation and we propose that assortative mating based on water color may be driving the divergence. Divergence resulting from ecologically driven selection highlights the importance of considering environmental heterogeneity in studies of speciation in tropical regions. Furthermore, we show that framing ecological speciation in a spatially explicit evolutionary landscape genetics framework provides an important first step in exploring a wide range of the potential effects of spatial dependence in natural selection., (© 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.)

Background: Divergence between populations in reproductively important features is often vital for speciation. Many studies attempt to identify the cause of population differentiation in phenotype through the study of a specific selection pressure. Holistic studies that consider the interaction of several contrasting forms of selection are more rare. Most studies also fail to consider the history of connectivity among populations and the potential for genetic drift or gene flow to facilitate or limit phenotypic divergence. We examined the interacting effects of natural selection, sexual selection and the history of connectivity on phenotypic differentiation among five populations of the Pacific leaping blenny (Alticus arnoldorum), a land fish endemic to the island of Guam., Results: We found key differences among populations in two male ornaments--the size of a prominent head crest and conspicuousness of a coloured dorsal fin--that reflected a trade-off between the intensity of sexual selection (male biased sex ratios) and natural selection (exposure to predators). This differentiation in ornamentation has occurred despite evidence suggesting extensive gene flow among populations, which implies that the change in ornament expression has been recent (and potentially plastic)., Conclusions: Our study provides an early snapshot of divergence in reproductively important features that, regardless of whether it reflects genetic or plastic changes in phenotype, could ultimately form a reproductive barrier among populations.

Detectable concentrations of the flame retardant hexabromocyclododecane (HBCD) have been reported in human tissues worldwide, but investigations to determine fetal exposure to this brominated flame retardant are lacking. This study was undertaken to determine the concentrations of α-, β- and γ-HBCD in human tissues (fetal liver and placenta) from Canada. Tissue samples were collected over a thirteen year period following elective pregnancy terminations in Montreal, Quebec, Canada. Samples were extracted using homogenisation with solvent, cleaned up using adsorption chromatography and analysis was performed with liquid chromatography-tandem mass spectrometry. Total HBCD concentrations ranged from below the limit of detection (

In most thermally treated products, a series of alkylated furan derivatives have been found, in particular 2-substituted alkylfurans such as 2-methylfuran. These methyl analogs are metabolically activated in a similar fashion as the parent furan, yielding highly reactive unsaturated dialdehydes. There is currently limited toxicological data available for 2-methyl furan exposure by any route that makes conducting a risk assessment difficult. In this pilot study, we report the general toxicology findings affecting tissue morphology, histopathology, clinical biochemistry, and hematology in a 28-day gavage study. The liver was the primary target organ that developed dose-dependent toxicity. Relative liver weights were increased by 42% at 25.0 mg/kg/body weight (bw)/day. Histological changes in the liver were observed at 0.4, 1.5, 3.0, 6.0, 12.0, and 25.0 mg/kg bw/day. These changes were not accompanied by clinical changes in the serum enzyme markers such as alanine transaminase, alkaline phosphatase, and aspartate transaminase. Clinical biochemistry markers for kidney were altered, but these were not accompanied by histological changes. The prostate was significantly decreased in size at the 25.0 mg/kg bw/day dose of 2-methyfuran. Some hematological parameters were also altered.

The first trimester of human fetal life, a period of extremely rapid development of physiological systems, represents the most rapid growth phase in human life. Interference in the establishment of organ systems may result in abnormal development that may be manifest immediately or programmed for later abnormal function. Exposure to environmental chemicals may be affecting development at these early stages, and yet there is limited knowledge of the quantities and identities of the chemicals to which the fetus is exposed during early pregnancy. Clearly, opportunities for assessing fetal chemical exposure directly are extremely limited. Hence, this review describes indirect means of assessing fetal exposure in early pregnancy to chemicals that are considered disrupters of development. Consideration is given to such matrices as maternal hair, fingernails, urine, saliva, sweat, breast milk, amniotic fluid and blood, and fetal matrices such as cord blood, cord tissue, meconium, placenta, and fetal liver. More than 150 articles that presented data from chemical analysis of human maternal and fetal tissues and fluids were reviewed. Priority was given to articles where chemical analysis was conducted in more than one matrix. Where correlations between maternal and fetal matrices were determined, these articles were included and are highlighted, as these may provide the basis for future investigations of early fetal exposure. The determination of fetal chemical exposure, at the time of rapid human growth and development, will greatly assist regulatory agencies in risk assessments and establishment of advisories for risk management concerning environmental chemicals.

