D) fixed by planktonic cyanobacteria in pelagic food webs remains unresolved, particularly for toxic cyanophytes that are selectively avoided by most herbivorous zooplankton. Current theory suggests that ND fixed during cyanobacterial blooms can enter planktonic food webs contemporaneously with peak bloom biomass via direct grazing of zooplankton on cyanobacteria or via the uptake of bioavailable ND  by palatable phytoplankton or microbial consortia. Alternatively, ND can enter planktonic food webs post-bloom following the remineralization of bloom detritus. Although the relative contribution of these processes to planktonic nutrient cycles is unknown, we hypothesized that assimilation of bioavailable ND  by palatable phytoplankton and subsequent grazing by zooplankton  would be the primary pathway by which ND was incorporated into the planktonic food web. Instead, in situ stable isotope measurements and grazing experiments clearly documented that the assimilation of ND by zooplankton outpaced assimilation by palatable phytoplankton during a bloom of toxic Nodularia spumigena Mertens. We identified two distinct temporal phases in the trophic transfer of ND from N. spumigena to the plankton community. The first phase was a highly dynamic transfer of ND to zooplankton with rates that covaried with bloom biomass while bypassing other phytoplankton taxa; a trophic transfer that we infer was routed through bloom-associated bacteria. The second phase was a slowly accelerating assimilation of the dissolved-ND pool by phytoplankton that was decoupled from contemporaneous variability in N. spumigena concentrations. These findings provide empirical evidence that ND can be assimilated and transferred rapidly throughout natural plankton communities and yield insights into the specific processes underlying the propagation of ND through pelagic food webs.The fate of diazotrophic nitrogen (N The filamentous N. spumigena is a toxic diazotrophic cyanobacterium that produces the hepatotoxin nodularin D, be it via uptake of ND exuded by N. spumigena during the bloom or remineralised post bloom. We expected evidence of a bloom-contemporary transfer of ND to zooplankton, although we hypothesized that this was likely to be small relative to post-bloom processes. We also hypothesized that direct grazing of zooplankton on N. spumigena would be negligible and we conducted grazing experiments to quantify the role of direct grazing. We used δ15N stable isotope data to track the fate of bloom-derived ND and generate estimates of mass-specific flux rates of ND through different functional groups of the plankton community. Patterns in ND flux rates and compositional changes in the plankton community were interpreted to infer the role of contemporaneous  and post-bloom processes  as delivery mechanisms of ND to the planktonic food web. Our study verifies the transfer of ND from cyanobacteria to multiple elements of the estuarine food web and identifies the specific trophic routing that facilitates the propagation of ND to higher trophic levels.Our current understanding of plankton trophodynamics and cyanobacterial physiology suggests that NNodularia spumigena Mertens The Gippsland Lakes are an interconnected chain of three lagoonal estuaries in southeastern Victoria  that are subject to anthropogenic eutrophication and experience episodic blooms of Bioassay experiments were conducted using the dilution method in situ temperatures under natural light conditions: ∼ 100 µmol photons m−2 s−1 illumination and a 14-hr light:10-hr dark cycle. The composition of the autotrophic community was analysed non-destructively in a Phytopam , which quantifies the relative contribution of green (chlorophytes), brown (diatoms and dinoflagellates) and cyan (cyanobacteria) groups to the total measured chlorophyll fluorescence . Regression of Phytopam output against direct cell counts showed the intensity of the cyan channel to be an excellent predictor of N. spumigena biovolume . During the experiments, there was evidence that N. spumigena fluorescence was contributing to the intensity of the green channel; therefore, the green channel was not considered in the data analysis. This was justified on the basis that chlorophytes made a negligible contribution to total phytoplankton biomass throughout this study. Dark-adapted fluorescence measurements were taken at the start of each incubation and every 1–2 days thereafter from each chamber. Incubations were maintained for 7–12 d; however, growth and grazing rate calculations were based on the initial response of each phytoplankton group  to minimize the influence of bottle-effects on rate measurements.Three replicate 100-ml incubations were prepared in 150 ml chambers by diluting ambient estuary water with 0.7 µm GF/F filtered water in ratios of 1∶0 , 1∶4 and 1∶19. Incubation chambers were maintained in a water bath at G, d−1) associated with color channels were calculated for each replicate incubation chamber. A least-squares linear regression was fitted to G (dependent variable) across the three different dilution regimes (independent variable) and the extrapolated intercept interpreted as the maximum predicted daily growth rate (Gmax) in the absence of zooplankton grazing. The difference between Gmax and the mean G observed in the undiluted control samples yields an estimate of grazing rate  by zooplankton on each phytoplankton group. Differences in rG between the cyan and brown groups were tested with a separate variance t-test due to evidence of heteroskedasticity in the data .Daily growth rates of phytoplankton  and Sedgewick Rafter chamber (larger taxa). Biovolumes of N. spumigena were estimated using the Sedgewick Rafter chamber on the Lugol’s preserved samples. Additional biovolume estimates were provided by ancillary sampling conducted by the Victorian Environmental Protection Agency (EPA).We targeted four unique components of the planktonic food web for stable isotope analysis: phytoplankton (0.7–63 µm), microzooplankton (80–149 µm), mesozooplankton (≥150 µm), and N. spumigena filaments in the sample and subsequent analysis verified that the day-specific δ15N of the 63 µm filtered phytoplankton was significantly enriched relative to ambient water samples during the bloom  but not after . Larger size classes of plankton were collected by towing an 80 µm plankton net for 3–5 min at ∼ 3 km h−1 at each location. High concentrations of N. spumigena filaments in net samples precluded the partitioning of plankton size classes in the field; therefore, whole plankton samples were stored in sample bottles with 0.7 µm filtered ambient water and held on ice for up to several hours until stored at −20°C in the laboratory.The 0.7–63 µm phytoplankton samples were collected by filtering a known volume of 63 µm prefiltered ambient water onto pre-combusted glass fiber filters . Prefiltering removed most N. spumigena filaments from other plankton in the water samples. A 150-µm sieve was used to separate the remaining plankton into the 80–149 µm microzooplankton and ≥150 µm mesozooplankton size classes. After filtering, light microscopy was used to verify that biomass of N. spumigena was a negligible component of the size-fractioned plankton samples. If substantial N. spumigena filaments were apparent , size-fractioned samples were diluted with deionized water and centrifuged at 2000–4000 rpm in a refrigerated swinging bucket centrifuge for 5–10 min. Non-N. spumigena plankton typically settled out first and formed the apex of the sediment; whereas, N. spumigena remained suspended in solution or formed a recognizable green upper layer on the sediment. The supernatant and any sedimented N. spumigena was discarded and the remaining sediment re-examined under a microscope. This procedure was repeated until the bulk of the sample biomass was composed of non-N. spumigena taxa. The sample material was then refiltered and triple-rinsed with deionized water to remove any particulates generated during the centrifugation process.In the laboratory, samples were thawed and a pipette used to siphon the positively buoyant Aulacoseira sp., Pseudo-nitzschia sp., Skeletonema costatum), with scattered mixotrophs  and heterotrophs  also present at low levels. Microzooplankton biomass was primarily heterotrophic zooplankton  with mixotrophic dinoflagellates and Coscinodiscus sp. contributing a small fraction to the total organic biomass. Biomass in the mesozooplankton was almost exclusively heterotrophic zooplankton .The size classes in this study generally correspond to taxonomically and functionally different plankton components δ-notation relative to ambient air with precision = 0.1‰.Samples were held in an oven at 60°C until completely dried (min 48 hrs), after which they were ground to a fine homogenous powder and packed into sterile tin capsules for N-isotope analysis. Isotope samples were analysed on a Hydra 20–22 isotope ratio mass-spectrometer and coupled ANCA-GSL2 elemental analyser  at Monash University. Isotope values are reported in the δ15N of plankton groups at each site from initial conditions (pre-bloom) to the termination of the bloom (22 September 2011–7 February 2012).Least-squares linear regression was used to assess the magnitude of observed declines in D) into the non-N. spumigena plankton groups were calculated using isotope mass balances. Mass-specific rate calculations were carried out using the mass-balance approach described by Montoya et al. δ15N was included in estimates of ND flux into the micro- and mesozooplankton groups, but not the phytoplankton group. The proportion of ND present in the biomass of plankton group i at day t was estimated by calculating the proportion of ND in each plankton sample, p, using a simple two-endmember mixing model:i,tδN is the nitrogen isotope value of plankton group i at time t, i,0δN is the nitrogen isotope value of the plankton group prior to the N. spumigena bloom, NodδN is the average nitrogen isotope value of N. spumigena, and Δ is the trophic fractionation value assigned to that particular plankton