Effects of diluted bitumen exposure on the survival, physiology, and behaviour of zebra finches (Taeniopygia guttata).

Diluted bitumen (dilbit) is an unconventional crude petroleum increasingly being extracted and transported to market by pipeline and tanker. Despite the transport of dilbit through terrestrial, aquatic, and coastal habitat important to diverse bird fauna, toxicity data are currently only available for fish and invertebrates. We used the zebra finch (Taeniopygia guttata) as a tractable, avian model system to investigate exposure effects of lightly weathered Cold Lake blend dilbit on survival, tissue residue, and a range of physiological and behavioural endpoints. Birds were exposed via oral gavage over 14-days with dosages of 0, 2, 4, 6, 8, 10, or 12 mL dilbit/kg bw/day. We identified an LD50 of 9.4 mL/kg/d dilbit, with complete mortality at 12 mL/kg/d. Mortality was associated with mass loss, external oiling, decreased pectoral and heart mass, and increased liver mass. Hepatic ethoxyresorufin-O-deethylase activity (EROD) was elevated in all dilbit-dosed birds compared with controls but there was limited evidence of sublethal effects of dilbit on physiological endpoints at doses < 10 mL/kg/d (hematocrit, hemoglobin, total antioxidants, and reactive oxygen metabolites). Dilbit exposure affected behavior, with more dilbit-treated birds foraging away from the feeder, more birds sleeping or idle at low dilbit doses, and fewer birds huddling together at high dilbit doses. Naphthalene, dibenzothiophene, and their alkylated congeners in particular (e.g. C2-napthalene and C2-dibenzothiophene) accumulated in the liver at greater concentrations in dilbit-treated birds compared to controls. Although directly comparable studies in the zebra finch are limited, our mortality data suggest that dilbit is more toxic than the well-studied MC252 conventional light crude oil with this exposure regime. A lack of overt sublethal effects at lower doses, but effects on body mass and composition, behaviour, high mortality, and elevated PAC residue at doses ≥ 10 mL/kg/d suggest a threshold effect.

Trade-off between aerobic performance and egg production in migratory macaroni penguins.

When successive stages of an organism's life-history overlap, conflicts and trade-offs can emerge due to competition among physiological pathways. For example, long periods of sustained locomotion in migrating birds are supported by the androgenic up-regulation of aerobic factors, such as new red blood cell production and hematocrit. However, towards the end of migration, many female birds begin up-regulating 17ß-estradiol (E2) to support vitellogenesis and egg production, but E2 secretion is known to have suppressive effects on red blood cell production (anti-erythropoiesis). We explored potential trade-offs between factors related to aerobic performance (hematocrit, reticulocyte index) and the expression of factors related to E2-mediated vitellogenesis (i.e. yolk precursor production) in female macaroni penguins (Eudyptes chrysolophus), a species in which the physiologies controlling egg production and migratory activity run simultaneously (e.g. females experience a migratory conflict). We collected blood samples from penguins immediately upon their return to the colony, prior to egg laying. Hematocrit was elevated when the penguins returned to the colony (50.05% ± 3.40 SD), which is similar to pre-laying values observed in other migratory bird species. Furthermore, mean reticulocyte levels were elevated (34.87% ± 2.34), which is the highest level yet recorded in birds. Similarly, both plasma vitellogenin and yolk-targeted very low density lipoprotein levels were upregulated (2.30 ± 0.06 µg Zn ml-1, and 9.70 ± 0.19 mmol l-1, respectively), indicating that penguins were reproductively active and producing eggs during migration and upon arrival on land. As predicted, a negative relationship between hematocrit and plasma vitellogenin was found, but we found no evidence to suggest that birds were experiencing reproductive anemia. Alternatively, we attribute the negative relationship to a hemodilution effect of yolk precursor secretion into circulation. It appears that female macaroni penguins are able to preserve hematocrit levels and new red blood cell production when migratory activity overlaps with reproductive processes.

The Effects of the Organic Flame-Retardant 1,2-Dibromo-4-(1,2-dibromoethyl) Cyclohexane (TBECH) on Androgen Signaling in Human Prostate Cancer Cell Lines.

The effects of the organic flame retardant 1,2-Dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ-/δ-TBECH alone led to a significant increase in prostate-specific antigen (PSA) mRNA accumulation, both the α-/-TBECH and γ-/δ-TBECH mixtures repressed androgen-inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.

The Flame-Retardant Tris(1,3-dichloro-2-propyl) Phosphate Represses Androgen Signaling in Human Prostate Cancer Cell Lines.