Arsenobetaine (ASB) is the major form of arsenic (As) in seafood sources such as molluscs and fish. Limited data demonstrated that ASB toxicity in mammals is minimal; however, data on possible reproductive effects are lacking. This study investigated the tissue distribution and developmental effects of ASB during pregnancy, early postnatal life, and development to adulthood. Pregnant rats were randomly assigned to 3 cohorts and gavaged daily from gestational day 8 (GD8) with ASB in deionized water at 0, 0.1, 1, or 10 mg/kg body weight (bw)/d. Cohort 1 dams were sacrificed on GD20 (n = 6 per dose group), cohort 2 dams and pups were sacrificed on postnatal day 13 (PND13; n = 4 dams per dose group), and cohort 3 pups (n = 2 dams per dose group) were sacrificed on PND90. Residue analysis detected significant levels of ASB in livers of cohort 1 dams and lower levels in cohort 1 GD20 fetuses, as well as in cohort 2 male and female offspring, indicating placental transfer from the maternal circulation in utero. Trace amounts of ASB in dams' milk were found only in the 10-mg/kg bw/d dose cohort 2 (PND13), demonstrating that lactational transfer was limited. ASB levels in liver varied during pregnancy, lactation, and postweaning, with levels falling rapidly as these physiological states progress. Although transfer of ASB through the placenta to the fetuses and to a limited extent through milk was confirmed, ASB exposure during pregnancy and lactation appeared to produce no teratogenic or deleterious effects on reproductive development.

Using next-generation sequencing technology, we describe the complete mitochondrial genomes for 5 Australian passerine birds (Epthianura albifrons, Petroica phoenicea, Petroica goodenovii, Petroica boodang, and Eopsaltria australis). We successfully assemble each mitogenome de novo using just 1/8th of a Roche GL FSX 454 pyrosequencing plate. From the assembled mitogenomes, we identify 2 different mitochondrial gene arrangements in the region spanning 5'-3' from Cytochrome B to 12s RNA. These gene arrangements represent 2 of the 4 known avian mitochondrial gene arrangements. Our results, together with other previously described avian mitogenomes, highlight that certain mitochondrial rearrangements appear to have arisen multiple times.

In this study, the presence of bisphenol A (BPA) in human placental and fetal liver samples collected from 1998 to 2008 was investigated to provide a more detailed analysis of the transfer of BPA across the placenta and fetal exposure to BPA. The average concentrations in placental samples were 12.6 ng g(-1) for free BPA, 17.2 ng g(-1) for BPA-glu, and 30.2 ng g(-1) for total BPA. The highest concentrations in placental samples were 165 ng g(-1) for free BPA, 178 ng g(-1) for BPA-glu, and 280 ng g(-1) for total BPA. Samples with higher levels of BPA-glu had higher levels of free BPA in general. Fetal age was observed to have a significant effect on BPA-glu levels in placental samples, but not on free or total BPA. The percentages of free BPA relative to total BPA for the placental samples varied considerably from 4.2% to 100%, suggesting that the ability of maternal liver and/or the placenta to conjugate BPA is highly variable during early to mid-gestation. The average concentrations in fetal liver samples were 9.02 ng g(-1) for free BPA, 19.1 ng g(-1) for BPA-glu, and 25.8 ng g(-1) for total BPA. The highest concentrations in fetal liver samples were 37.7 ng g(-1) for free BPA, 93.9 ng g(-1) for BPA-glu, and 123 ng g(-1) for total BPA. The percentages of free BPA level relative to total BPA for all fetal liver samples varied from 12.4% to 99.1%, indicating extensive variability in the ability of the human feto-placental unit to glucuronidate BPA., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Natural selection and ecological adaptation are ultimately responsible for much of the origin of biodiversity. Yet, the identification of divergent natural selection has been hindered by the spatial complexity of natural systems, the difficulty in identifying genes under selection and their relationship to environment, and the confounding genomic effects of time. Here, we employed genome scans, population genetics and sequence-based phylogeographic methods to identify divergent natural selection on population boundaries in a freshwater invader, the Amazonian pufferfish, Colomesus asellus. We sampled extensively across markedly different hydrochemical settings in the Amazon Basin and use 'water colour' to test for ecological isolation. We distinguish the relative contribution of natural selection across hydrochemical gradients from biogeographic history in the origin and maintenance of population boundaries within a single species and across a complex ecosystem. We show that spatially distinct population structure generated by multiple forces (i.e. water colour and vicariant biogeographic history) can be identified if the confounding effects of genetic drift have not accumulated between selective populations. Our findings have repercussions for studies aimed at identifying engines of biodiversity and assessing their temporal progression in understudied and ecologically complex tropical ecosystems., (© 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.)

