Dynamics underlie the drug recognition mechanism by the efflux transporter EmrE.

The multidrug efflux transporter EmrE from Escherichia coli requires anionic residues in the substrate binding pocket for coupling drug transport with the proton motive force. Here, we show how protonation of a single membrane embedded glutamate residue (Glu14) within the homodimer of EmrE modulates the structure and dynamics in an allosteric manner using NMR spectroscopy. The structure of EmrE in the Glu14 protonated state displays a partially occluded conformation that is inaccessible for drug binding by the presence of aromatic residues in the binding pocket. Deprotonation of a single Glu14 residue in one monomer induces an equilibrium shift toward the open state by altering its side chain position and that of a nearby tryptophan residue. This structural change promotes an open conformation that facilitates drug binding through a conformational selection mechanism and increases the binding affinity by approximately 2000-fold. The prevalence of proton-coupled exchange in efflux systems suggests a mechanism that may be shared in other antiporters where acid/base chemistry modulates access of drugs to the substrate binding pocket.

Acid-base and hematological regulation in chicken embryos during internal progressive hypercapnic hypoxia.

Development of the capacity to mitigate potential disturbances to blood physiology in bird embryos is incompletely understood. We investigated regulation of acid-base and hematology in day 15 chicken embryos exposed to graded intrinsic hypercapnic hypoxia created by varying degrees of water submersion. Metabolic acidosis with additional respiratory or metabolic acidosis occurred at 2 h according to magnitude of submersion. Acid-base disturbance was partially compensated by metabolic alkalosis at 6 h, but compensatory metabolic alkalosis was absent at 24 h. Following submersion with only air cell exposed to air, both hypercapnic respiratory acidosis and metabolic acidosis occurred within 10 min. Subsequently, both forms of acidosis created lethal levels of [HCO3-] at ∼120 min. Blood hematology showed small but significant effects associated with induced acid-base disturbance. Increased Hct occurring during partial egg submersion lasting 24 h was attributed to an increase in MCV. By day 15 of development chicken embryos are able to partially compensate for and withstand all but severe induced internal hypoxic hypercapnia.

Pectoralis Major Tendon Tears During Airborne Operations: Are These Injuries Isolated?

INTRODUCTION: Pectoralis major tendon tears are an injury pattern often treated in military populations. Although the majority of pectoralis major tendon tears occur during eccentric loading as in bench press weightlifting, military service members may also experience this injury from a blunt injury and traction force produced by static line entanglement during airborne operations. Although these injuries rarely occur in isolation, associated injury patterns have not been investigated previously. MATERIALS AND METHODS: After obtaining institutional review board approval, medical records were reviewed for all patients who underwent surgical repair of a pectoralis major tendon tear sustained during static line parachuting at a single institution. Radiology imaging, operative notes, and outpatient medical records were examined to determine concomitant injury patterns for each patient identified over a 4-year study period. RESULTS: Twenty-five service members met the study inclusion criteria. All patients underwent presurgical magnetic resonance imaging. Of these 25 service members, 10 (40%) presented with a total of 13 concomitant injuries identified on physical exams or imaging studies. The most common associated injuries were injuries to the biceps brachii and a partial tear of the anterior deltoid. Biceps brachii injuries consisted of muscle contusion proximal long head tendon rupture, proximal short head tendon rupture, partial muscle laceration, and complete muscle transection. Additional concomitant injuries included transection of coracobrachialis, a partial tear of the inferior subscapularis tendon, antecubital fossa laceration, an avulsion fracture of the sublime tubercle, and an avulsion fracture of the coracoid process. CONCLUSIONS: Military static line airborne operations pose a unique risk of pectoralis major tendon tear. Unlike the more common bench press weightlifting tear mechanism, pectoralis major tendon tears associated with static line mechanism present with a concomitant injury in 40% of cases, with the most common associated injury occurring about the biceps brachii. Treating providers should have a high index of suspicion for concomitant injuries when treating pectoralis major tendon tears from this specific mechanism of injury.

Understanding Noise Exposure During Cast Removal: The Effect of Cast Saw Type and Casting Material.