The effects of six organophosphate flame retardants (OPFRs) tris(2-butoxyethyl) phosphate, tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, tris(methylphenyl) phosphate, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), and triethyl phosphate on the activities of androgen receptor (AR), estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) were assessed in human prostate and endometrial cancer cells. OPFRs had no effect on ER or AhR target gene activation in ECC-1 cells. The effect of TDCIPP on mRNA and protein accumulation of AR target genes was examined further. AR-inducible gene and protein expression were significantly altered by TDCIPP exposure and repressed PSA levels in conditioned media of prostate cancer cells. We demonstrated that TDCIPP has no affinity for the AR ligand binding domain (AR-LBD) and exerts its antiandrogenic effects in a noncompetitive fashion. Thus, the clinical relevance of TDCIPP exposure on prostate cancer detection and progression to a therapeutically refractile state ought to be investigated further.

Social cues regulate reciprocal switching of hypothalamic Dio2/Dio3 and the transition into final follicle maturation in European starlings (Sturnus vulgaris).

With final maturation of ovarian follicles, birds are committed to a major energetic investment: egg laying. Follicles develop in a 2-step process: 1) initial development of regressed follicles stimulated by long days and 2) yolk incorporation into hierarchical follicles, ovulation, and oviposition. We know little about how females transduce environmental cues into neuroendocrine signals regulating the second step. The present study measures gene expression in tissues within the hypothalamo-pituitary-gonadal axis. Females were housed in seminatural enclosures experiencing natural changes in photoperiod and environmental cues (eg, temperature, rainfall, etc), without males or with constant access to males (January to April). By April, females with males had begun to lay eggs, whereas those without males had not. In a second study, females without males for 3.5 months were then given access to males for 7 days. Restricting male access completely inhibited final follicle maturation, whereas 7-day male access stimulated full vitellogenesis and follicle maturation. Few gene expression changes were attributable to constant male access (January to March), but naïve females given 7-day male access had increased type 2 deiodinase (DIO2) and decreased DIO3 synthesis in the hypothalamus, potentially influencing local thyroid hormone metabolism, increased expression of LH receptor and aromatase in follicles and vitellogenin in liver. Our data suggest that initial follicle development may be more heavily influenced by photoperiod, but the second step (final maturation) is sensitive to other cues such as social interactions. This is the first demonstration of a social effect on the Dio2/Dio3 system, previously thought only responsive to photoperiod cues.

Effects of the bioaccumulative polybrominated diphenyl ether flame retardant congener BDE-47 on growth, development, and reproductive success in zebra finches.

This study investigated the effects of the polybrominated diphenyl ether congener, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on the growth and development, and subsequent breeding success of exposed zebra finches (Taeniopygia guttata). Using oral dosing procedures and treatments adjusted by weight, we treated newly hatched chicks daily for the first 20-days-post-hatch (dph) with varying treatments of BDE-47 (0, 5, 50, and 500 ng/g bw/day). Weight and tarsal measurements were monitored from hatch to 90 dph, but no differences were observed between treatment groups at any age. Treated females that reached sexual maturity were mated with untreated males; however, again no treatment effects were observed on breeding success. Analysis of tissue samples at 21 dph did indicate that debromination of BDE-47 had occurred resulting in BDE-28 and BDE-17 metabolites.

Maternal effects underlie ageing costs of growth in the zebra finch (Taeniopygia guttata).

Maternal effects provide a mechanism to adapt offspring phenotype and optimize the mother's fitness to current environmental conditions. Transferring steroids to the yolk is one way mothers can translate environmental information into potential adaptive signals for offspring. However, maternally-derived hormones might also have adverse effects for offspring. For example, recent data in zebra finch chicks suggested that ageing related-processes (i.e. oxidative stress and telomere loss) were increased after egg-injection of corticosterone (CORT). Still, we have few experimental data describing the effect of maternal effects on the growth-ageing trade-off in offspring. Here, we chronically treated pre-laying zebra finch females (Taeniopygia guttata) with 17-ß-estradiol (E2) or CORT, and followed offspring growth and cellular ageing rates (oxidative stress and telomere loss). CORT treatment decreased growth rate in male chicks and increased rate of telomere loss in mothers and female offspring. E2 increased body mass gain in male offspring, while reducing oxidative stress in both sexes but without affecting telomere loss. Since shorter telomeres were previously found to be a proxy of individual lifespan in zebra finches, maternal effects may, through pleiotropic effects, be important determinants of offspring life-expectancy by modulating ageing rate during embryo and post-natal growth.

