Is there potential for estradiol receptor signaling in lophotrochozoans?

Estrogen receptors (ERs) are thought to be the ancestor of all steroid receptors and are present in most lophotrochozoans studied to date, including molluscs, annelids, and rotifers. A number of studies have investigated the functional role of estrogen receptors in invertebrate species, although most are in molluscs, where the receptor is constitutively active. In vitro experiments provided evidence for ligand-activated estrogen receptors in annelids, raising important questions about the role of estrogen signalling in lophotrochozoan lineages. Here, we review the concordant and discordant evidence of estradiol receptor signalling in lophotrochozoans, with a focus on annelids and rotifers. We explore the de novo synthesis of estrogens, the evolution and expression of estrogen receptors, and physiological responses to activation of estrogen receptors in the lophotrochozoan phyla Annelida and Rotifera. Key data are missing to determine if de novo biosynthesis of estradiol in non-molluscan lophotrochozoans is likely. For example, an ortholog for the CYP11 gene is present, but confirmation of substrate conversion and measured tissue products is lacking. Orthologs CYP17 and CYP19 are lacking, yet intermediates or products (e.g. estradiol) in tissues have been measured. Estrogen receptors are present in multiple species, and for a limited number, in vitro data show agonist binding of estradiol and/or transcriptional activation. The expression patterns of the lophotrochozoan ERs suggest developmental, reproductive, and digestive roles but are highly species dependent. E2 exposures suggest that lophotrochozoan ERs may play a role in reproduction, but no strong dose-response relationship has been established. Therefore, we expect most lophotrochozoan species, outside of perhaps platyhelminths, to have an ER but their physiological role remains elusive. Mining genomes for orthologs gene families responsible for steroidogenesis, coupled with in vitro and in vivo studies of the steroid pathway are needed to better assess whether lophotrochozoans are capable of estradiol biosynthesis. One major challenge is that much of the data are divided across a diversity of species. We propose that the polychaetes Capitella teleta or Platyneris dumerilii, and rotifer Brachionus manjavacas may be strong species choices for studies of estrogen receptor signalling, because of available genomic data, established laboratory culture techniques, and gene knockout potential.

Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation.

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.

Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings.

Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2°C, 5°C, or 8°C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption ( [Formula: see text] ) were measured across embryonic development, and [Formula: see text] was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and [Formula: see text] compared to control embryos measured at the same temperature (2°C, 5°C, or 8°C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered [Formula: see text] or fH. Incubation at 2°C or 5°C through gastrulation significantly lowered [Formula: see text] (42% decrease) and fH (20% decrease) at 8°C, incubation at 2°C significantly lowered [Formula: see text] (40% decrease) and fH (30% decrease) at 5°C, and incubation at 5°C and 8°C significantly lowered [Formula: see text] at 2°C (27% decrease). Through the latter half of development, [Formula: see text] and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2°C, [Formula: see text] was higher in groups incubated at 5°C or 8°C through organogenesis, compared to 2°C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos.

Experimental exposure of juvenile chinook (Oncorhynchus tshawytscha) to bleached kraft mill effluent: hepatic CYP1A induction is correlated with DNA adducts but not with organochlorine residues.

Juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed for 28 days to 0-16% treated effluent from an elemental chlorine free (ECF) bleached kraft pulp mill. Fresh effluent was diluted with river water drawn from upstream of the effluent diffuser. Fish were tested for biochemical responses to identify if the effluent would cause significant effects at concentrations spanning those present in the Fraser River, BC, Canada during winter conditions. Hepatic ethoxyresorufin-O-deethylase (EROD) activity was increased significantly at all effluent concentrations and hepatic cytochrome P450 1A (CYP1A) protein was increased in all but 2% effluent. Hepatic DNA adduct concentrations were increased significantly at 8 and 16% effluent. These data indicate that there is a significant increase in all three responses at concentrations similar to those found in the receiving waters (4%) and that a dose-response relationship exists between BKME concentration and the responses measured. Carcasses contained low (< 0.2 pg g(-1)) concentrations of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Polychlorinated biphenyls (PCBs) were at higher concentrations, accounting for 77% of the total contaminant burden, expressed as TEQ, but contaminants do not appear to have bioaccumulated in this study. It is likely that the PCB concentrations were due to contaminated commercial fish feed. Bile did not contain detectable levels of polycyclic aromatic hydrocarbons (PAHs) as measured by synchronous fluorescence spectrometry.

Measuring the potency of pulp mill effluents for induction of hepatic mixed-function oxygenase activity in fish.

A bioassay protocol was optimized for measuring the potency of effluents or waterborne chemicals for inducing mixed-function oxygenase (MFO) activity of rainbow trout (Oncorhynchus mykiss). Measurements of ethoxyresorufin O-deethylase (EROD) can be made with an established endpoint assay using large volumes of reagents and tissue. However, a new kinetic microplate assay offers significant savings in time, reagents, and sample volumes. Data are distributed lognormally and must be log transformed before statistical analyses. EROD activity increases with exposure time to pulp mill effluent, and a 4-d exposure provides a near-maximal response. Optimum fish size conforms to standard practices in fish toxicology; loading rates should not exceed 1 g of fish per liter of test solution per day. Feed should be withheld from test fish 48 h before testing to reduce the variance of measured activity, and anaesthetizing fish with MS-222 does not affect their response to MFO inducers. Pulp mill effluents do not lose their potency during 2-3 wk of exposure at temperatures ranging from -20 to 13 degrees C, whether stored in plastic or glass. Steel containers were associated with slight losses in potency. Bioassays of MFO induction in fish exposed to liquid effluents are practical and conform to standard practice for testing the lethality of waterbone chemicals. The results are sufficiently precise that differences among means based on live fish per treatment can be discriminated statistically when activity changes by threefold or more.