group.Daily uptake rates of diazotrophic N (NV (d−1), was calculated as:Δt is the interval in days between samples and i,tp is the proportion of the total nitrogen derived from ND in plankton group i at day = t. Mass-specific uptake of ND, i,tρ (nmol N mg−1 d−1), was then calculated as:The specific rate of nitrogen uptake, Δ = 3.4 ‰ ρ estimates increased by 14.9±8.2 (SD) nmol N mg−1 d−1 (microzooplankton) and 27.8±13.0 nmol N mg−1 d−1 (mesozooplankton) for each 1 ‰ increase in the value of the Δ parameter. These patterns were consistent: estimates of ND flux increased (decreased) for a plankton group if the group was assigned to a higher (lower) estimated trophic position or if the value of the fractionation term was increased (decreased). Although the absolute value of our flux estimates are contingent on the value of Δ, the relative changes and the overall patterns among plankton groups were unaffected by the presence or absence of the fractionation term. In the absence of empirical trophic position estimates for zooplankton in this study and based on the flexible feeding ecologies of these taxa, we opted to include Δ while using a conservative estimate of 1 trophic exchange between zooplankton groups and their putative food source.We used a nominal one-trophic level enrichment of δ15ND can also occur during assimilation of dissolved ND by phytoplankton 4+ in the water column 4+ and NO3−, particularly when N-limited; thus, the mass-balance of the combined uptake of total DIN by phytoplankton would not reflect the kinetic discrimination of 15ND during nitrification. Taken together, these conditions suggest that declines in δ15N values across plankton groups were primarily driven by assimilation of ND into their tissues rather than a reduction in the concentration of 15N in the DIN pool.Fractionation of D through planktonic food webs; yet ultimately, the planktonic web is only one of several interacting components of the larger aquatic food web. We investigated the transfer of bloom-associated ND to the demersal food web by analyzing the white muscle and liver tissue δ15N composition of a commercially caught finfish, black bream Acanthopagrus butcheri, collected over the course of the study. The δ15N composition of fish muscle and liver tissue equilibrate to the isotopic composition of diet (with fractionation) at different rates δ15N value of these tissues during a time-series can provide an indicator of the timing and relative magnitude of ND flux from the pelagic food web to the demersal food web. All fish were collected with gill nets from areas affected by the N. spumigena bloom and immediately frozen. White muscle from the dorsal musculature and liver tissue were dried at 60°C, then pulverized and analysed for δ15N on the isotope ratio mass-spectrometer as previously described.The focus of this study is on the flux of NN. spumigena throughout Lake King during the summer months of November 2011 to February 2012. N. spumigena was present in low concentrations (c. 0.5 mm3 L−1) at both sites in Lake King by late September (study day = 21) but substantial biovolumes did not develop until mid-November , the secondary bloom of N. spumigena had collapsed at both sites and biovolumes declined to concentrations of ∼ 0.2 mm3 L−1; by May N. spumigena was no longer detectable in the water column.There was a large bloom of N. spumigena bloom, the autotrophic plankton community was dominated by the cyanobacterium Eucapsis sp., the diatom Coscinodiscus sp. and a chlorophyte Kirchneriella sp. Copepods, primarily adult Sulcanus conflictus  and a mixed assemblage of unidentified adult-stage calanoids and nauplii, were the most abundant zooplankton taxa during the same pre-bloom period. There was a shift in the plankton community coincident with the N. spumigena bloom that was marked by declines in most of the dominant pre-bloom taxa and the appearance of previously unobserved taxa. The autotrophic plankton community of the mid-summer peak bloom period was numerically dominated by N. spumigena with the remainder composed of a suite of diatoms , dinoflagellates  and a small number  of unidentified chlorophytes. Non-N. spumigena phytoplankton typically contributed an average of <5% to total autotrophic cell counts during the bloom. The cyclopoid copepod Oncaea media replaced S. conflictus as the most abundant copepod species with copepod nauplii, bivalve veligers, and ciliates  also present. Following the final collapse of the N. spumigena bloom, the diatoms Pseudo-nitzschia cf. pungens and S. costatum, dinoflagellates Prorocentrum minimum, Prorocentrum lima, and C. furca, and unidentified chlorophytes dominated the phytoplankton. Abundant post-bloom zooplankton included the ciliates Favella sp. and Mesodinium rubrum, several cyclopoid copepods including O. media, and a mixed assemblage of copepod nauplii.Prior to the Gr (d−1), between N. spumigena and a combined diatom and dinoflagellate group. For N. spumigena, growth rate peaked at G = 0.34±0.12 (SD) d−1 on study day = 106, coincident with the first peak in bloom biovolume. Among the diatoms and dinoflagellates, G peaked later in the time series at 0.46±0.15 on study day = 161 following the collapse of the N. spumigena bloom. Estimates of specific grazing rates (N. spumigena (Gr = −0.25±0.23) and preferentially selected  the diatoms and dinoflagellates (Gr = 0.62±0.97). These taxon-specific patterns in grazing rate were consistent over the course of the bloom cycle  ‰ and −0.1±0.5 ‰ for the northern and southern site, respectively. Negative δ15N values in this range of are typical of nitrogen-fixing cyanobacteria such as N. spumigenaN. spumigena, δ15N values of the other plankton groups were enriched, with pre-bloom isotope values spanning 8.8–11.8 ‰ , 3.9±1.8 ‰ in the 80–149 µm size class (hereafter “microzooplankton”) and 4.9±0.7 ‰ in the ≥150 µm size class (hereafter “mesozooplankton”) by early February. During the bloom, daily rates of change in δ15N among plankton groups ranged from −0.03 to −0.07 ‰ d−1 and were consistent within size classes between sites.Plankton isotope dynamics followed very similar trends between the two sampling locations . Through8–11.8 ‰ . FollowiD flux ρ (µmol diazotrophic N mg dry wt−1 day−1) into plankton groups indicated rapid and dynamic transfer of ND into all measured compartments of the planktonic food web (ρ over time in the phytoplankton showed ND-flux accelerated consistently throughout the bloom and most of the post-bloom periods (study days 89–196) at rates of 0.97±0.08 (SE) nmol ND mg−1 d−2 at the north site  and 1.32±0.20 nmol ND mg−1 d−2 at the south site . Mean flux rates of ND into the phytoplankton peaked at ρ = 126.8 and 135.6 nmol N mg−1 d−1 at the north and south sites respectively, approximately 20 to 30 d after the collapse of the secondary bloom. Flux rates had declined to 68.0 (north site) and 104.8 nmol ND mg−1 d−1 (south site) by the end of the time-series. Flux into the micro- and mesozooplankton groups differed from that into the phytoplankton in both the timing and magnitude of peak uptake rates  d (microzooplankton) and 21±11 d (mesozooplankton). Mean peak ρ values for the microzooplankton ranged from 211.4–217.1 nmol ND mg−1 d−1 at the north site and 151.8–191.8 nmol ND mg−1 d−1 at the south site. Mesozooplankton displayed comparable uptake maxima, with ρ values of 288.7–295.0 nmol ND mg−1 d−1 at the north site and 288.4–258.9 nmol ND mg−1 d−1 at the south site. Similar to the phytoplankton, ρ had declined by the end of the time-series at both sites to 60.5–109.8 nmol ND mg−1 d−1 in the microzooplankton and 60.4–65.8 nmol ND mg−1 d−1 in the mesozooplankton.Estimates of mass-specific Nfood web . Least-ske rates . Unlike δ15N values for black bream displayed different patterns over the course of the time-series  to 9.9±0.1 ‰ after the post-bloom depletion (study days: 181–202). Post-hoc tests of differences between bloom and post-bloom periods for each tissue showed no difference for muscle tissue ; whereas, the decline in liver δ15N was significant between bloom periods  and nutrient addition experiments showed N-limitation among diatoms and dinoflagellates  that indicate a rapid transfer of ND from filamentous cyanobacteria to epiphytic bacteria . Bacterivory is very common among the protozoan and certain metazoan  taxa that constitute the microzooplankton N. spumigena-associated bacteria has been inferred from experimental mesocosms D to the microzooplankton. In turn, mesozooplankton are effective predators of microzooplankton ρ that we observed between the micro- and mesozooplankton suggests a close coupling between these two functional groups. Thus, nutrient transfer from N. spumigena to consortial bacteria followed by heterotrophic transfers from bacteria to microzooplankton to mesozooplankton represents the most likely trophic pathway supporting the flux of ND into heterotrophic zooplankton taxa.In the absence of direct grazing, we infer that ND in the zooplankton functional groups versus the autotrophic producers throughout the bloom and immediate post-bloom phases  estuary. Further work is needed to quantify the total flux of ND during different bloom phases as well as the allocation of assimilated ND to the somatic and metabolic demands of higher trophic level consumers within the estuarine food web.Results from the tissue analysis of black bream provide initial evidence that the post-bloom uptake of remineralized ND constitutes a potentially important subsidy for production among higher trophic levels in coastal and oceanic pelagic food webs D during the bloom and the post-bloom weeks  the short-term ‘snapshot’ characteristics of the sampling regimes. Other studies have noted the transfer of ND-fixation products from diazotrophic cyanobacteria to consortial bacteria D from cyanobacteria to zooplankton. Our results provide strong support for this consensus and emphasize the trophic linkages between N. spumigena-associated bacterial communities and the eukaryotic elements of the planktonic food web Click here for additional data file.


