Dietary yolk supplements and rate of yolk deposition do not influence sex ratios in Japanese Quail.

Birds appear to have the ability to bias offspring sex ratios in response to environmental and social conditions. The mechanisms responsible remain unknown, however one previous study indicated a link between rates of ovarian follicle growth and the sexes of resulting eggs. This could indicate that either follicles destined to retain male or female follicles grow at different rates, or the rate of ovarian follicle growth determines the sex chromosome that will be retained to influence the sex of the resulting offspring. We tested for evidence of both possibilities by staining yolk rings that are indicative of daily growth. First, we tested for a correlation between the number of yolk rings and the sexes of the resulting germinal discs collected from each egg, and second, we tested whether experimentally decreasing follicle growth rates with a dietary yolk supplement impacts the sexes of the resulting germinal discs. There was no significant correlation between the number of yolk rings and the sexes of the resulting embryos, and decreasing follicle growth rates did not impact sexes of resulting germinal discs. These results indicate that offspring sex is not related to the rate of ovarian follicle growth in quail.

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda).

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.

Differences in morphology and in composition and release of parotoid gland secretion in introduced cane toads (Rhinella marina) from established populations in Florida, USA.

Cane toads are highly toxic bufonids invasive in several locations throughout the world. Although physiological changes and effects on native predators for Australian populations have been well documented, Florida populations have received little attention. Cane toads were collected from populations spanning the invaded range in Florida to assess relative toxicity, through measuring morphological changes to parotoid glands, likelihood of secretion, and the marinobufagenin (MBG) content of secretion. We found that residual body indices increased in individuals from higher latitude populations, and relative parotoid gland size increased with increasing toad size. There was no effect of latitude on the allometric relationship between gland size and toad size. We observed an increase in likelihood of secretion by cane toads in the field with increasing latitude. Individuals from southern and northern populations did not vary significantly in the quantity of MBG contained in their secretion. Laboratory-acclimated cane toads receiving injections of epinephrine were more likely to secrete poison with increasing dose, although there was no difference in likelihood of secretion between southern and northern populations. This suggests that differences between populations in the quantities of epinephrine released in the field, due to altered hypothalamic sensitivity upon disturbance, may be responsible for the latitudinal effects on poison secretion. Our results suggest that altered pressures from northward establishment in Florida have affected sympathetic sensitivity and defensive mechanisms of cane toads, potentially affecting risk to native predators.

Stress and immunity: Field comparisons among populations of invasive cane toads in Florida.

Cane toads (Rhinella marina) were introduced worldwide and have become invasive in multiple locations, representing a major driver of biodiversity loss through competition (food, shelter, territory), predation, and the poisoning of native species. These toads have been used in Australia as a model for studies concerning invasion biology and ecoimmunology, as longer-established (core) and invasion front (edge) populations show altered stress and immune response profiles. Although cane toads were also introduced into the United States in the 1950s, these patterns have yet to be evaluated for the populations spanning Florida. Toads introduced into Florida have dispersed primarily northward along a latitudinal gradient, where they encounter cooler temperatures that may further impact stress and immune differences between core and edge populations. In this study, we sampled cane toads from nine different locations spanning their invasion in Florida. Cane toads from southern populations showed higher plasma bacterial killing ability and natural antibody titers than the toads from the northern populations, indicating they have a better immune surveillance system. Also, southern toads were more responsive to a novel stressor (1 hr restraint), showing a higher increase in corticosterone levels. These results indicate that possible trade-offs have occurred between immune and stress responses as these toads have become established in northern cooler areas in Florida.

Innate immunity of Florida cane toads: how dispersal has affected physiological responses to LPS.