Postmigratory body condition and ovarian steroid production predict breeding decisions by female gray-headed albatrosses.

Carryover effects have been documented in many migratory bird species, but we know little about the physiological mechanisms that mediate those effects. Here we show that the energetic, endocrine, and aerobic characteristics of postmigratory female gray-headed albatrosses (Thalassarche chrysostoma) can affect their decision to breed. All females in this study, whether breeding or not, were secreting ovarian steroids when they arrived at the breeding colony at Bird Island, South Georgia, which suggests that all were responding to seasonal cues. However, deferring, nonbreeding birds were characterized by a steroid profile of high progesterone (P4) and low testosterone (T), whereas breeding birds showed the opposite pattern. Deferring birds also had low body mass, hematocrit, and hemoglobin. These results suggest that postmigratory condition can influence patterns of ovarian steroidogenesis and that the maintenance of high P4 without subsequent conversion to T favors breeding deferral. Whereas breeding females normally convert P4 to T, which is a key deterministic step toward 17ß-estradiol synthesis, vitellogenesis, and follicle development, deferring females did not make this conversion and instead maintained high levels of P4, perhaps due to inhibition of the hydroxylase-lyase enzyme complex, thus rendering them infertile for the current season. Results are discussed within the context of the biennial breeding system of this species, and comparisons with other biennially and annually breeding albatrosses are made.

Individual variation in body burden, lipid status, and reproductive investment is related to maternal transfer of a brominated diphenyl ether (BDE-99) to eggs in the zebra finch.

Avian eggs are exposed to hydrophobic contaminants through maternal transfer. How maternal transfer of contaminants within a species is influenced by individual variation in characteristics such as body burden, yolk precursor levels, or reproductive investment is not understood. The authors investigated sources of variation in the maternal transfer of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) in zebra finches (Taeniopygia guttata). The authors dosed adult female zebra finches with levels of BDE-99 relevant to exposure in wild birds (0, 33.7 or 173.8 ng/g body wt/d) for three weeks prior to pairing. Maternal BDE-99 and very-low-density lipoprotein (VLDL) in plasma were measured during egg formation and at clutch completion, and BDE-99 was measured in the corresponding egg. The lipid-normalized egg-to-maternal tissue BDE-99 relationship decreased with increasing maternal burden. Individual variation in maternal VLDL was related to BDE-99 transfer to the eggs when BDE-99 was at background levels in control birds, but not when BDE-99 was elevated in dosed birds. The decrease in maternal plasma BDE-99 over the laying period was only significant (p < 0.05) in the high-dose birds. Finally, the decrease in BDE-99 in maternal plasma during egg-laying was significantly positively correlated with clutch mass in the high-dose group. These results suggest that the relationship between maternal and egg contaminant levels can be highly variable. This has significant implications for using eggs as indicators of adult or environmental concentrations.

Early exposure to 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) affects mating behavior of zebra finches.

2,2',4,4',5-Pentabromodiphenyl ether (BDE-99) is a brominated flame retardant congener that has pervaded global food chains, being reported in avian egg and tissue samples throughout the world. Its effects on birds are not well known, but there is evidence in exposed mammals that it directly mediates and causes neurotoxicity, alters thyroid hormone homeostasis, and lowers sex steroid hormone concentrations. In birds, those processes could disrupt the song-control system and male mating behavior. In this study, the effects of nestling exposure to environmentally relevant levels of BDE-99 were assessed in a model songbird species, the zebra finch (Taeniopygia guttata). A tissue residue study in which zebra finch nestlings were orally exposed to 0, 2.5, 15.8, or 50.7 ng BDE-99/g body weight (bw) per day over the 21-day nesting period validated dosing methods and confirmed dose levels were environmentally relevant (332.7 ± 141.0 to 4450.2 ± 1396.2 ng/g plasma lipid). A full-scale study exposing nestlings to 0, 2.5, 15.8, 50.7, or 173.8 ng BDE-99/g bw/day was carried out to investigate long-term effects of BDE-99 on the adult song-control nuclei volumes, song quality, and male mating behavior. Early exposure to BDE-99 had significant effects on male mating behavior and the response of clean experienced females to exposed males. There was no effect on male song-control nuclei or song quality, and there were nondose-dependent effects on female song-control nuclei. The results demonstrate that early exposure to environmentally relevant levels of BDE-99 affects the behavior of zebra finches.

Migratory carryover effects and endocrinological correlates of reproductive decisions and reproductive success in female albatrosses.