Variation in 13C abundance can therefore be obscured by variation in percent lipids. Past attempts to establish an algebraic equation to correct carbon isotope ratios for lipid content in eggs have been unsuccessful, possibly because they relied partly on data from coastal or migratory species that may obtain egg lipids from different habitats than egg protein. We measured carbon, nitrogen and sulphur stable isotope ratios in 175 eggs from eight species of aquatic birds. Carbon, nitrogen and sulphur isotopes were enriched in lipid-extracted egg samples compared with non extracted egg samples. A logarithmic equation using the C∶N ratio and carbon isotope ratio from the non extracted egg tissue calculated 90% of the lipid-extracted carbon isotope ratios within ±0.5‰. Calculating separate equations for eggs laid by species in different habitats  improved the fit. A logarithmic equation, rather than a linear equation as often used for muscle, was necessary to accurately correct for lipid content because the relatively high lipid content of eggs compared with muscle meant that a linear relationship did not accurately approximate the relationship between percent lipids and the C∶N ratio. Because lipid extraction alters sulphur and nitrogen isotope ratios , we suggest that isotopic measurement on bulk tissue followed by algebraic lipid normalization of carbon stable isotope ratio is often a good solution for homogenated eggs, at least when it is not possible to complete separate chemical analyses for each isotope.Stable isotope ratios are biogeochemical tracers that can be used to determine the source of nutrients and contaminants in avian eggs. However, the interpretation of stable carbon ratios in lipid-rich eggs is complicated because  Stable isotope analysis is a useful technique for tracing the origin of nutrients in tissues with applications in environmental chemistry, paleoecology, migration biology and diet reconstruction Agencies in a number of countries systematically collect and archive bird eggs for toxicological and chemical analyses because eggs obtained early in the season can be re-laid, collection of 10–20 eggs has little impact on bird populations numbering in the thousands or millions, and the lipid-rich matrix accumulates many of the lipophilic toxins of interest 13C∶12C, measured relative to the PeeDee Belemnite standard and denoted δ13C) and nitrogen  ratios. Carbon isotopes can be used to identify habitat, as δ13C varies systematically with degree of aquatic and anthropogenic input. Nitrogen isotopes are primarily used to determine trophic level, as δ15N increases predictably with trophic level. Increasingly, sulfur isotope ratios  are also used to distinguish nutrients originating from marine environments. Stable isotope ratios change in a systematic fashion as nutrients are assimilated from an animal's food (i.e. the discrimination factor). An accurate knowledge of the discrimination factor is necessary to quantitatively predict nutrient origin The most common stable isotope ratios used by ecologists are those involving carbon  is used as an index of lipid content because nitrogen abundance is high in proteins and low in lipids, and C∶N ratios are readily calculated from data obtained during stable isotope analysis There are two methods for correcting for lipid content 13C that would have been present in a tissue following lipid extraction from the value measured in non-extracted tissue. Such models are accurate in muscle tissue in a wide variety of animals Lipid normalization models are equations that use the C∶N ratio or percent lipids to calculate the value of δ13C in eggs collected from three species of wild birds varied widely between lipid-extracted and non-extracted tissue, and was not predicted by C∶N ratio 13C compared to marine-derived lipids Using lipid normalization models in eggs, however, has been more problematic . For ins13C values. For tissues that have a relatively low percent lipids, such as muscle, a linear equation appears to hold , three seabirds that forage primarily on the continental shelf during the breeding season , one offshore-foraging marine seabird (Leach's storm-petrel Oceanodrama leucorhoa), a freshwater aquatic raptor  and an aquatic bird that also uses the terrestrial environment (great blue heron Ardea herodias). For the purposes of analysis, we classified bird species into three habitat types: offshore (Leach's storm-petrel), continental shelf (rhinoceros auklet and pelagic cormorant) and those in habitats influenced by the terrestrial environment, such as lakes or estuaries . Egg contents were homogenized and frozen until analysis.We randomly selected archived egg contents from the Environment Canada's Specimen Bank http://stableisotopefacility.ucdavis.edu). On a separate sample, we extracted the lipids using a Soxhlet apparatus with petroleum ether as the solvent http://www.uweilab.ca) for sulphur analysis.After thawing, samples were freeze-dried. A small sample (1 mg) was removed, encapsulated in tin, and sent to the Stable Isotope Facility at University of California, Davis  interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer . Samples were combusted at 1000°C in a reactor packed with chromium oxide and silvered copper oxide. Following combustion, oxides were removed in a reduction reactor (reduced copper at 650°C). The helium carrier then flowed through a water trap (magnesium perchlorate). N2 and CO2 were separated on a Carbosieve GC column  before entering the IRMS. Samples were analyzed for 34S/32S using a Europa Roboprep-20/20 EA-IRMS. During analysis, samples were interspersed with several replicates of at least two different laboratory standards. The final delta values were expressed relative to international standards Vienna PeeDee Belemnite, Vienna Cañon Diablo Troilite and air for carbon, sulphur and nitrogen, respectively.Samples were analyzed for 13C, we also collected egg yolk from glaucous-winged gulls (Larus glaucescens), a nearshore marine seabird that also uses the terrestrial environment. We used chloroform∶methanol extraction to remove lipids from egg yolk. Lipid-extracted and non-extracted egg yolks were encapsulated and sent to the UC Davis Stable Isotope Laboratory for carbon and nitrogen analyses. All data for both projects are presented in To examine the effect of a different lipid extraction process on variation in δ13C (δ13Clipid extracted - δ13Cnon extracted), Δδ15N (δ15Nlipid extracted - δ15Nnon extracted), Δδ34S (δ34Slipid extracted – δ34Snon extracted) and the C∶N ratio. Linear models are appropriate for tissues with low percent lipids because as long as percent lipids is low a linear model approximates the nonlinear function; for tissues with high percent lipids , the C∶N ratio does not change linearly with percent lipids  necessitating a non-linear function 13Clipid-extracted and δ13Cnon-extracted. We removed eight values, all from the same Soxhlet run, because the C∶N ratio after lipid extraction was >5.0. All raw data are available in We used R 2.14.2 for all statistical analyses. We used linear regression and non-linear regression based on previous published logarithmic models 13C and the C∶N ratio  differed by more than 0.5‰ for the logarithmic model and 12 (7%) differed by more than 0.5‰ from the complete model with habitat and species interactions included. Nonetheless, the best-supported model included separate terms in the intercept (but not the slope) for each habitat . Average repeatability  for duplicates (separately homogenized) was 0.26 for δ34S, 0.12 for δ13C and 0.31 for δ15N.Lipid-extracted egg samples were more enriched in carbon, sulphur and nitrogen than non-extracted egg samples . Across ∶N ratio , Fig. 1.upported . For the habitat . In cont13C and the C∶N ratio for all of the species with sample sizes greater than two , except ospreys .There was a statistically-significant relationship between Δδ13C values by ∼2‰, δ15N values by ∼1‰ and δ34S by ∼0.5‰. The degree of enrichment in egg δ13C values (Δ13C) was correlated with the percent lipids, as inferred by the C∶N ratio. Indeed, across the entire dataset, and within all marine species, the C∶N ratio strongly predicted Δ13C. As more than 90% of the samples were estimated within ±0.5‰ by the best-fit algebraic equation (logarithm model), our algebraic equation is a robust method of calculating lipid-extracted δ13C values given δ13C values measured on non-extracted egg tissue from marine birds.Lipid extraction enriched egg δ13C signatures; egg protein, in contrast, may be derived from the breeding grounds 13C may be particularly large for coastal birds switching between freshwater and marine prey bases 13C values were derived from wintering grounds and their protein δ13C values were mainly derived from breeding grounds, that would explain why the lipid-extracted and non-extracted samples were consistently different 13C in eggs is accurate for birds relying on resources acquired concurrently with reproduction for egg synthesis, such as income breeders, but not for coastal or migratory birds that may bring lipid reserves from a habitat with a different δ13C value. Specifically, algebraic corrections are applicable where lipids and proteins would be expected to be acquired in isotopically similar landscapes, with capital and income breeding being extremes of a continuum with actual reproductive strategies ranging from high to low reliance on stored nutrients Our models for egg tissue were as accurate as those for muscle tissue 13C. As the embryo develops, there may be differential uptake of varying nutrients and, therefore, variation in 13CO2 expiration. For instance, chicken yolk δ15N was depleted by 1.0‰ after 15 days of development and chicken albumin δ13C was depleted by 0.2‰ after 3 days of development 13C was excreted differentially to 12C in yolk relative to plasma, that could increase the variance in Δδ13C relative to lipid content. As we did not know the age of the