BACKGROUND: Cast removal can be associated with considerable noise exposure, especially impacting the pediatric patient and provider. Although noise generation from cast saws has been deemed safe by the U.S. Occupational Safety and Health Administration and the U.S. Centers for Disease Control and Prevention standards, there are no current studies on the effects of cast material on noise levels generated. METHODS: A simulated casting model utilizing plaster, fiberglass, and plaster with fiberglass overwrapping was used for experimental testing. Four different casting conditions were tested, with 5 samples in each group. Samples were tested using 2 different cast saws: a standard cast saw and a quiet saw. Each saw was used for 30 seconds of continuous cutting for each sample, measuring peak, mean, and minimum sound levels in decibels with sound level meters. Noise levels were measured at 18, 36, and 72 in (20, 91, and 183 cm) from the saw, comparing saw and cast types against ambient noise and baseline cast-saw noises. Between-group comparisons were performed using univariate analyses. RESULTS: Mean noise generation differed between casting materials, with plaster material demonstrating significantly greater noise levels than fiberglass casts at all distances for each saw type. Increasing fiberglass thickness significantly increased the mean noise levels with standard (18-in distance for 10 and 5 ply: 87.4 and 85.8 dB; p = 0.0004) and quiet cast saws (78.3 and 76.1 dB; p = 0.041. Additionally, the quiet cast saw provided a 5.7 to 10.6 dB reduction in mean and peak noise levels, varying by casting material and distance. CONCLUSIONS: Occupational noise exposure can be mitigated with the use of fiberglass casting material that is not >5 ply in thickness, with a quiet cast saw for removal. The use of a quiet cast saw substantially decreased noise exposure to patients and staff members over standard orthopaedic cast saws.

Effect of increased embryonic temperature during developmental windows on survival, morphology and oxygen consumption of rainbow trout (Oncorhynchus mykiss).

Temperature is a crucial environmental factor that influences physiological functions in fishes, and increased temperature during development can shape an organism's phenotype. An active line of inquiry in comparative developmental physiology is whether short-term exposure to thermal changes have lasting phenotypic effects. This is the first study to apply a developmental 3-dimensional critical window experimental design for a vertebrate, using time, temperature, and phenotypic response (i.e., variables measured). Rainbow trout (Oncorhynchus mykiss) are an anadromous species for which resident populations occupy freshwater environments that are likely impacted by variable and rising temperatures, particularly during embryonic development. To assess thermal effects on fish development, we examined trout hatchling phenotypes following rearing in constant temperatures (5, 10, 15 and 17.5 °C) and following exposure to increased temperature above 5 °C during specific developmental windows. Time to 50% hatch, hatchling mass and body length showed general trends of decreasing with increasing constant temperature, and survival was highest in constant 10 °C incubation. Thermally shifting embryos into 17.5 °C during gastrulation and organogenesis reduced survival at hatch compared to 10 °C, and exposure to 15 and 17.5 °C only late in development produced lighter and shorter hatchlings. Oxygen consumption rate (V̇o2) at organogenesis differed between embryos incubated constantly in increased temperature or exposed only during organogenesis, but generally we found limited temperature effects on V̇o2 that may be due to high data variability. Collectively, these results suggest that survival of rainbow trout hatchlings is most sensitive to 17.5 °C exposure during gastrulation and organogenesis, while warm water exposure later in development has greater impacts on morphology. Thus, trait-specific critical windows of thermal sensitivity exist for rainbow trout embryos that alter the hatching phenotype.

Temperature tolerance and oxygen consumption of two South American tetras, Paracheirodon innessi and Hyphessobrycon herbertaxelrodi.