Physiological tradeoffs occur in organisms coping with their environments, which are likely to increase as populations reach peripheries of established ranges. Invasive species offer opportunities to study tradeoffs that occur, with many hypotheses focusing on how immune responses vary during dispersal. The cane toad (Rhinella marina) is a well-known invasive species. Populations near the expanding edge of the Australian invasion have altered immune responses compared to toads from longer-established core populations, although this has not been well-documented for Florida populations. In this study, cane toads from a northern edge [New Port Richey (NPR)] and southern core (Miami) population in Florida were collected and injected with lipopolysaccharide (LPS) to compare immune responses. Core population individuals injected with LPS showed greater metabolic increases compared to their baseline rates that were higher compared to those from the edge population. In addition, LPS-injected core individuals had different circulating leukocyte profiles compared to saline-injected cane toads while edge individuals did not. There was a significant interaction between plasma bacteria-killing capability (BKA) and treatment, such that BKA decreased with time in saline compared to LPS-injected individuals, and saline-injected toads from the edge population had lower BKA compared to LPS-injected edge toads at 20 h post-injection. There was also a significant interaction between location and time on circulating corticosterone (CORT) levels following injections with saline or LPS, with CORT decreasing more with time in core population toads. The differential CORT response indicates that differential stress responses contribute to the tradeoffs observed with immunity and dispersal.

Differential gene expression to an LPS challenge in relation to exogenous corticosterone in the invasive cane toad (Rhinella marina).

The cane toad (Rhinella marina) is an invasive amphibian in several parts of the world. Much of the research performed on assessing the dispersal potential of invasive species has focused immunity. Invaders are predicted to rely less on pro-inflammatory immunity, allowing them to allocate energy to dispersal. Elevated stress may play a role in regulation of immune responses used by invasive species. RNA sequencing of spleen tissue from cane toads subjected to an acute LPS challenge revealed genes coding for cytokines involved in typical innate responses such as phagocytic cell recruitment, extravasation, inflammation, and lymphocyte differentiation were significantly upregulated, while toads receiving transdermal application of corticosterone in addition to an LPS injection showed downregulation of genes involved with cell mediated immunity. These results indicate hormonal changes associated with acute stress may alter investment into mounting cell-mediated or humoral responses while allowing for prolonged phagocytic innate responses in this invasive species.

Natural tissue concentrations in adult Ambystoma maculatum and larval DNA damage from exposure to arsenic and chromium.

Arsenic (As) and chromium (Cr) are two contaminants that are detected in aquatic and terrestrial habitats. Using the spotted salamander, Ambystoma maculatum, to assess impacts from these contaminants may be advantageous as adults live and breed in such environments. Adult amphibians typically exhibit elevated tissue concentrations of contaminants present in their environment, while larval stages were found to exhibit increased sensitivity to pollutants. From January through March of 2015, during the spring breeding season, 5 adults and approximately 32 egg masses were collected from a local breeding site. Field levels of As and Cr ranged from 5.99 to 8.88 µg/L and 1.45 to 2 µg/L, respectively, while mean adult As tissue concentrations were 56.74 µg/g dry weight for heart, 0.92 µg/g for liver, and 1.21 µg/g for tail tissue. Mean tissue concentrations for Cr were 87.64 µg/g for heart, 1.47 µg/g for liver, and 6.92 µg/g for tail. Developing larvae that were collected from the field and exposed in a lab setting for 12 d to 0.2 or 20 mg/L of either As or Cr displayed little DNA damage attributed to As, but marked damage due to exposure to 20 mg/L Cr when assessed using the comet assay. Exposure to a mixture of either 0.25:0.1 or 25:10 mg/L As and Cr resulted in significant DNA damage at the lower concentration of 0.25:0.1 mg/L. As adult spotted salamanders were found to possess high concentrations of these contaminants in cardiac tissue, and larvae were shown to be susceptible to DNA damage from increased exposures, assessing impacts and potential declines of amphibian populations exposed to As and Cr is needed.

Developmental and interactive effects of arsenic and chromium to developing Ambystoma maculatum embryos: Toxicity, teratogenicity, and whole-body concentrations.