embryo, this issue could play a role in our dataset, although collectors attempted to obtain eggs shortly after laying.Stage of embryonic development also may have impacted the relationship between C∶N ratio and ΔδAt the early stages of development (prior to significant growth of the embryo), the egg interior primarily consists of the cysteine-rich protein albumin (egg white) and lipid-rich yolk. Many authors have separated the two components and analyzed stable isotopes separately on each component 13C than nearshore foragers (rhinoceros auklets and pelagic cormorants), which had higher Δδ13C than birds foraging in terrestrial-influenced habitats . Ancient murrelets may forage farther offshore than pelagic cormorants, but for the current classification scheme they appeared to group with those species feeding in terrestrial-influenced habitats. Perhaps there is a larger difference in the carbon isoscape in winter (where lipids were derived) relative to breeding (where non-lipids were derived) habitat for birds feeding offshore than for those feeding in the terrestrial environment. Alternatively, within the capital-income breeding strategy continuum, Leach's storm-petrels may rely more on endogenous stores for egg production and the terrestrial-influenced species may rely more on exogenous stores for egg production 13C for terrestrial birds  than waterfowl. Either variation in habitat or breeding strategy  may explain why Δδ13C differed between terrestrial birds and waterfowl in that study.Pelagic foragers (Leach's storm-petrels) had higher Δδ13C and δ15N, at least in fish and invertebrates 13C in lipid-extracted tissue varied among three different solvents, with chloroform-methanol . Furthermore, pure gull albumin had a C∶N ratio of 3.72±0.06 and lipid-extracted whole egg had a C∶N ratio of 3.72±0.04 in the gull dataset using the chloroform-methanol extraction process, whereas lipid-extracted whole egg had a C∶N ratio of 4.33±0.50 in the seabird dataset using the petroleum ether extraction process. Thus, we believe that chloroform-methanol extractions remove more, or at least different, lipids (and other molecules) than petroleum ether, and therefore cause a larger Δδ13C.We believe a solvent effect is present for bird eggs. The arithmetic correction for Δδ13C (and δ15N/δ34S) will vary among extraction techniques and, finally, to avoid overestimation of δ15N or δ34S  it would be necessary to complete two different analyses, doubling expenses. Lastly, lipid extraction removes at least some amino acids, which can insert some (unwanted) variability into diet estimation. For studies of nutrient allocation or prey use, very accurate and unbiased measurements of δ13C may be necessary, but for toxicological studies that primarily use stable isotope ratios as correlative, predictive variables, the algebraic approach may be sufficient; across all species, lipid-extracted and algebraically-corrected values were highly correlated (R2 = 0.99) so that in correlative studies essentially no information would be lost by using the algebraic approach. Furthermore, where the algebraic equations do not provide an accurate index of lipid extracted δ13C because lipids and proteins are derived from different environments, the algebraic equations will give the value more associated with lipophilic contaminants; the stable isotope ratios measured on lipid-extracted tissue  will not reflect the diet associated with the prey from which the lipophilic contaminants are derived. We, therefore, urge the use of algebraic corrections, rather than lipid extraction, in toxicological studies.Our lipid normalization equations will be of use in accounting for the effect of lipids in toxicological studies of purely marine birds, although we urge the establishment of separate equations for each species. There are many reasons why lipid extraction is sub-optimal: chloroform is not allowed at many laboratories for health reasons, the Soxhlet apparatus used in the current studies cost over $10 000, the apparatus created a bottleneck of only eight samples processed each day, δThe most-widely used equation is the Post et al. Table S1Stable isotope data analyzed in the manuscript.(XLSX)Click here for additional data file.


Here, we investigate the free energy landscape of αβ-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy  and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αβ-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αβ-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated. Microtubules are composed of αβ-tubulins that play an instrumental role in regulating intracellular trafficking and formation of the mitotic spindle during mitosis and cell division. Structural studies have shown that tubulin exists in a “straight” conformation compatible with that in the microtubule lattice and a “bent” conformation thought to represent the unassembled state. There is current debate as to whether the straight-to-bent conformational change in tubulin is the cause or consequence of tubulin's assembly into the microtubule lattice. Here, we use free-energy molecular dynamics simulations to qualitatively understand the conformational landscape of tubulin in the unassembled state and upon lateral binding. We predict that soluble tubulin exists primarily in a bent conformation; our simulation results show that tubulin primarily adopts an intermediately bent conformation in agreement with structural data. We also show that lateral binding of two tubulins shifts the equilibrium in favor of the “straight” state, supporting the hypothesis that the straight-to-bent conformational change is the consequence of tubulin's incorporation into the microtubule lattice via lateral interactions. We also show that colchicine binding shifts the population of tubulin in favor of a bent state, further implicating our work in drug discovery. Microtubules (MTs) are dynamic cytoskeletal polymers formed by the polymerization of αβ-tubulin, a globular heterodimer comprised of two structurally related 55 kDa α- and β-subunits. Microtubules play a vital role in intracellular trafficking and cell division; these functions are influenced by the complex dynamics of the MT plus end, which undergoes stochastic periods of assembly and disassembly. The role of αβ-tubulin conformational changes in the processes of assembly and disassembly has been the subject of great interest and some controversy Tubulin has been shown to exist in two extreme conformations: a “straight” conformation observed in antiparallel zinc-induced tubulin sheets Multiple rotational and translational motions in the α- and β-subunits differentiate the “straight” and “bent” conformations. We describe the ∼12° intradimer conformational change of the heterodimer, as described originally by Knossow et al., by the movement of the H7 central helices in the α- and β-subunits see . It is nThe role of nucleotide state and tubulin conformation has been debated in the context of two competing models of microtubule assembly 2+-induced protofilaments and “bent” tubulin from the T2R-colchicine complex. In contrast to unrestrained molecular dynamics simulations, we explore tubulin conformations only along a coordinate connecting these two states, which we quantify using an intradimer rotation angle , and the bent tubulin structure observed in the α1β1 heterodimer of the T2R-colchicine complex   were incorporated using those in Nogales et al.'s earlier Zn2+-induced protofilament model  and neutralized with NaCl. The GROMACS 4.5.5 g_mindist module was used to ensure that there was no overlap between the system with its periodic image during equilibration. Each system was energy-minimized using steepest descent while applying Ferguson's flexible SPC water model These two modified endpoint structures were then energy-minimized and equilibrated using the GROMACS 4.5.5 package For the straight pair endpoint, two straight tubulins laterally paired in the α-α, β-β manner were taken from Wells et al.'s cryo-EM-derived “N” model of the MT lattice 1β1 heterodimer from the T2R-colchicine complex was used as the bent endpoint, preserving the colchicine molecule. Solvation, minimization, and equilibration of this structure were performed in analogous fashion as for the unpolymerized bent tubulin. The GlycoBioChem PRODRG2 server The αR relative to this path can be expressed in terms of two collective variables, s(R), which represents the progress of the dynamics along the conformational change coordinate, and z(R), which represents the progress away from the reaction path, as follows i is a discrete index ranging from 1 to P, the total number of structures along the reaction coordinate, λ is a prefactor in the exponential term that defines both s(R) and z(R).  