Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

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.     

Fine-Scale Ecological and Genetic Population Structure of Two Whitefish (Coregoninae) Species in the Vicinity of Industrial Thermal Emissions

Thermal pollution from industrial processes can have negative impacts on the spawning and development of cold-water fish. Point sources of thermal effluent may need to be managed to avoid affecting discrete populations. Correspondingly, we examined fine-scale ecological and genetic population structure of two whitefish species (Coregonus clupeaformis and Prosopium cylindraceum) on Lake Huron, Canada, in the immediate vicinity of thermal effluent from nuclear power generation. Niche metrics using δ¹³C and δ¹⁵N stable isotopes showed high levels of overlap (48.6 to 94.5%) in resource use by adult fish captured in areas affected by thermal effluent compared to nearby reference locations. Isotopic niche size, a metric of resource use diversity, was 1.3- to 2.8-fold higher than reference values in some thermally affected areas, indicative of fish mixing. Microsatellite analyses of genetic population structure (F_st, STRUCTURE and DAPC) indicated that fish captured at all locations in the vicinity of the power plant were part of a larger population extending beyond the study area. In concert, ecological and genetic markers do not support the presence of an evolutionarily significant unit in the vicinity of the power plant. Thus, future research should focus on the potential impacts of thermal emissions on development and recruitment.     

Variable effects of goitrogens in inducing precocious metamorphosis in sea lampreys (Petromyzon marinus)

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO(4)], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO(4)]) are anionic competitors of iodide uptake. Because, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO(4), NaClO(4), and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO(4) treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event.     

Gender differences in glucoregulatory responses to intense exercise.

We compared glucoregulatory responses to intense exercise (14 min at 88% maximum O(2) uptake) between genders (16 men, 12 women). Analysis of covariance of maximum O(2) uptake showed no gender effect, with 82% of variance due to fat-free mass (FFM). Glycemia rose comparably during exercise but was higher in women during recovery (P = 0.02). Glucose production [rate of appearance (R(a)); in mg/min] increased markedly in both; stepwise multiple regression and analysis of covariance of R(a) (peak and incremental area under the curve) showed no effect of gender, body weight, or FFM. Glucose uptake [rate of disappearance (R(d))] increased less than R(a) and slower in women. R(d) area under the curve related to FFM (P = 0.01) but not gender or body weight. Norepinephrine and epinephrine responses (13-18x baseline) were the same and correlated significantly with R(a). Exercise insulin and glucagon changes were slight, but postexercise hyperinsulinemia was greater in women (P = 0.018), along with higher R(d). Therefore, intense exercise glucoregulation is qualitatively similar between genders, with a "feed-forward" regulation of R(a) (consistent with catecholamine mediation). However, women have a lesser R(d) response, related to FFM. This combination leads to greater recovery-period hyperglycemia and hyperinsulinemia.