The unparalleled diversity of tropical ecosystems like the Amazon Basin has been traditionally explained using spatial models within the context of climatic and geological history. Yet, it is adaptive genetic diversity that defines how species evolve and interact within an ecosystem. Here, we combine genome scans, population genetics and sequence-based phylogeographic analyses to examine spatial and ecological arrangements of selected and neutrally evolving regions of the genome of an Amazonian fish, Triportheus albus. Using a sampling design encompassing five major Amazonian rivers, three hydrochemical settings, 352 nuclear markers and two mitochondrial DNA genes, we assess the influence of environmental gradients as biodiversity drivers in Amazonia. We identify strong divergent natural selection with gene flow and isolation by environment across craton (black and clear colour)- and Andean (white colour)-derived water types. Furthermore, we find that heightened selection and population genetic structure present at the interface of these water types appears more powerful in generating diversity than the spatial arrangement of river systems and vicariant biogeographic history. The results from our study challenge assumptions about the origin and distribution of adaptive and neutral genetic diversity in tropical ecosystems. In addition, they have important implications for measures of biodiversity and evolutionary potential in one of the world's most diverse and iconic ecosystems., (© 2012 Blackwell Publishing Ltd.)

Accurately quantifying biodiversity is fundamental for both evolutionary theory and conservation strategies. DNA-based studies are exposing high cryptic diversity irrespective of taxonomic group or environmental setting, and increasing the ever-growing estimates of global biodiversity. This has severe implications for under-sampled and species-rich tropical regions, such as the Amazon Basin. We used biogeographic predictions derived from geomorphological history and contemporary hydrochemical and genetic data to examine cryptic diversity in the Amazonian driftwood catfish Centromochlus existimatus. Using both nuclear and mitochondrial DNA markers, five deeply divergent cryptic lineages are reported, for which at least three are sympatric in distribution. These lineages appear relatively old, with divergence times dating back to middle Miocene. Diversification events appear to be chronologically associated with the formation of the modern Amazon River system, and perhaps influenced by hydrochemical gradients between tributaries. The cause of apparent morphological stasis in the C. existimatus species complex is speculated within the context of hydrochemistry and non-visual mating cues and a full taxonomic revision is recommended. Our findings suggest that the diversity of Amazonian ichthyofauna is vastly underestimated and highlight the relevance of biogeographic predictions to guide sampling efforts in ecologically complex and under-studied ecosystems.

The present study examined, using rats as a model, the effects of sex and age of exposure to dietary soya components on serum total and soya-specific antibody content. In Expt 1, Sprague-Dawley rats at 50 d of age were fed diets containing 20 % casein or 20 % alcohol-washed soya protein isolate (SPI) with or without supplemental isoflavones (ISF, 250 mg/kg diet) for 70, 190 or 310 d. The offspring were fed the same diets as their parents. In Expt 2, juvenile Sprague-Dawley rats at 30 d of age were fed diets containing 20 % casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10 or 20 %) for 90 d. Exposure of rats to dietary SPI before the age of 28 d increased serum total IgA and IgM, and induced the production of SPI-specific IgA, IgG, IgM and IgE antibodies. Feeding of juvenile or adult rats with SPI elevated serum total IgA in females, while the opposite occurred in males, and markedly stimulated the production of SPI-specific IgM in females and IgG in males. Our data suggest that the effects of soya proteins and ISF on the production of serum total and SPI-specific antibodies appear to be sex dependent and also related to the age of exposure to soya in rats. However, the physiological significance of these immune responses remains to be determined.