Temperature is a primary factor affecting species' ability to thrive in a particular ecological niche, but thermal conditions have changed dramatically in recent decades. Fishes shift their thermal tolerance range with a maximum and minimum temperature correlated to their recent thermal acclimation history, and species can show a reduced temperature quotient (Q10) following chronic thermal acclimation. Neon tetra (Paracheirodon innesi) and Black Neon tetra (Hyphessobrycon herbertaxelrodi) are popular hobbyist aquarium fishes, and both species are examples of freshwater teleosts native to South American river systems that are potentially affected by changing thermal conditions. We acclimated these species to three different constant temperatures (26 °C, 29 °C, and 31 °C) for 15.4 ±â€¯2.1 days, then measured acute critical thermal maxima (CTMax) and acute oxygen consumption rate (Mo2) at each acclimation temperature. We also estimated chronic lethal thermal maximum (CLT) for both species following a 2-week acclimation to 30.4 °C. Mean CTMax of both species were found to increase with acclimation temperature from 38.5 to 39.6 °C for Neon tetra and from 39.5 to 41.0 °C for Black Neon tetra, gaining 0.24 (Neon tetra) or 0.29 °C (Black Neon tetra) of tolerance per 1 °C of acclimation. However, Black Neon tetra demonstrated consistently higher CTMax (1.0-1.4 °C). CLT was lower for Neon tetra (33.5 °C), compared to Black Neon tetra (35.9 °C). Mean Mo2 were statistically similar across acclimation temperatures within species; Q10 between 26-31 °C were 1.92 and 1.22 for Neon and Black Neon tetra, respectively. Neon and Black Neon tetras physiologically acclimated to changing thermal demands, and although they demonstrate robust CTMax responses, CLT responses indicated both species are unable to survive temperatures 4-5 °C above current average natural values. The demonstrated metabolic plasticity and CTMax values provide a moderate cushion for both species to combat changing temperatures due to climate change, but CLT values suggest vulnerability to projected climate trends.

Immediate and Persistent Effects of Temperature on Oxygen Consumption and Thermal Tolerance in Embryos and Larvae of the Baja California Chorus Frog, Pseudacris hypochondriaca.

The developmental environment has significant immediate effects on phenotypes, but it may also persistently or permanently shape phenotypes across life history. This study examined how developmental temperature influenced embryonic and larval phenotypes of Baja California chorus frog (Pseudacris hypochondriaca), an abundant amphibian in southern California and northern Baja California. We collected egg clutches from native ponds in northern San Diego County within 24 h of fertilization, and clutches were separated and distributed between constant temperatures of 10, 15, 20, and 25°C for incubation. Oxygen consumption rate ( V ˙ O2), developmental stage, and embryo and yolk masses were measured throughout development. Time to 50% hatch, survival at 50% hatch, and hatch duration were determined. Development rate was strongly affected by temperature, with warmer temperatures reducing time to hatch and hatch duration. Survival to hatch was high across all temperatures, >90%. Mass-specific V ˙ O2 of embryos either remained constant or increased throughout development, and by hatching energy demand was significantly increased at higher temperatures. There were limited temperature effects on growth, with embryo and yolk dry mass similar between temperatures throughout embryonic development. To examine long-term effects of embryonic temperature, we reared hatchlings from each temperature until onset of larval feeding. Once feeding, larvae were acclimated to 20 or 25°C (>2 weeks). Following acclimation to 20 or 25°C, we measured larval mass-specific V ˙ O2 and critical thermal maximum (CTMax) at a common developmental stage (Gosner stages 32-36, "hindlimb toe differentiation"). Embryonic temperature had persistent effects on larval V ˙ O2 and CTMax, with warmer temperatures generally resulting in similar or higher V ˙ O2 and CTMax. This partially supported a "warmer is better" effect of embryonic incubation temperature. These results suggest that in a thermally robust amphibian species, temperature may program the phenotype during early development to construct traits in thermal tolerance and energy use that may persist. Overall, P. hypochondriaca displays a thermally robust phenotype, and it is possible that amphibians that possess a wider range of phenotypic plasticity will be relatively more successful mitigating effects of climate change.

Rapid metabolic compensation in response to temperature change in the intertidal copepod, Tigriopus californicus.

