Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

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Mammalian cytochrome P4501 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2 and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.

The timing, success and energetics of fish embryonic development are strongly influenced by temperature. However, it is unclear if there are developmental periods, or critical windows, when oxygen use, survival and hatchling phenotypic characteristics are particularly influenced by changes in the thermal environment. Therefore, we examined the effects of constant incubation temperature and thermal shifts on survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis) embryos. We incubated whitefish embryos at control temperatures of 2, 5, or 8 °C, and shifted embryos across these three temperatures at the end of gastrulation or organogenesis. We assessed hatch timing, mass at hatch, and yolk conversion efficiency (YCE). We determined cost of development, the amount of oxygen required to build a unit of mass, for the periods from fertilization-organogenesis, organogenesis-fin flutter, fin flutter-hatch, and for total development. An increase in incubation temperature decreased time to 50 % hatch (164 days at 2 °C, 104 days at 5 °C, and 63 days at 8 °C), survival decreased from 55 % at 2 °C, to 38 % at 5 °C, and 17 % at 8 °C, and hatchling yolk-free dry mass decreased from 1.27 mg at 2 °C to 0.61 mg at 8 °C. Thermal shifts altered time to 50 % hatch and hatchling yolk-free dry mass and revealed a critical window during gastrulation in which a temperature change reduced survival. YCE decreased and cost of development increased with increased incubation temperature, but embryos that hatched at 8 °C and were incubated at colder temperatures during fertilization-organogenesis had reduced cost. The relationship between cost of development and temperature was altered during fin flutter-hatch, indicating it may be a critical window during which temperature has the greatest impact on energetic processes. The increase in cost of development with an increase in temperature has not been documented in other fishes and suggests whitefish embryos are more energy efficient at colder temperatures.

Cytochrome P4501 (CYP1) and CYP3A proteins are primarily responsible for the metabolism of 17b-estradiol (E 2 ) in mammals. We have cloned and heterologously expressed CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP1D1, and CYP3A65 from zebrafish (Danio rerio) to determine the CYP-mediated metabolism of E 2 in a non-mammalian species. Constructs of each CYP cDNA were created using a leader sequence from the bacterial ompA gene to allow appropriate expression in Escherichia coli without 5 0 modification of the gene. Membrane vesicles were purified, and functional CYP protein was verified using carbon monoxide difference spectra and fluorescent catalytic assays with the substrates 7-ethoxyresorufin and 7-benzyloxy-4-(trifluoromethyl)-coumarin. Rates of in vitro E 2 metabolism into 4-hydroxyE 2 (4-OHE 2 ), 2-hydroxyE 2 (2-OHE 2 ), and 16a-hydroxyE 1 (16a-OHE 1 ) metabolites were determined by gas chromatography/mass spectrometry. The 2-OHE 2 metabolite was produced by all CYPs tested, while 4-OHE 2 was only detected following incubation with CYP1A, CYP1B1, CYP1C1, and CYP1C2. The 16a-OHE 1 metabolite was only produced by CYP1A. The highest rates of E 2 metabolism were from CYP1A and CYP1C1, followed by CYP1C2. CYP1B1, CYP1D1, and CYP3A65 had low rates of E 2 metabolism. E 2 metabolism by zebrafish CYP1A, CYP1C1, and CYP1C2 produced similar ratios of 4-OHE 2 to 2-OHE 2 as previous studies with mammalian CYP1As. CYP1B1 formed the highest ratio of 4-OHE 2 to 2-OHE 2 metabolites. Contrary to mammals, these results suggest that fish CYP1A and CYP1C proteins are primarily responsible for E 2 metabolism, with only minor contributions from CYP3A65 and CYP1B1. Similar to mammals, 2-OHE 2 is the predominant metabolite from CYP-mediated E 2 metabolism in fish, suggesting that all vertebrate species produce the same major E 2 metabolite.

The development of diagnostic markers has been a long-standing interest of population geneticists as it allows clarification of taxonomic uncertainties. Historically, there has been much debate on the taxonomic status of species belonging to the Mytilus species complex (M. edulis, M. galloprovincialis and M. trossulus), and whether they are discrete species. We analysed reference pure specimens of M. edulis, M. galloprovincialis and M. trossulus, using Restriction site associated DNA (RAD) sequencing and identified over 6,000 SNP markers separating the three species unambiguously. We developed a panel of diagnostic SNP markers for the genotyping of Mytilus species complex as well as the identification of hybrids and interspecies introgression events in Mytilus species. We validated a panel of twelve diagnostic SNP markers which can be used for species genotyping. Being able to accurately identify species and hybrids within the Mytilus species complex is important for the selective mussel stock management, the exclusion of invasive species, basic physiology and bio-diversity studies.

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