Furan is a heterocyclic organic compound formed during heat treatment for processing and preservation of various types of food. Rodent studies have previously shown that furan is a hepatocarcinogen. Those studies were conducted over a high dose range, which induced tumors at nearly 100% incidence at all doses. This ninety-day gavage study in mice was conducted to extend the dose to a lower range (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg body weight [bw] per day) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects, including those affecting clinical biochemistry, hematology, tissue morphology, and histopathology. The liver was the primary target organ with dose-dependent toxicity. Liver weights were increased at the 8.0 mg/kg bw dose in females only. Levels of the serum enzyme alanine transaminase, representative of liver damage, were increased three-fold at the highest dose. Histological changes in the liver were observed at 2.0 and 8.0 mg/kg bw in both sexes. Although clinical parameters were also altered for the kidney, these differences were not accompanied by histological changes. Based on these clinical biochemical and histological changes, a no-observed adverse effect level of 0.12 mg/kg bw per day of furan in mice is suggested.

Background: Ecosystem engineers facilitate habitat formation and enhance biodiversity, but when they become invasive, they present a critical threat to native communities because they can drastically alter the receiving habitat. Management of such species thus needs to be a priority, but the poorly resolved taxonomy of many ecosystem engineers represents a major obstacle to correctly identifying them as being either native or introduced. We address this dilemma by studying the sea squirt Pyura stolonifera, an important ecosystem engineer that dominates coastal communities particularly in the southern hemisphere. Using DNA sequence data from four independently evolving loci, we aimed to determine levels of cryptic diversity, the invasive or native status of each regional population, and the most appropriate sampling design for identifying the geographic ranges of each evolutionary unit., Results: Extensive sampling in Africa, Australasia and South America revealed the existence of "nested" levels of cryptic diversity, in which at least five distinct species can be further subdivided into smaller-scale genetic lineages. The ranges of several evolutionary units are limited by well-documented biogeographic disjunctions. Evidence for both cryptic native diversity and the existence of invasive populations allows us to considerably refine our view of the native versus introduced status of the evolutionary units within Pyura stolonifera in the different coastal communities they dominate., Conclusions: This study illustrates the degree of taxonomic complexity that can exist within widespread species for which there is little taxonomic expertise, and it highlights the challenges involved in distinguishing between indigenous and introduced populations. The fact that multiple genetic lineages can be native to a single geographic region indicates that it is imperative to obtain samples from as many different habitat types and biotic zones as possible when attempting to identify the source region of a putative invader. "Nested" cryptic diversity, and the difficulties in correctly identifying invasive species that arise from it, represent a major challenge for managing biodiversity.

A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography-mass spectrometry (GC-MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. β-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77 ng/g for placenta samples and 1.2 ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8 ng/g in beef and calf livers to 17.1 ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60 ng/g to as high as 64 ng/g with an average of 9.5 ng/g and a median of 3.0 ng/g, and conjugated BPA was as high as 7.8 ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27 ng/g with an average of 8.5 ng/g and a median of 3.2 ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20 ng/g with an average of 3.9 ng/g and a median of 1.5 ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Background/aims: There has been great interest in the potential beneficial and adverse health effects of dietary isoflavones. Determination of tissue concentrations of isoflavone metabolites provides an insight into the potential bioactivity of dietary isoflavones. However, data on the distribution of isoflavones in animal models fed dietary isoflavones are limited. In this study, additional data on the distribution of isoflavones in serum and/or tissues of rats and pigs fed dietary isoflavones were generated., Methods: Rats (male and female) were fed a casein control diet (containing no isoflavones) and an isoflavone-supplemented diet (containing an alcohol-washed soy protein isolate plus NOVASOY, providing a total of 1,047 mg/kg of total isoflavones). Female pigs were fed a control diet (without soy) containing 17.5 mg/kg of isoflavones, a soy diet containing 582.8 mg/kg of isoflavones or a soy diet supplemented with a daily dose of 2.3 g (equivalent to 42.0 and 14.5 mg/kg of body weight at the onset and end of treatment, respectively) of crystalline genistein. The concentrations of isoflavones in serum and tissues (liver and mammary gland) and in tissues (liver and mammary gland) of pigs were determined via a sensitive and rapid method using liquid chromatography/mass spectrometry., Results: Rats fed the control diet containing no isoflavones had nondetectable levels of isoflavone metabolites in serum, liver and mammary gland samples. Rats fed the isoflavone-supplemented diet had the greatest levels of equol, followed by genistein, daidzein and glycitein, respectively, in their serum, livers and mammary glands. The concentrations of total isoflavones (daidzein, equol and genistein plus glycitein) in serum were significantly (p < 0.05) greater in male rats vs. female rats, but the reverse was true in the case of livers. Concentrations of daidzein, equol, genistein and glycitein were lowest (p < 0.05) in the livers of pigs fed the control diet, and in the mammary glands of female pigs there was only an effect of feeding soy plus genistein on the concentrations of daidzein and equol (p <0.05)., Conclusions: The current data therefore show gender as well as species differences in the tissue distribution of isoflavones., (Copyright © 2011 S. Karger AG, Basel.)