Physiological mechanisms mediating carryover effects, wherein events or activities occurring in one season, habitat, or life-history stage affect important processes in subsequent life-history stages, are largely unknown. The mechanism most commonly invoked to explain carryover effects from migration centres on the acquisition and utilization of resources (e.g. body mass, or individual 'condition'). However, other mechanisms are plausible, e.g. trade-offs reflecting conflict or incompatibility between physiological regulatory systems required for different activities or life-history stages (migration vs. reproduction). Here we show that in female black-browed albatrosses (Thalassarche melanophris) the decision to reproduce or to defer reproduction, made prior to their arrival at breeding colonies after long-distance migration, is associated with condition-related (body mass, hematocrit, hemoglobin concentrations) and hormonal (progesterone, testosterone, estrogen-dependent yolk precursors) traits. In contrast, reproductive success showed little association with condition but showed significant associations with the steroidogenic processes underlying follicle development. Specifically, success was determined by reproductive readiness via differences in steroid hormones and hormone-dependent traits. Successful albatrosses were characterized by high progesterone and high estradiol-dependent yolk precursor levels, whereas failed albatrosses had high testosterone and low yolk precursor levels. Results are discussed with reference to migratory carryover effects and how these can differentially affect the physiologies influencing reproductive decisions and reproductive success.

Hematological changes associated with egg production: estrogen dependence and repeatability.

The 'cost of reproduction' (i.e. the trade-off between current reproduction and future fecundity and/or survival) is a central concept in life history theory, yet we still know very little about the physiological mechanisms underlying such costs. Recently it has been recognized that reproduction itself or the regulatory (hormonal) mechanisms underlying reproduction might result in 'costs' (cf. resource-allocation based mechanisms). As one example, it has been suggested that the decrease in hematocrit observed during egg production in birds might be due to antagonistic pleiotropic effects of estrogens. This could generate costs of reproduction by reducing oxygen-carrying capacity during subsequent aerobically demanding stages such as chick-provisioning. Here we show that the reduction in hematocrit during egg-laying is dependent on receptor-mediated actions of endogenous estrogens: blocking estrogen receptors using the anti-estrogen tamoxifen reduces the decrease in hematocrit during egg production in female zebra finches (Taeniopygia guttata) such that hematocrit at the 1-egg stage is not significantly different than pre-breeding, baseline values. We also show that both pre-breeding hematocrit and the decrease in hematocrit associated with egg production are repeatable, and that females with the highest pre-breeding hematocrit values tend to show the largest decreases in hematocrit during egg production. We suggest that hematological changes during egg production are a good candidate mechanism for a regulatory-network based trade-off involving antagonistic pleiotropic effects of estrogens, which otherwise have essential reproductive functions.

Laying-sequence-specific variation in yolk oestrogen levels, and relationship to plasma oestrogen in female zebra finches (Taeniopygia guttata).

We investigated the relationship between plasma and yolk oestrogens in laying female zebra finches (Taeniopygia guttata) by manipulating plasma oestradiol (E2) levels, via injection of oestradiol-17beta, in a sequence-specific manner to maintain chronically high plasma levels for later-developing eggs (contrasting with the endogenous pattern of decreasing plasma E2 concentrations during laying). We report systematic variation in yolk oestrogen concentrations, in relation to laying sequence, similar to that widely reported for androgenic steroids. In sham-manipulated females, yolk E2 concentrations decreased with laying sequence. However, in E2-treated females plasma E2 levels were higher during the period of rapid yolk development of later-laid eggs, compared with control females. As a consequence, we reversed the laying-sequence-specific pattern of yolk E2: in E2-treated females, yolk E2 concentrations increased with laying-sequence. In general therefore, yolk E2 levels were a direct reflection of plasma E2 levels. However, in control females there was some inter-individual variability in the endogenous pattern of plasma E2 levels through the laying cycle which could generate variation in sequence-specific patterns of yolk hormone levels even if these primarily reflect circulating steroid levels.

Impact of oil-sands based wetlands on the growth of mallard (Anas platyrhynchos) ducklings.

Identifying the potential effects of industrially formed wetlands on waterfowl populations is important for assessing the suitability of such wetlands in industrial reclamation strategies. Mallard ducklings were held in situ on two industrially formed wetlands and one reference wetland in northern Alberta, Canada. Duckling mass and skeletal size were measured at regular intervals over 33 d, and blood was collected to investigate the analysis of plasma metabolites (triglyceride and glycerol) as an indicator of physiological condition. In repeated-measures analysis of variance (ANOVA), multivariate ANOVA, and subsequent multiple-comparisons tests, body mass and skeletal size were significantly lower in ducklings maintained on the industrial wetland after 2, 5, 9, and 13 d of exposure. In this situation, plasma metabolite analysis did not provide additional information on mass-independent condition. We conclude that if the observed differences in growth and size translate into a decreased survival of juvenile waterfowl inhabiting these wetlands, then populations of these birds in the area could be negatively affected. We emphasize the importance of field-based ecological research in toxicological studies of wildlife.