Animals living in the intertidal zone must adapt to thermal variability, including adjustments in metabolism. We examined metabolic responses to temperature in the copepod, Tigriopus californicus, which inhabits supratidal splash pools along the Pacific coast of North America. We maintained three populations of T. californicus at 20 °C, one from southern California (San Diego, "SD") and two from Oregon (Fogarty Creek, "FCN", Boiler Bay, "BOB") and examined possible geographic patterns in metabolism. We measured oxygen consumption rate (V̇o2) at 20 °C and following 48 h (chronic) acclimation to 25, 27.5 and 30 °C. V̇o2 was temperature-independent, with temperature quotients (Q10) values ≤1 in all populations, indicative of metabolic compensation. We detected no variation in V̇o2 or survival between populations. To explore the time course of metabolic compensation, we performed an acute acclimation experiment in which V̇o2 was measured at 20 °C, following immediate exposure to 25 °C, and following 2 h, 4 h and 6 h exposure to 25 °C. This acute acclimation experiment revealed that V̇o2 increased immediately in SD and FCN, but was no longer different than 20 °C levels by 2 h and 4 h at 25 °C, respectively. BOB showed no significant change in V̇o2, which may indicate complete temperature-independent metabolism or different mechanisms of compensation between populations. This study demonstrates a time course of rapid metabolic compensation in response to temperature that occurs in a small intertidal animal, and suggests intertidal invertebrates can thermally acclimate within a few hours of a significant temperature change.

Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) utilize overwintering embryonic development (up to 180 days), and such stenothermic, cold-water embryos may be particularly susceptible to thermal shifts. We incubated whitefish embryos in temperature treatments that were constant temperature (2.0 ±â€¯0.1 °C, 5.0 ±â€¯0.1 °C, and 8.0 ±â€¯0.1 °C; mean ±â€¯SD) or variable temperature (VT, mean = 5.0 ±â€¯0.3 °C). In the VT, a daily 2 °C temperature change followed a continuous pattern throughout development: 2-4-6-8-6-4-2 °C. Hatchling survival proportion from fertilization to hatch was significantly impacted by incubation temperature (P < 0.001): 2 °C (0.88 ±â€¯0.01) and 5 °C (0.91 ±â€¯0.01) showed higher survival than both the VT (0.83 ±â€¯0.02) and 8 °C groups (0.15 ±â€¯0.06), which were statistically distinct from each other. Time to hatch (dpf) was significantly different across all treatments (P < 0.001): 8 °C (68 ±â€¯2 dpf), VT (111 ±â€¯4 dpf), 5 °C (116 ±â€¯4 dpf), 2 °C (170 ±â€¯3 dpf). Likewise, hatchling yolk-free dry mass (mg) and total body length (mm) were significantly different across all treatments (P < 0.001): 8 °C (0.66 ±â€¯0.08 mg; 11.1 ±â€¯0.08 mm), VT (0.97 ±â€¯0.06 mg; 11.7 ±â€¯0.05 mm), 5 °C (1.07 ±â€¯0.03 mg; 12.0 ±â€¯0.02 mm), 2 °C (1.36 ±â€¯0.04 mg; 12.8 ±â€¯0.05 mm). Oxygen consumption rate (V̇o2) was significantly affected by the interaction between treatment and measurement temperature (P < 0.001). Hatchling VT whitefish showed mean V̇o2 that was higher compared to the 2 °C group measured at 2 °C, and lower compared to the 2 °C and 5 °C group measured at 8 °C. This study demonstrates that the VT incubation treatment produced fewer (increased mortality), smaller embryos that hatched earlier than 2 °C and 5 °C embryos. The plasticity of V̇o2 for this stenothermic-incubating fish species under variable incubation conditions reveals a metabolic cost to cycling thermal incubation conditions.

Chronic captopril treatment reveals the role of ANG II in cardiovascular function of embryonic American alligators (Alligator mississippiensis).