Rodent studies have shown that furan is a hepatocarcinogen. Previous studies conducted with high doses showed tumors at nearly 100% incidence at all doses. In this paper, a ninety-day gavage experiment conducted with lower doses (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg bw) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects including gross changes and histopathology, clinical biochemistry, hematology, and immunotoxicology is reported. As indicated by changes in serum biomarkers, increased liver weights and gross and histological lesions, the liver is the major target organ affected by furan. There were no changes in body weights, food consumption, or histology in other organs. Some of the serum electrolyte markers, including phosphorus, were altered. There was a significant increase in serum thyroxine and triidothyronine in males. This increase was not accompanied by histological thyroid changes. Immunophenotypic analysis showed that thymic lymphocyte maturation was altered in male rats. Although altered clinical biochemistry and hematological parameters were observed at a dose of > 0.5 mg/kg bw, mild histological lesions in the liver were observed at > 0.12 mg/kg bw. Based on this finding, a furan dose of 0.03 mg/kg bw was proposed as the no-observed adverse effect level for hepatic toxicity.

Steroid-5alpha-reductases (SRD5alpha) and steroid-5beta-reductase (SRD5beta) represent a convergence in evolution: they share similar biological functions, but do not have a common ancestor. In vertebrates, SRD5alpha and SRD5beta are involved in C-19 and C-21 steroid biosynthesis, bile acid biosynthesis and erythropoiesis. We compare and contrast the history, evolution, tissue distribution, enzyme characteristics and biological functions of SRD5alpha and SRD5beta and suggest possible future directions for research efforts. Both, the unique and overlapping roles that SRD5alpha and SRD5beta play in steroid hormone metabolism, are indicated. We also present the phylogeny of the SRD5alpha. The main SRD5alpha subfamilies obtained include, not only the well-known SRD5alpha type 1, type 2 and type 3, but also the synaptic glycoprotein (GPSN2)/trans-2,3-enoly-CoA reductase group. Phylogenetic analysis indicated that a eukaryotic ancestor likely underwent duplication events to generate these three subfamilies (type 1/2, type 3 and GPSN2 ancestors); both SRD5alpha type 1/2 and GPSN2 subfamilies may have evolved by ancient duplication events at the early stage of vertebrate and chordate evolution., (Copyright 2009 Elsevier Inc. All rights reserved.)

Steroidogenic enzymes and their steroid products play critical roles during gonadal differentiation in amphibians; however their roles during embryogenesis remain unclear. The objective of this study was to investigate the expression and activity of aromatase (cyp19; estrogen synthase) and 5 beta-reductase (srd5 beta; 5 beta-dihydrotestosterone synthase) during amphibian embryogenesis. Expression and activity profiles of cyp19 and srd5 beta were first established during Silurana (Xenopus) tropicalis embryogenesis from Nieuwkoop-Faber (NF) stage 2 (2-cell stage; 1h post-fertilization) to NF stage 46 (beginning of feeding; 72 h post-fertilization). Exposures to fadrozole (an aromatase inhibitor; 0.5, 1.0 and 2.0 microM) and finasteride (a putative 5-reductase inhibitor; 25, 50 and 100 microM) were designed to assess the consequences of inhibiting these enzymes on gene expression in early amphibian larval development. Exposed embryos showed changes in both enzyme activities and sex steroid- and thyroid hormone-related gene expression. Fadrozole treatment inhibited cyp19 activity and increased androgen receptor and thyroid hormone receptor (alpha and beta) mRNAs. Finasteride treatment inhibited srd5 beta (activity and mRNA), decreased cyp19 mRNA and activity levels and increased estrogen receptor alpha mRNA. Both treatments altered the expression of deiodinases (thyroid hormone metabolizing enzymes). We conclude that cyp19 and srd5 beta are active in early embryogenesis and larval development in Silurana tropicalis and their inhibition affected transcription of genes associated with the thyroid and reproductive axes., ((c) 2009 Elsevier Inc. All rights reserved.)