Anthropogenic activity has contributed to elevated environmental concentrations of arsenic (As) and chromium (Cr). The spotted salamander, Ambystoma maculatum, may be useful for identifying developmental effects produced by exposure to these contaminants as adults breed and larvae develop in water that may contain As or Cr. Three sample sets among 700 developing larvae were exposed to a range of As, Cr, or 2.5:1 mixture of As:Cr concentrations, respectively. From these 700 larvae, samples containing approximately 24 larvae showed different patterns of whole-body As and Cr from individual and mixture exposure. Whole-body As concentrations were 20.27 and 45.4 µg/g dry weight for larvae exposed to 20 mg/L As and 25:10 mg/L As:Cr, respectively, while whole-body Cr concentrations were 24.8 and 22 µg/g dry weight for larvae exposed to 20 mg/L Cr and 25:10 As:Cr, respectively. Observed malformations included edema, tail kinking, facial deformities, and abnormal bending. Twelve-day lethal concentrations for As and Cr in Ambystoma maculatum larvae were 261.17 mg/L and 71.93 mg/L, respectively, while 12-d effective concentrations to induce malformations were 158.82 and 26.05 mg/L, giving teratogenic indices of 1.64 and 2.76 for individual metal exposure. Exposure to a mixture of As and Cr resulted in a response addition and yielded lower lethal and effective concentration values with a teratogenic index of 2.78, indicating that these contaminants are developmentally toxic at lower concentrations when exposed as a mixture. Data demonstrate that As and Cr affect development of amphibian larvae, and that Ambystoma maculatum may be a useful indicator of environmental toxicity for these metals.

Assessing differences in toxicity and teratogenicity of three phthalates, Diethyl phthalate, Di-n-propyl phthalate, and Di-n-butyl phthalate, using Xenopus laevis embryos.

Phthalates, compounds used to add flexibility to plastics, are ubiquitous in the environment. In particular, the diethyl (DEP), di-n-propyl (DnPP), and di-n-butyl (DBP) phthalates were found to exert detrimental effects in both mammalian and non-mammalian studies, with toxic effects varying according to alkyl chain length. Embryos of Xenopus laevis, the African clawed frog, have been used to assess toxicity and teratogenicity of several compounds and serves as a model for assessing adverse and teratogenic effects of ortho-phthalate esters. The purpose of this study was to develop a model for comparison of developmentally toxic effects of ortho-phthalate esters using Xenopus embryos. In this study developing Xenopus laevis embryos were exposed to increasing concentrations of DEP, DnPP, and DBP using the 96-h Frog Embryo Teratogenesis Assay-Xenopus (FETAX), with 96-h lethal concentrations, effective concentrations to induce malformations, teratogenic indices, and concentrations to inhibit growth determined. DEP, DnPP, and DBP showed enhanced toxicity with increasing ester length. Developing Xenopus laevis exposed to DEP, DnPP, and DBP showed similar malformations that also occurred at lower concentrations with increasing alkyl chain length. Teratogenic risk did not change markedly with alkyl chain length, with data showing only DBP to be teratogenic.

Measuring the Refractive Index of Bovine Corneal Stromal Cells Using Quantitative Phase Imaging.

The cornea is the primary refractive lens in the eye and transmits >90% of incident visible light. It has been suggested that the development of postoperative corneal haze could be due to an increase in light scattering from activated corneal stromal cells. Quiescent keratocytes are thought to produce crystallins that match the refractive index of their cytoplasm to the surrounding extracellular material, reducing the amount of light scattering. To test this, we measured the refractive index (RI) of bovine corneal stromal cells, using quantitative phase imaging of live cells in vitro, together with confocal microscopy. The RI of quiescent keratocytes (RI = 1.381 ± 0.004) matched the surrounding matrix, thus supporting the hypothesis that keratocyte cytoplasm does not scatter light in the normal cornea. We also observed that the RI drops after keratocyte activation (RI = 1.365 ± 0.003), leading to a mismatch with the surrounding intercellular matrix. Theoretical scattering models showed that this mismatch would reduce light transmission in the cornea. We conclude that corneal transparency depends on the matching of refractive indices between quiescent keratocytes and the surrounding tissue, and that after surgery or wounding, the resulting RI mismatch between the activated cells and their surrounds significantly contributes to light scattering.