of the straight and bent tubulin molecules. The conformational space apo tubulin structures along a linear path. These thirteen apo structures were then liganded with GTP, GDP, and the Mg2+ ion as follows: 1) each of the two endpoint structures was globally aligned with its nearest apo intermediate; 2) GTP, GDP, and the Mg2+ ion were superimposed onto the nearest intermediate structure, which was then solvated, minimized, and equilibrated using the methods described in the previous section. This cycle was iterated from both endpoints for each successive tubulin intermediate. The final snapshots of the fifteen equilibrated tubulin configurations  were used as the initial P = 15 umbrella nodes along the reaction path for the calculating the potential of mean force for unpolymerized tubulin  the equilibrated colchicine-bound fully bent tubulin complex was globally aligned with its nearest apo intermediate; 2) colchicine, GTP, GDP, and Mg2+ from the equilibrated colchicine-bound fully bent tubulin were then superimposed onto the nearest apo intermediate; and 3) solvation, minimization, and equilibration were performed on the successive holo tubulin intermediate as described above. This cycle was repeated for each successive intermediate from the bent to straight endpoints until the colchicine-bound straight tubulin had been fully equilibrated. The final snapshots for the nine equilibrated colchicine-bound tubulin configurations  were used as the initial P = 9 umbrella nodes along the reaction coordinate for calculating the potential of mean force for colchicine-bound tubulin  and z(R) are expressed as:s and z, and Ri is each umbrella position i within the configurational space R.Harmonic biasing potentials are placed at each umbrella position along a collective variable. The harmonic biasing potentials for P = 15 equidistantly-spaced umbrella windows along the s(R) coordinate. However, this discretization proved later insufficient to characterize the free energy along s(R), so we increased the number of umbrella windows to P = 35 while restraining each umbrella node by P = 13 equidistantly-spaced umbrella windows to P = 50  collective variable, we did not place any harmonic restraints on the z(R) path for the unpolymerized tubulin and laterally-paired tubulin dimers.For the unpolymerized tubulin, we initially defined P = 9 umbrella windows along s(R). To achieve adequate sampling among successive umbrella windows along s(R), we optimized P = 31 nodes. To prevent colchicine unbinding, especially from the conformations with lower intradimer curvature (0°≤θ≤6°), we restrained the dynamics along the z(R) coordinate to We used the initial nine colchicine-bound tubulin morphing intermediates to define All simulations for the unpolymerized tubulin, laterally-paired tubulins, and colchicine-bound tubulin were performed in the canonical (NVT) ensemble at 1.013 bar and 300 K for 4 ns using the PLUMED 1.3 s(R). For the unpolymerized tubulin and laterally-paired tubulins, we used the g_wham module s(R) path, based on s(R) path accordingly Nb bootstrapped free energy profiles, and Nb bootstrapped free energy profiles. For each umbrella position ξi, we generated a new bootstrapped trajectory ξb,i(t) yielding a new histogram hb,i(ξ). WHAM was then executed on this new set of histograms for Nb iterations to compute a bootstrapped free energy profile Wb,k(ξ).  at the initial position, ξi = 0, in s(R) so that the uncertainty at ξi = 0 is zero.) The uncertainty values along the s(R) coordinate are reported for the unpolymerized tubulin and laterally-bound tubulin pair in The weighted histogram analysis (WHAM) wham-2d program  space remained quite close to the s(R) path, even at s(R) positions corresponding to lower tubulin curvature, confirming that the harmonic restraining potential s(R) path. Using GROMACS g_wham, we then projected this 2D free energy profile onto s(R) at z(R) = 0, using 1000 bins and a tolerance of 0.001, and computed the statistical uncertainty s(R) path using the same bootstrapping methods described above.For colchicine-bound tubulin, we calculated the two-dimensional probability distribution and free-energy surface using the All calculations of the intradimer rotation angles of each bent tubulin heterodimer with respect to the straight were performed using UCSF Chimera version 1.7 2+; any other protein chains , solvent molecules, and counter ions other than Mg2+ were also removed. The solvent-accessible surface area of each tubulin heterodimer (αβSASA) and the solvent-accessible surface area of each α- and β-subunit, αSASA and βSASA, excluding the other subunit, were calculated in PyMOL using a solvent probe radius of 1.2 Å and a solvent density of 4. The buried surface area of each tubulin heterodimer was determined accordingly: BSA = (αSASA+βSASA)−αβSASA.To compute buried surface area, the crystal structures were first pre-processed so that each heterodimer is only complexed with GDP and GTP-MgFor each of the 31 umbrella positions along the conformational change coordinate, five colchicine-bound tubulin structures corresponding to the peak of each histogram were extracted from the last 3 ns of the restrained molecular dynamics trajectories. The SiteMap module We also analyzed the hydrogen bonding, hydrophobic interactions, and steric clashes of the colchicine-tubulin complexes at all 31 umbrella positions. For each of the five representative structures from each umbrella position, we used the Maestro command-line “Display Hydrophobic Interactions” script  of various intermediately bent tubulin structures, as an alternative way of quantifying differences between “straight” and “bent” structures see . These BThe α- and β-subunits of our bent endpoint structure have intramonomer rotations of ∼8° and ∼11°, respectively. To examine whether the rotation of the individual the α- and β-subunits is related to the rotation of the entire heterodimer, we calculated the rotation of each α- and β-subunit of the X-ray crystallographic structures of tubulin shown in Having characterized the surprisingly ‘flat’ free energy profile for tubulin along a bending coordinate, we next examined how this potential of mean force is modulated by (1) lateral association of two tubulin heterodimers (with implications for the lattice model of microtubule assembly), and (2) binding a small molecule drug, colchicine (with implications for microtubule-binding therapeutics).1β1 heterodimer from the T2R-colchicine:tubulin complex exhibits a ∼8.3° twist angle, which is somewhat large compared to the degree of twisting exhibited by the newer X-ray crystallographic structures of tubulins, whose twist angles range from ∼5–7° , which again is separated from the intermediately bent conformation by a small free energy barrier , in this case at ∼4°. The global free energy minimum along the PMF has an intradimer rotation angle (0.5°) very close to that observed in structures of zinc-induced protofilament sheets (1JFF). Although we find it geometrically infeasible for both tubulins to be in a fully bent state, both can exist in an intermediately bent state when laterally associated, and this state represents ∼25% of the population.  Overall, comparison of the ‘free’ and laterally-paired tubulin PMFs suggests that lateral association shifts the bending equilibrium by ∼1.5 kcal/mol toward the straight conformation, providing direct  support for the lattice model of microtubule assembly.Specifically, at the earliest stages of tubulin assembly, the lattice model predicts that lateral contacts will be disadvantaged because the binding interface is disrupted in the bent conformation. These results suggest that even forming a single lateral pair is sufficient to largely shift the equilibrium toward the straight conformation, which then makes subsequent lateral additions more thermodynamically favorable.kf = 1×106 M−1s−1. Reverse rate constants for nucleation N species (in which each species is represented by a heterodimer) have been described.39 Using these kinetic parameters and a nucleus size of N heterodimers, Rice et al. generated 500-second polymerization profiles for a range of straightening penalties from 0 to 1.6 kBT . Using our data that the ensemble of bent states are ∼1 kcal/mol more energetically favorable than the straight conformer, Rice et al.'s model suggests that a ∼1 kcal/mol straightening penalty increases the time required to assemble into a 4-species nucleus by ∼10-fold. That a modest straightening penalty of 1 kcal/mol is sufficient to decrease the rate of MT assembly highlights the sensitivity of MT assembly kinetics to the initial stages of nucleation and elongation.To begin to explore the functional consequences of an assembly-dependent conformational change in tubulin that underlies the lattice model, Rice et al. had used a simple two-step nucleation-elongation model to approximate the kinetics of the early stages of MT assembly. In keeping with experimental data, kinetic parameters were set to capture the low affinity of subunit addition during nucleation and a high affinity of subunit addition during elongation: the monomer concentration was set to 10 µM, the bimolecular on-rate constant to 3±29 Å3) than for fully bent tubulin (937 Å3±31 Å3 at θ = 12°). The pocket cannot collapse entirely in these simulations because colchicine is restrained to remain in the binding pocket. The colchicine-bound tubulin structures with ∼8° intradimer curvature also appear to be stabilized by hydrogen bonds with the backbone amide of α-Val 181 and side chain amine of β-Lys 350  to understanding how drugs or drug candidates modulate the free energy landscape of the target proteins. We expect this type of approach to be particularly relevant to understanding and ultimately designing allosteric modulators, and more broadly to how the chemical structure and binding mode of a drug dictate the ways in which it modulates the energy landscape.”Structural and functional studies have previously demonstrated the existence of the straight-bent equilibrium for tubulin, and its relevance to microtubule assembly. Our new contribution here is to compute the free energy along a conformational coordinate connecting two limiting straight and bent structures. We had hypothesized that we would observe two free energy basins, corresponding to the straight and bent states, with a free energy barrier separating these, and that the bent state would correspond to the lowest (most favorable) free energy, consistent with the lattice model of microtubule assembly. The computed ‘potential of mean force’ along the chosen bending coordinate largely supports this hypothesis, but also suggests that the bent state is best understood as an ensemble of states with a significant range of intradimer rotation angles (4–13°). The global free energy minimum in the PMF lies at 6° intradimer rotation, whereas the colchicine-bound structure that we (and others) have used to define the ‘fully bent’ state lies at 12°, and is slightly higher in free energy. Our colchicine simulations also support the observation that colchicine biases towards conformations more bent than the 6° rotation angle calculated for free tubulin. Recent