Angiotensin II (ANG II) is a powerful vasoconstrictor of the renin-angiotensin system (RAS) that plays an important role in cardiovascular regulation in adult and developing vertebrates. Knowledge of ANG II's contribution to developmental cardiovascular function comes from studies in fetal mammals and embryonic chickens. This is the first study to examine the role of ANG II in cardiovascular control in an embryonic reptile, the American alligator (Alligator mississippiensis). Using chronic low (~ 5-mg kg embryo-1), or high doses (~ 450-mg kg embryo-1) of captopril, an angiotensin-converting enzyme (ACE) inhibitor, we disrupted the RAS and examined the influence of ANG II in cardiovascular function at 90% of embryonic development. Compared to embryos injected with saline, mean arterial pressure (MAP) was significantly reduced by 41 and 72% under low- and high-dose captopril treatments, respectively, a greater decrease in MAP than observed in other developing vertebrates following ACE inhibition. Acute exogenous ANG II injection produced a stronger hypertensive response in low-dose captopril-treated embryos compared to saline injection embryos. However, ACE inhibition with the low dose of captopril did not change adrenergic tone, and the ANG II response did not include an α-adrenergic component. Despite decreased MAP that caused a left shifted baroreflex curve for low-dose captopril embryos, ANG II did not influence baroreflex sensitivity. This study demonstrates that ANG II contributes to cardiovascular function in a developing reptile, and that the RAS contributes to arterial blood pressure maintenance during development across multiple vertebrate groups.

Dynamics of acid-base and hematological regulation in day 15 chicken embryos (Gallus gallus domesticus) exposed to graded hypercapnia and hypoxia.

Most experiments examining acid-base regulation of chicken embryos have employed static, single time point measurements rather than dynamic, multiple time point measurements that might reveal additional components of developing acid-base regulation. Thus, we studied blood acid-base balance and hematology of day 15 chicken embryos under 24h exposure to graded hypercapnia (1%-7% CO2) accompanied by graded hypoxia (20% O2 down to 13% O2). Across all hypercapnic/hypoxic environments, respiratory acidosis occurred 2h after exposure in proportion to the magnitude of hypercapnia. An additional metabolic alkalosis occurred in ≥16% O2, and metabolic acidosis in ≤14% O2. As exposure progressed, compensatory metabolic alkalosis occurred in all groups at 6h, but partial metabolic compensation could not be preserved as hypoxia increased (≤18% O2). Across all hypercapnic/hypoxic groups, hematocrit, mean corpuscular volume and red blood cell concentration significantly increased by 24h, most likely due to hypoxia rather than hypercapnia. Overall, day 15 chicken embryos cannot maintain even partial compensation in their acid-base physiology after 24h exposure to hypercapnic/hypoxic environments of ≥5% CO2+≤15% O2.

Shoulder Pain-Is It From the Shoulder, Neck, or Both?

A thorough history and physical examination, revisited on subsequent follow-up, was necessary to properly diagnose pain emanating from both the shoulder and upper arm.

Lipid content and fatty acid profile during lake whitefish embryonic development at different incubation temperatures.

Lipids serve as energy sources, structural components, and signaling molecules during fish embryonic development, and utilization of lipids may vary with temperature. Embryonic energy utilization under different temperatures is an important area of research in light of the changing global climate. Therefore, we examined percent lipid content and fatty acid profiles of lake whitefish (Coregonus clupeaformis) throughout embryonic development at three incubation temperatures. We sampled fertilized eggs and embryos at gastrulation, eyed and fin flutter stages following chronic incubation at temperatures of 1.8, 4.9 and 8.0°C. Hatchlings were also sampled following incubation at temperatures of 3.3, 4.9 and 8.0°C. Fertilized eggs had an initial high percentage of dry mass composed of lipid (percent lipid content; ~29%) consisting of ~20% saturated fatty acids (SFA), ~32% monounsaturated fatty acids (MUFA), ~44% polyunsaturated fatty acids (PUFA), and 4% unidentified. The most abundant fatty acids were 16:0, 16:1, 18:1(n-9c), 20:4(n-6), 20:5(n-3) and 22:6(n-3). This lipid profile matches that of other cold-water fish species. Percent lipid content increased during embryonic development, suggesting protein or other yolk components were preferentially used for energy. Total percentage of MUFA decreased during development, which indicated MUFA were the primary lipid catabolized for energy during embryonic development. Total percentage of PUFA increased during development, driven largely by an increase in 22:6(n-3). Temperature did not influence percent lipid content or percent MUFA at any development stage, and had inconsistent effects on percent SFA and percent PUFA during development. Thus, lake whitefish embryos appear to be highly adapted to low temperatures, and do not alter lipids in response to temperature within their natural incubation conditions.