Prior experience with photostimulation enhances photo-induced reproductive development in female European starlings: a possible basis for the age-related increase in avian reproductive performance.

Reproductive performance in female birds improves with age, and this is generally attributed to experiences obtained during breeding. In temperate-zone species, experience with photostimulation during the first breeding year may prime the hypothalamo-pituitary-gonadal axis to respond to photic cues more rapidly or robustly in subsequent years. To test this idea, we captured 32 photorefractory juvenile (hence naive to photostimulation) female European starlings (Sturnus vulgaris) and held half of them (naive group) on a photoperiod of 8L:16D for 32 wk and the other half (experienced group) on 8L:16D for 12 wk, 16L:8D for 12 wk, and then 8L:16D for 8 wk. When we subsequently transferred all birds to 16L:8D, the increase in body mass, which may presage egg laying in the wild, was more robust in experienced than in naive females. Experienced females also showed a more robust elevation in plasma concentrations of the yolk-precursor protein vitellogenin, although naive females showed an initial rapid but transient rise in vitellogenin that we attribute to their extended exposure to short-day photoperiods prior to photostimulation. Finally, the photo-induced increase in diameter of the largest ovarian follicle, in plasma concentrations of luteinizing hormone, and in the number of septo-preoptic fibers relative to the number of cell bodies immunoreactive to GnRH was greater in experienced than in naive females. Thus, prior experience with photostimulation enhances some initial phases of photo-induced reproductive development and may explain, in part, why reproductive performance improves with age in temperate-zone birds.

The metabolic cost of avian egg formation: possible impact of yolk precursor production?

Little is known about the energy costs of egg production in birds. We showed in previous papers that, during egg production, European starlings (Sturnus vulgaris) undergo a 22% increase in resting metabolic rate (RMR) and that the maintenance and activity costs of the oviduct are responsible for 18% of the variation in elevated laying RMR. Therefore, other energy-consuming physiological mechanisms must be responsible for the remaining unexplained variation in elevated laying RMR. Yolk precursor [vitellogenin (VTG) and very-low-density lipoprotein (VLDL)] production is likely to be costly because it signifies a marked increase in the biosynthetic activity of the liver. We documented the pattern of yolk precursor production in response to daily injections of 17beta-estradiol (E2) in zebra finches (Taeniopygia guttata). Based on this pattern we carried out an experiment in order to evaluate the metabolic costs of producing VTG and VLDL. Our E2 treatment resulted in a significant increase in plasma VTG and VLDL levels within the natural breeding range for the species. Although RMR was measured during the period of active hepatic yolk precursor production, it did not differ significantly within individuals in response to the treatment or when comparing E2-treated birds with sham-injected birds. This could mean that yolk precursor production represents low energy investment. However, we discuss these results in light of possible adjustments between organs that could result in energy compensation.

Seasonal dynamics of flight muscle fatty acid binding protein and catabolic enzymes in a migratory shorebird.

We developed an ELISA to measure heart-type fatty acid binding protein (H-FABP) in muscles of the western sandpiper (Calidris mauri), a long-distance migrant shorebird. H-FABP accounted for almost 11% of cytosolic protein in the heart. Pectoralis H-FABP levels were highest during migration (10%) and declined to 6% in tropically wintering female sandpipers. Premigratory birds increased body fat, but not pectoralis H-FABP, indicating that endurance flight training may be required to stimulate H-FABP expression. Juveniles making their first migration had lower pectoralis H-FABP than adults, further supporting a role for flight training. Aerobic capacity, measured by citrate synthase activity, and fatty acid oxidation capacity, measured by 3-hydroxyacyl-CoA-dehydrogenase and carnitine palmitoyl transferase activities, did not change during premigration but increased during migration by 6, 12, and 13%, respectively. The greater relative induction of H-FABP (+70%) with migration than of catabolic enzymes suggests that elevated H-FABP is related to the enhancement of uptake of fatty acids from the circulation. Citrate synthase, 3-hydroxyacyl-CoA-dehydrogenase, and carnitine palmitoyl transferase were positively correlated within individuals, suggesting coexpression, but enzyme activities were unrelated to H-FABP levels.