structures of tubulin co-crystallized with various small molecules and macromolecules support the prediction that tubulin can adopt a wide range of bent states.Overall, the PMF predicts that the ensemble of bent states, taken as a whole, has a free energy that is lower than that of the straight state by about 1 kcal/mol, a modest value that is nonetheless sufficient to have a significant impact on polymerization kinetics, as represented (crudely) by a simple nucleation-elongation model. We emphasize that the precise value should not be over-interpreted, because the statistical uncertainty estimated for the PMF is ∼0.5 kcal/mol; in addition, the underlying molecular mechanics force field model also has limitations that could lead to systematic errors, the magnitude of which are difficult to estimate. An additional limitation is that the PMF only probes one linear coordinate connecting two structures that we chose as the extreme straight and bent states. There could exist other ‘off-pathway’ states, involving conformational changes orthogonal to and independent of the coordinate probed here, which could also have functional relevance. It is also highly likely that tubulin is capable of adopting bent conformations whose curvature is in the direction opposite of that of 1SA0, as explored by Voth and co-workers qualitative predictions of the PMFs agree well with a variety of experimental observations, including the fact that the intradimer rotation and surface area buried between the α and β chains in experimental structures correspond well with low free energy portions of the PMF. We also demonstrate that tubulin binding to the drug colchicine, and binding to another tubulin in a lateral interaction, both perturb the PMF in ways consistent with expectations. Colchicine binding strongly biases the equilibrium toward the bent state, to the extent that the straight state is thermodynamically infeasible; we attribute this shift largely to simple steric considerations, where the binding pocket shrinks as tubulin straightens. Lateral tubulin binding shifts the conformational equilibrium in the opposite manner, such that the most extreme bent states become infeasible and the straight conformation corresponds to the global free energy minimum . This result is consistent with the lattice model of assembly, and additionally suggests that a single lateral interaction is sufficient to “pay” the free energy cost of straightening tubulin.Nonetheless, the Our PMFs of unpolymerized tubulin and laterally-paired tubulins are based on tubulins bound with GDP at the β-subunit E-site. We had chosen to use GDP-bound tubulin because these were the only X-ray crystallographic structures available at the commencement of this study, and it would have been risky to artificially build in GTP. We have not considered in this work the role of nucleotide state at this site, a factor that has been considered in some prior MD simulations. Although SAXS experiments reveal that GTP- and GDP-bound tubulin adopt very similar, bent states in solution and that nucleotide choice does not influence colchicine binding, we cannot rule out the possibility that nucleotide state could modulate the PMF in some way, e.g., GTP shifting the equilibrium of the M-loop in a way that might additional favor lateral interactions. In principle, free energy simulations  could be used to address this issue in future work.Text S1Supporting Information. This section documents the supplementary figures and tables referenced throughout the text.(DOCX)Click here for additional data file.


Brief heat stimuli that excite nociceptors innervated by finely myelinated (Aδ) fibers evoke an initial, sharp, well-localized pain ("first pain") that is distinguishable from the delayed, less intense, more prolonged dull pain attributed to nociceptors innervated by unmyelinated (C) fibers ("second pain"). In the present study, we address the question of whether a brief, noxious heat stimulus that excites cutaneous Aδ fibers activates a distinct set of forebrain structures preferentially in addition to those with similar responses to converging input from C fibers. Heat stimuli at two temperatures were applied to the dorsum of the left hand of healthy volunteers in a functional brain imaging (fMRI) paradigm and responses analyzed in a set of volumes of interest (VOI).Brief 41°C stimuli were painless and evoked only C fiber responses, but 51°C stimuli were at pain threshold and preferentially evoked Aδ fiber responses. Most VOI responded to both intensities of stimulation. However, within volumes of interest, a contrast analysis and comparison of BOLD response latencies showed that the bilateral anterior insulae, the contralateral hippocampus, and the ipsilateral posterior insula were preferentially activated by painful heat stimulation that excited Aδ fibers.These findings show that two sets of forebrain structures mediate the initial sharp pain evoked by brief cutaneous heat stimulation: those responding preferentially to the brief stimulation of Aδ heat nociceptors and those with similar responses to converging inputs from the painless stimulation of C fibers. Our results suggest a unique and specific physiological basis, at the forebrain level, for the "first pain" sensation that has long been attributed to Aδ fiber stimulation and support the concept that both specific and convergent mechanisms act concurrently to mediate pain. There is substantial evidence that pain is mediated by two classes of nociceptive afferent fibers, finely myelinated Aδ fibers and unmyelinated C fibers ,2. FolloIn the present study, the aim was to identify the cerebral mechanisms that could be uniquely involved in mediating the brief, sharp "first pain" that is mediated by Aδ fibers. This was done by comparing responses to a noxious heat stimulus that excites cutaneous Aδ fibers with responses to converging input from C fibers. We used brain evoked potentials to identify the brief heat stimuli that preferentially excite Aδ and C fibers and we used functional magnetic resonance imaging (fMRI) to compare quantitatively the brain responses to each of these stimuli. We used contrast imaging and temporal analysis to detect the preferential activation of brain structures by Aδ fibers.We found that two sets of forebrain structures mediate the initial sharp pain evoked by brief cutaneous heat stimulation. One set responding preferentially to the brief stimulation of Aδ heat nociceptors and those with similar responses to converging inputs from the stimulation of C fibers.Seven right handed volunteers  participated in the experiment. All subjects were free of medication and had not consumed caffeine or other psychoactive drugs on the day of testing. All subjects gave informed consent before testing. The experimental protocol was conducted in accordance with the Helsinki declaration and was approved by the Internal Review Boards of the Veterans Affairs Medical Center (Ann Arbor) and the University of Michigan, Ann Arbor, MI, USA.contact-heat evoked potential stimulator . The thermode was strapped to the hand and not moved between stimuli. The thermode contacted a cutaneous area of 573 mm2 and is comprised of a heating thermofoil  covered with a 25 micron layer of thermoconductive plastic (Kapton®). Two thermocouples embedded 10 microns within this conductive coating provide an estimate of the skin temperature at the thermode surface. The thermofoil-skin interface temperature is measured and software-controlled 150 times per second. Heat stimuli were delivered at intensities of 41°C and 51°C. From a contact baseline temperature (35°C), the average time from onset to peak temperature is on average 190 ± 24 ms for 41°C and 250 ± 8 ms for 51°C. The full-width-half-maximum (FWHM) of the heat pulse is 350 ms. As reported previously, the stimulus parameters selected here reliably evoke quantifiable contact heat evoked potentials (CHEPs) mediated through C fibers accompanied by a warm sensation (41°C) or mediated through Aδ fibers accompanied by a sharp, pricking pain (51°C) [We used a n (51°C) ,17. WithEvoked potentials (EPs) and functional magnetic resonance images (fMRI) were acquired in two sessions one to four weeks apart. All subjects participated in both sessions and received up to 200 stimuli in each session; 20 runs of 10 stimuli divided into 4 runs of five different modalities  : 1 - 'not perceived', 2 - 'perceived but not painful', 3 - 'barely painful', 4 - 'moderately painful' and 5 - 'very painful'. Before data acquisition, the subject was instructed to pay close attention to the stimulus site and to rate the intensity of each stimulus approximately two seconds after it was perceived (EP) or when a visual cue appeared (fMRI). The ratings were performed verbally (EP) or via a five-button claw attached the right hand (fMRI). The five-button claw was a custom designed key pad strapped to the hand having one button for each finger . Pain intensity ratings and EP latencies were not normally distributed and therefore compared with the non-parametric Wilcoxon test. The Mann-Whitney U test was used for post-hoc comparisons; p < 0.05 was considered statistically significant. These statistical measures were carried out using SPSS .Before recordings, the subjects were presented with the different stimuli and practiced subjective ratings. Recordings were made from the vertex (Cz), referenced to bilateral earlobes (A1+A2), using a standard EEG cap and Neuroscan software . The electrooculogram (EOG) was recorded from supra- and infraorbital electrodes for offline artifact rejection. The impedance was maintained below 5 kΩ. The signals were amplified 100 000 times, sampled at 500 Hz, bandpass filtered at 0.1 - 30 Hz and notch filtered at 60 Hz. Subjects were seated in a padded reclining chair, room temperature was 22-23°C, and skin temperature was always above 30°C. The subjects were instructed to keep their eyes open, to focus on a fixed point at the wall and