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos.

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2°C, 5°C or 8°C water) and weekly, 1-h heat shocks (+3°C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148d at 2°C, 92d at 5°C, 50d at 8°C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3°C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8°C and 5°C embryos were significantly smaller and had larger yolks than 2°C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.

Salt sensitivity of the morphometry of Artemia franciscana during development: a demonstration of 3D critical windows.

A 3D conceptual framework of 'critical windows' was used to examine whether the morphometry of Artemia franciscana is altered by salinity exposure during certain key periods of development. Artemia franciscana were hatched at 20 ppt (designated control salinity) and were then exposed to 10, 30, 40 or 50 ppt either chronically (days 1-15) or only on days 1-6, 7-9, 10-12 or 13-15. On day 15, maturity was assessed and morphometric characteristics, including mass, total body length, tail length and width, length of the third swimming appendage and eye diameter, were measured. Maturation and morphometry on day 15 were influenced by the exposure window and salinity dose. Artemia franciscana were generally larger following exposure to 10 and 40 ppt during days 1-6 and 7-9 when compared with days 10-12 and 13-15, in part due to a higher percentage of mature individuals. Exposure to different salinities on days 1-6 produced the greatest differences in morphometry, and thus this appears to be a period in development when A. franciscana is particularly sensitive to salinity. Viewing the developmental window as three-dimensional allowed more effective visualization of the complex interactions between exposure window, stressor dose and the magnitude of morphometric changes in A. franciscana.

Solution Structure of CCL19 and Identification of Overlapping CCR7 and PSGL-1 Binding Sites.

CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1's enhancement of resting T-cell recruitment are discussed.

Hypercapnic thresholds for embryonic acid-base metabolic compensation and hematological regulation during CO2 challenges in layer and broiler chicken strains.

Time specific acid-base metabolic compensation and responses of hematological respiratory variables were measured in day 15 layer (Hyline) and broiler (Cornish Rock) chicken embryos during acute hypercapnic challenges (3, 6, 10 and 20% CO2). Control acid-base status and hematology differed between two strains. Broiler embryos were relatively respiratory acidotic and had higher hematocrit (Hct) and hemoglobin concentration. The partial metabolic compensation for respiratory acidosis produced by ≤ 10% CO2 exposures occurred in proportion to CO2 concentrations in both strains, but metabolic compensation for 20% CO2 respiratory acidosis was depressed at 2, 6 and 24h, particularly in broiler embryos. Exposure to ≤ 10% CO2 induced the same hematological responses across CO2 concentrations; i.e., Hct and mean corpuscular volume (MCV) increased while RBC concentration remained unchanged. In response to 20% CO2 exposure, Hct and MCV increased dramatically in both stains. Consequently, altered acid-base and hematology responses to 20% CO2 exposure compared to ≤ 10% CO2 suggest that the hypercapnic threshold to compensation for acidosis and regulation of hematology is >10% CO2.

Developmental critical windows and sensitive periods as three-dimensional constructs in time and space.

A critical window (sensitive period) represents a period during development when an organism's phenotype is responsive to intrinsic or extrinsic (environmental) factors. Such windows represent a form of developmental phenotypic plasticity and result from the interaction between genotype and environment. Critical windows have typically been defined as comprising discrete periods in development with a distinct starting time and end time, as identified by experiments following an on and an off protocol. Yet in reality, periods of responsiveness during development are likely more ambiguous that depicted. Our goal is to extend the concept of the developmental critical window by introducing a three-dimensional construct in which time during development, dose of the stressor applied, and the resultant phenotypic modification can be utilized to more realistically define a critical window. Using the example of survival of the brine shrimp (Artemia franciscana) during exposure to different salinity levels during development, we illustrate that it is not just stressor dose or exposure time but the interaction of these two factors that results in the measured phenotypic change, which itself may vary within a critical window. We additionally discuss a systems approach to critical windows, in which the components of a developing system--whether they be molecular, physiological, or morphological--may show differing responses with respect to time and dose. Thus, the plasticity of each component may contribute to a broader overall system response.

Embryonic critical windows: changes in incubation temperature alter survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis).

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.