Correction: Quantification of Collagen Ultrastructure after Penetrating Keratoplasty - Implications for Corneal Biomechanics.

[This corrects the article on p. e68166 in vol. 8.].

Quantification of collagen ultrastructure after penetrating keratoplasty - implications for corneal biomechanics.

PURPOSE: To quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK), and evaluate their possible implications for corneal biomechanics. METHODS: A pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left)/28 (right) years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal)×0.25 mm (vertical) intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas. RESULTS: Marked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident. CONCLUSIONS: Abnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the long term.

Efficacy of intraoperative cell salvage systems in pediatric idiopathic scoliosis patients undergoing posterior spinal fusion with segmental spinal instrumentation.

STUDY DESIGN: Retrospective case-control study. OBJECTIVE: Determine whether intraoperative cell salvage system use during pediatric posterior spinal fusion (PSF) with segmental spinal instrumentation for idiopathic scoliosis decreases intraoperative and perioperative (intraoperative plus postoperative) allogeneic blood transfusion. SUMMARY OF BACKGROUND DATA: Intraoperative cell salvage and reinfusion can reduce or obviate perioperative allogeneic blood transfusion. Despite these benefits, their efficacy in pediatric PSF is unclear. Reported complications include transient hematuria, altered hemostasis, and electrolyte imbalance. METHODS: A total of 54 consecutive idiopathic scoliosis patients were studied: 21 non-cell saver and 33 cell saver patients. Data included age, body mass index, Cobb angle, perioperative hemoglobin levels, mean arterial pressure, surgical time, levels fused, perioperative estimated blood loss, and perioperative transfusions. A chi2 and t tests were performed for intraoperative and perioperative allogeneic transfusion between groups. A regression analysis was performed between selected covariates and allogeneic transfusion. Relative risk analysis examined significant covariates regarding allogeneic transfusion rate. RESULTS: Allogeneic transfusion rates were lower in the cell saver group (6% vs. 55% intraoperative and 18% vs. 55% perioperative, P < 0.05). Mean allogeneic transfusion volumes (mL/kg) were also lower (0.4 vs. 9.1 intraoperative and 1.9 vs. 11.1 perioperative, P < 0.05). Multivariate analysis confirmed these differences were independent of perioperative blood loss, and also demonstrated that surgical time and blood loss were significantly related to allogeneic transfusion volume. The allogeneic transfusion relative risk was 2.04 in patients with surgery >6 hours and 5.87 in patients not receiving cell saver blood. All patients with surgeries >6 hours and estimated blood loss >30% of total blood volume received cell saver system blood. CONCLUSION: Cell saver use decreased allogeneic transfusion, particularly in surgeries >6 hours with estimated blood loss >30% of total blood volume. This study confirms the utility of routine cell saver use during PSF with segmental spinal instrumentation for idiopathic scoliosis.

XPS study of molybdenum sulfide catalyst exposed to CO and H2.

Amorphous MoS(3) (air-dried precipitate), crystalline MoS(2) (made in the laboratory under nitrogenous atmosphere), and commercial molybdenum disulfide catalysts have been investigated by X-ray photoelectron spectroscopy (XPS). The chemical species present on the surface and the S/Mo atomic ratios of laboratory catalysts were compared to those present on a commercial molybdenum disulfide catalyst before and after treatment with CO and H(2). Pretreatment of MoS(2) with CO followed by H(2) showed that CO was adsorbed on the surface with no change in the chemical oxidation state of Mo and S. However, the results indicated that CO was not adsorbed when the sequence of gas exposures was reversed.