to avoid blinking. During offline analysis, the continuous EEG signal was split into epochs relative to stimulus onset using EEGLAB 6 . Each epImaging was performed in a 3 Tesla GE Signa scanner using a standard 16 rung bird-cage head coil. The head was packed with foam pads and strapped with a headband to avoid head movement. In each run 96 whole T2*-weighed spiral-out brain scans were acquired . The field of view was 24 cm with a 64 × 64 matrix. Repetition time (TR) was 1500 ms, echo time (TE) 24 ms and flip angle 60°. T1-weighed structural scans for anatomical localization  were also obtained . The stimulation paradigm was controlled by computer software . The software also stored subjective ratings via the 5-button claw strapped to the subject's right hand. The computer running E-prime was triggered by the onset of the scanner radio frequency (RF) pulse. The computer then delivered trigger signals to the stimulator. Exact stimulus onset times were stored for later use during the statistical modeling.Messages were presented to the subject via head mounted MRI compatible LCD display . Before each stimulus, a 'READY' screen was displayed for 2 to 6 s. At the offset of 'READY' the stimulus was delivered. The following 'WAIT' screen lasted 5 s before 'RATE STIMULUS' was displayed for 4 s, at which time the subject rated the stimulus by pushing one of the five buttons Figure . After eBased on the previous experience in this laboratory and the results of published pain activation studies ,7, volumThe volumes were motion corrected (MCFLIRT) and spatially smoothed (5-mm FWHM Gaussian kernel) and high-pass filtered (50 s). General linear model analysis was carried out using FEAT  Version 5.4, part of FSL (FMRIB's Software Library) . The triand C-fiber input. A later peak latency was taken as a contribution from C-fibers. Thus, VOI were defined as differentiating between Aδ and C fiber inputs if the peak BOLD latency at 51°C stimulation was shorter than the peak BOLD latency at 41°C stimulation.For each VOI and stimulus intensity, single trial averages were calculated from the individual BOLD responses. First, an average BOLD response of the significant voxels  was calculated across the four runs per stimulus intensity for each subject. Second, an average BOLD response was calculated across subjects, resulting in 72 BOLD responses (36 VOI × 2 stimulus intensities). The BOLD responses were extracted from the functional images in subject space by inverse transformation of the 36 VOI mask images. Voxelwise normalization over the time course was achieved by dividing the signal intensity at each time point by the voxel's mean intensity. All calculations were done using custom made Matlab software  . We idenA major difference between the time series analysis and the mixed effects analysis at group level is that no statistical comparison is made between the two stimulus modalities in the time series analysis. This distinction means that a higher BOLD amplitude following one stimulus modality compared to another does not necessarily mean higher activation. A statistical comparison between modalities, such as the mixed effects analysis above, is necessary to conclude that one stimulus gives more reliable activation than another.In summary, the identification of Aδ-predominant structures based on the functional imaging data was accomplished by combining three criteria: i) mixed effects analysis, identifying main effects (at 41°C and 51°C), ii) differences in the amplitude of the responses to different stimuli (51 > 41°C and 51 < 41°C); and iii) by latency analysis of the averaged BOLD responses  at 41°C and 51°C.Contact heat evoked potentials (CHEPs) were identifiable in all but one subject. The grand average responses at Cz after ten 41°C stimuli (major positive peak latency: 554 ms) and ten 51°C stimuli (352 ms) across subjects are shown in Figure The average rating of the 51°C stimuli was 2.9 ± 0.8  on the 5-point NRS Figure . This waWe sought to determine whether the cutaneous stimulation of Aδ fibers differentially activates structures that are anatomically distinct from structures activated by the cutaneous heat stimulation of C fibers.Of the 36 structures analyzed , larger activation at 51°C than 41°C (contrast of stimulus responses), and shorter 51°C latency than 41°C latency . Table Our findings show that two distinct sets of forebrain structures participate in elaborating the perception of "first pain" that follows the preferential excitation of Aδ heat nociceptors: 1) those preferentially responsive to noxious stimulation of Aδ fibers and 2) those with similar responses to converging inputs from the innocuous stimulation of C fibers. We used cerebral potentials to identify stimuli that predominantly stimulate Aδ or C fibers. We then used these stimuli in an fMRI contrast paradigm to identify structures that were preferentially active during the pain associated with the brief excitation of Aδ heat nociceptors as compared with the innocuous warmth associated with the brief excitation of heat-sensitive C fibers. Because this image contrast reflects both heat pain and preferential Aδ excitation, the relative contribution to the contrast of each of these variables is unknown. An experiment comparing painless and painful selective excitation of both Aδ and C heat receptors could address this question but would be challenged seriously by the strong association of Aδ heat receptors with pain and the temporal summation required to evoke heat pain mediated only by C fibers ,14,15,27By selecting stimulus temperatures and measuring the latencies of contact heat evoked potentials, we assured that nociceptive Aδ fibers were stimulated preferentially by 51°C and that C fibers were stimulated preferentially by 41°C. This assessment is based on the psychophysical measurements and the CHEP results, which were essentially identical to data presented in Granovsky et al.  for hairWe did not elicit a clearly painful sensation with contact heat stimuli that evoked a cerebral potential mediated by C fibers without evidence for Aδ fiber excitation. The inability to evoke pain reliably, if at all, with single, brief C fiber selective stimuli is in accord with previous studies ,31,33-37only during the 51°C stimulation, so the activity in this structure appears to be uniquely associated with the experience of "first pain".Because we were unable to directly compare and contrast structures active during "second pain" alone with those activated during "first pain", it is possible that two of the structures we have identified as responding preferentially during "first pain", the bilateral anterior insulae and contralateral hippocampus, would also be active during "second pain" because these structures also showed responses to the innocuous C fiber stimulus; this does not, of course, preclude their function as critical determinants in the mediation of "first pain". The ipsilateral P Ins, however, responded It is not surprising that the posterior insula is differentially and perhaps uniquely responsive during what may be considered among the most intense, salient, alerting, and well localized pain experiences. The posterior insula is connected primarily with cortical areas related to somatosensory, auditory, and visual sensory functions ,40. The Several studies suggest that the anterior insula is an essential component of the cortical network mediating early aspects of pain perception including the anticipation of pain; this is consistent with the differential, but not exclusive, response of these structures during "first pain". The anterior insula is predominantly connected with cortical areas related to limbic, olfactroy, gustatory, and viscero-autonomic functions; it receives input from entorhinal cortex, and sends projections to the entorhinal, periamygdaloid, and anterior cingulate cortices . All resThe entorhinal cortex, the main input pathway to the hippocampus, has strong reciprocal connections with the dysgranular and agranular cortex of the anterior insula , providiQiu and colleagues  used verThese findings show that two sets of forebrain structures mediate the initial sharp pain evoked by brief cutaneous heat stimulation: those responding preferentially to the brief stimulation of Aδ heat nociceptors and those with similar responses to converging inputs from the painless stimulation of C fibers. Our results suggest a unique and specific physiological basis, at the forebrain level, for the "first pain" sensation that has long been attributed to Aδ fiber stimulation and support the concept that both specific and convergent mechanisms act concurrently to mediate pain.aMCC: anterior midcingulate cortex; A Ins: anterior insula; BOLD: blood level oxygen dependent (response); CHEP: contact heat evoked potential; dPCC: dorsal posterior cingulate cortex; EEG: electroencephalography; EOG: electrooculography; EP: evoked potentials; fMRI: functional magnetic resonance imaging; HippHiInf: hippocampus - high inferior; HippInf: hippocampus - inferior; HippLowSup: hippocampus - low superior; HippSup: hippocampus - superior; HyInf: hypothalamus - inferior; HySup: hypothalamus - superior; LS1: lateral primary somatosensory cortex; LThal: lateral thalamus; MS1: medial primary somatosensory cortex; MThal: medial thalamus; pACC: pregenual anterior cingulate cortex; P Ins: posterior insula; pMCC: posterior midcingulate cortex; POFC: posterior orbitofrontal cortex; S2: secondary somatosensory cortex; VOI: Volumes of interest.The authors declare that they have no competing interests.DAM carried out the data acquisition  and drafted the manuscript. LHG contributed significantly to software programming, study design and statistical analysis of the fMRI data. TDT participated in the design and analysis of the evoked potential data. KLC participated in the design of the study, in data analysis and contributed significantly to the manuscript. All authors read and approved the final manuscript.