Background: There are conflicting reports regarding the effects of atrazine (ATZ) on amphibian development. Therefore, further studies are needed to examine the potential mechanisms of action of ATZ in amphibians., Objectives: Our aim in this study was to determine whether low concentrations of ATZ affect gonadal development and metamorphosis in the Northern leopard frog, Rana pipiens., Methods: Tadpoles were exposed in outdoor mesocosms to nominal concentrations of 0.1 and 1.8 microg/L of formulated ATZ from Gosner stage 27 (G27) to metamorphic climax (G42). Exposure to 17alpha-ethinylestradiol (EE2; 1.5 microg/L) provided a positive control for induction of testicular oocytes in males. Endocrine-related gene expression and gonadal histopathology were examined at G42 and in a subset of premetamorphic G34 tadpoles that failed to metamorphose., Results: Gonadal gross morphology revealed that the 1.8-microg/L ATZ treatment produced 20% more females compared with the control. Histologic analysis revealed that 22% of EE2-treated males had testicular oocytes, whereas none were observed in any animals from the control or either ATZ groups. ATZ increased brain estrogen receptor alpha mRNA to 2.5 times that of the control at premetamorphosis and altered liver levels of 5beta-reductase activity at metamorphosis. In contrast, brain aromatase mRNA level and activity did not change. ATZ treatments significantly reduced metamorphic success (number of animals reaching metamorphosis) without affecting body weight, snout-vent length, or age at metamorphosis. Gene expression analysis indicated that ATZ decreased the expression of deiodinase type 3 in the tail at premetamorphosis., Conclusions: Our study indicates that exposure to low concentrations of ATZ in experimental mesocosms alters gonadal differentiation and metamorphosis in developing R. pipiens.

Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17alpha-ethinyl estradiol (EE, 10 microg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.

There are limited reports on the bioavailability and pharmacokinetics of isoflavones in elderly humans and aged animals. The present study was conducted to assess the effect of glycosidation of isoflavones on their bioavailability and pharmacokinetics in aged (20 month old) male Fischer-344 (F-344) rats. The F-344 rat, developed by the National Institute on Aging, is an inbred rat model that is commonly used for aging studies and resembles many features of aging humans. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycons (daidzein, genistein and glycitein), and a mixture of synthetic glucosides (daidzin, genistin, and glycitin) were tested. Following administration, blood samples were collected at different times (0-48 h post-oral gavage and 0-8 h post-IV dosing). Plasma isoflavones and 7-hydroxy-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein) were measured by LC/MS. The extent of absorption was determined by comparing the area under the curve (AUC) of the plasma-concentration time curve after intravenous (IV) administration with that following oral administration. The extent of bioavailability was then calculated as: %bioabailability = (AUC(or)/AUC(IV))x(Dose(IV)/Dose(or))x100. Bioavailabilities for genistein were significantly (p = 0.013) higher for the aglycon (35 +/- 9%) compared with the glucoside forms (11 +/- 3%). In contrast, the bioavailabilities for glycitein were significantly (p = 0.011) higher in Novasoy (27 +/- 13%) and the glucoside form (21 +/- 10%) compared with the aglycon (8 +/- 3%). No significant differences in the bioavailability of daidzein were observed in aged rats dosed with aglycon, glucoside or Novasoy. However, aged rats were able to produce equol as early as 8 h post-dosing. In summary, the source of isoflavones had significant effects on genistein and glycitein bioavailability in aged male rats.