A series of experiments are carried out to investigate the factors which impact on the morphology of the Cu2O samples. It is observed that ethylenediamine (EDA) serves as a molecular template in the formation of hollow structure. Octahedral Cu2O with solid structure is prepared without EDA. When EDA is added, Cu2O sample with hollow structure is formed. Different morphologies of Cu2O such as spherical and octahedral could be obtained by adjusting the concentration of EDA and NaOH. The temporal crystal growth mechanism is proposed. Furthermore, the photocatalytic activities of the as-prepared Cu2O nanoparticles are evaluated by monitoring two dyes (methyl orange and congo red) using UV-visible spectrophotometer. Results show that the order of photocatalytic activity of Cu2O with different morphologies is as follows: hollow octahedral morphology > hollow sphere morphology > solid octahedral morphology. The hollow octahedral Cu2O nanoparticles would be a promising material on applications for photocatalytic degradation of organic pollutants.Herein, we report that octahedral and spherical Cu Thus, developing effective and facile methods for the synthesis of hollow Cu2O crystals especially with hollow octahedral morphology has become a key focus.Cuprous oxide , and the reducing agent hydrazine hydrate were involved. In this reaction, EDA served as a molecular template in the formation of hollow structure. The morphology of Cu2O nanoparticles can be easily tuned from spherical to octahedral with hollow structure by adjusting the concentration of EDA and NaOH. Moreover, the photocatalytic activities of the prepared Cu2O nanocrystals were investigated by methyl orange and congo red photodegradation.In this study, we mainly focused on a facile synthesis of hollow octahedral and spherical Cu−1) and varying amounts of EDA  were added to a glass reactor (capacity 50 mL). Afterwards, 3 to 10 mL of Cu(NO3)2 (0.10 mol L−1) aqueous solution was added. Followed by a thorough mixing of all reagents, the reactor was then placed in a water bath with temperature controlled over 25°C to 100°C. Finally, hydrazine  was added to reduce the Cu2+ ion to Cu2O. After 15 to 60 min, the cuprous oxide products with orange-red color were obtained. The resultant products were washed and harvested with centrifugation-redispersion cycles and dried at 60°C for 4 h in a vacuum oven. Further details on the synthesis can be found in supporting information 1 in Additional fileAll reagents purchased from the Shanghai Chemical Company  were of analytical grade and used without further purification. In each synthesis, 20 mL of NaOH (0.1 to 15 mol Lθ ranging from 20° to 80°). The morphological investigations of scanning electron microscopy (SEM) images were taken on a field emission scanning electron microscope . The transmission electron microscopy (TEM) images and electron diffraction patterns of the samples were captured on a JEOL/2100 F transmission electron microscope  at an accelerating voltage of 200 kV.The crystallographic structure of the products were determined with X-Ray diffraction (XRD) ; test conditions: Cu target at a scanning rate of 7°/min with 22O samples was performed by constantly monitoring the photocatalytic decolorization of two dyes (methyl orange and congo red) in aqueous solution under visible light irradiation (ordinary household table lamp) by the changes in UV-visible (vis) absorption spectra. The typical procedure was as follows: 0.01 g of the prepared sample was dispersed into 15 mL of the corresponding dye aqueous solution . Before illumination, the suspension was magnetically stirred in the dark for over 2 h to ensure adsorption equilibrium of the corresponding dye on the surface of the Cu2O crystal. Then, 300 μL of hydrogen peroxide was added to the solution, and the photocatalytic reaction was carried out with a 40-W daylight lamp (15 cm above the sample) used as a light source (ordinary household table lamp). The corresponding dye aqueous solution was then taken out in 20-, 40-, and 60-min intervals and centrifugated to exclude Cu2O. Finally, the corresponding dye aqueous solution was diluted fivefold and measured by a UV–vis spectrophotometer .The evaluation of the morphology-related photocatalytic properties of these Cu2O hollow crystals is based on the reduction of the Cu(EDA)22+ complex by hydrazine. The detailed experimental conditions on the synthesis of hollow spherical and octahedral Cu2O crystals were listed in supporting information 1 (A1-A2) in Additional file2O with hollow spherical and octahedral structures were displayed in Figure−1 and the concentration of EDA was 150 μL/30 mL , hollow spherical Cu2O particles with narrow size distribution were formed. Figure2O crystals, which suggested that the product exhibits a uniform, regular shape with an average diameter of about 300 to 350 nm. The crystal orientation and crystallinity of Cu2O hollow spheres were further studied with selected area electron diffraction (SAED) methods. The inset of Figure2O hollow sphere. As indicated by the clear diffraction spots, all of the hollow spheres were nearly single crystalline, although there are a little of intercrystallite mismatches . In the XRD pattern of hollow octahedral samples, very weak diffraction peaks of [002] and [−111] of CuO appeared. As the amount of the reducing agent is many times more than the stoichiometric ratio, CuO should not be formed in the synthesis procedure. CuO might be formed by oxidation of oxygen in water or air in the centrifugation-redispersion procedure. In any case, it proved Cu2O crystals were prepared.Formation of Cu2O, a series of reactions with different concentrations of EDA in 15 mol L−1 and 0.1 mol L−1NaOH solutions were investigated. Figure2O prepared with different concentrations of EDA in 15 mol L−1NaOH solutions. The detailed experimental conditions were listed in supporting information 1 (B1-B4) in Additional file2O crystals about 1,000 nm in size were prepared when EDA was unused. The inset TEM morphology proved the as-prepared Cu2O crystals to be a solid structure. The structure of Cu2O crystals turned to hollow when the concentration of EDA was 70 μL/28 mL , weak diffraction peaks of Cu for [111] and [211] crystal surfaces (JCPDS: 04–0836) appeared from the solid octahedral sample 42− complex could be formed; thus, no precipitation formed when EDA was unused. As shown in Figure2O crystals were prepared when the concentration of EDA was 40 μL/24 mL , the concentration of EDA played an important role on the morphological evolution of Cu2O. In conclusion, octahedral morphology benefited from the small concentration of EDA, and spherical morphology benefited from the relatively large concentration of EDA. In addition, NaOH concentration is another important factor. When the concentration of NaOH solution was 0.1 mol L−1, all the as-prepared products were of uniform hollow structure and about 400 nm in size.The morphological evolution of Cu2O nanocrystals is not very clear. We believe that EDA plays an important role as soft template for hollow structure formation. A classic rolling mechanism inspired by the natural phenomena of a piece of foliage or a piece of wet paper curling into scrolls during its drying process might be applicable for hollow Cu2O nanocrystal growth, which was used to explain the formation processes of metal chalcogenide nanotubes and nanorods surface on sample e. In addition, sample d has similar photocatalytic activity to samples e and f, but they had significant differences in the total surface areas. Here, the UV–vis absorption spectra result will give explanation.To investigate the reason why hollow octahedral CuO sample. The hol−4 g mL−1 for each. The UV–vis absorption spectra , UV–vis absorption intensity and utilization efficiency of visible light also contributed to the photocatalytic activities.The sample concentration for UV–vis absorption spectra test was 2 × 10a Figure suggesteFinally, different crystal surfaces have significant impact on the catalytic activity. It is reported that the photocatalytic activity of the [111] surface is much higher than other surfaces. Thus, octahedral nanoparticles (mainly coverd by [111] surfaces) have a higher photocatalytic activity than cubic-shaped nanoparticles and spherical nanoparticles (mainly coverd by [100] or [110] surfaces).2O samples could ascribe to the larger surface area, high utilization efficiency of visible light, and many [111] surfaces.In summary, these results demonstrated that the photocatalytic activities of the microcrystals were related to their morphology. The highest photocatalytic activity of hollow octahedral Cu2O samples with hollow structures had been synthesized by a simple chemical reduction method. In this reaction, EDA played a key role in the formation of hollow structures. Different morphologies of Cu2O could be obtained by adjusting the concentration of EDA and NaOH. The photocatalytic activities of these as-prepared Cu2O microcrystals with different morphologies, such as solid octahedral, hollow sphere, and hollow octahedral, were investigated by photodegradation of two dyes (methyl orange and congo red). The results demonstrated that hollow octahedral Cu2O possessed the highest photocatalytic activity, which could ascribe to the larger surface area, high utilization efficiency of visible light, and many [111] surfaces. The hollow octahedral Cu2O would be a promising material on applications for photocatalytic degradation of organic pollutants.In summary, octahedral and spherical CuThe authors declare that they have no competing interests.LLF designed and carried out the whole study. CLZ and GG participated in the discussion of this research. DXC gave the instruction of the study. All authors read and approved the final manuscript.Supporting information. A document showing supporting information 1 to 6 for facile synthesis of hollow Cu2O octahedral and spherical nanocrystals and their morphology-dependent photocatalytic properties.Click here for file