Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0±0.13 for micronuclei and 5.8±2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety. Ionizing radiation leads to chromosome damage of the type seen in cancer cells. Ionizing radiation is also an effective method for treating tumors because it can be localized to the tumor and is a potent inducer of DNA double-strand breaks, a highly toxic form of DNA damage. While much has been learned about x-ray and gamma-ray effects on cells and whole organisms, less is known about the biological effects of neutrons. Neutrons are highly energetic uncharged particles that induce more severe DNA damage than photons and are therefore more effective than photons in controlling radioresistant tumors. The relative biological effectiveness (RBE) of neutrons has been reported to be as low as 1 and perhaps higher than 10 depending on the tissue type, neutron energy and the biological endpoint being measured One of the major paradigm shifts in the field of radiation biology was the discovery of non-targeted effects such as the bystander effect in which cells in the vicinity of radiation-damaged cells behave as though they were also irradiated The bystander effect has been observed repeatedly in mammalian cell lines, including human skin fibroblasts, epithelial cells and leukemic cells in response to ionizing photons in vivoMost bystander effect studies have been performed using x-rays Here we used the cytokinesis-block micronucleus assay to address the question whether neutrons induce a bystander effect in normal human lymphoblastoid cell lines. We also assessed the RBE of fast neutrons produced by 50.5 MeV protons incident on a Be target (∼17 MeV average neutron energy) compared to cobalt-60 gamma radiation. For the endpoints of micronuclei and nucleoplasmic bridges, we found no evidence to indicate that neutrons induce a bystander effect in normal human lymphoblastoid cells. The measurements indicate that the neutron RBE for directly damaged cells compared to cobalt-60 gamma rays is 2.0±0.13 for micronuclei and 5.8±2.9 for bridges.The Nuclear Division Indices (NDI) for all experimental conditions were high enough to enumerate micronuclei and bridges, with the exception of cells irradiated with the highest two neutron doses (3 Gy and 4 Gy). Here, radiation-induced cell cycle delays precluded obtaining sufficient numbers of scoreable binucleated cells . AlthougFor both cell lines, direct irradiation with neutrons resulted in clear dose-responsive increases in the number of micronuclei  and nuclCells treated with ICCM from neutron irradiated cells did not show significant increases in micronuclei frequencies compared to sham treated controls for either replicate experiment and for either cell line . As expeThe neutron beam used in these experiments is contaminated with photons at a level of approximately 5%. Even though a bystander effect was not observed with the neutron exposures, we sought to determine whether photons might cause a bystander effect at the doses employed in these experiments. We cultured GM15510 and GM15036 cells in ICCM obtained from the corresponding cell line that had been irradiated with cobalt-60 at doses equivalent to 5% of the neutron doses. The results, shown in To reaffirm that the experimental conditions used here are capable of seeing a direct (i.e. non-bystander) effect of gamma ray exposure, experiments identical to those performed with neutrons were carried out with high doses of cobalt-60. Direct exposure to these photons with doses from 0 to 4 Gy showed a clear dose responsive increase in the number of micronuclei and the number of bridges per 1000 binucleated cells for both cell lines . The frePhotons have been known to induce bystander effects in human cells for many years. However, since the neutron exposures and the low-dose photon exposures did not induce a bystander effect, it was important to verify that the cells and the experimental conditions in these experiments, including the serum used in the culture media, are capable of demonstrating a bystander effect if one exists. As expected, GM15510 and GM15036 cells cultured in ICCM obtained from cells exposed to high doses of cobalt-60 gamma rays showed a 2 to 3 fold increase in micronuclei frequencies compared to sham treated controls, clearly indicating induction of a bystander effect . CompareIf the mechanisms of action for direct and non-targeted damage are independent, the total cellular response to ionizing radiation is the sum of the direct and the indirect exposure effects. The bystander components in the total dose response of cobalt-60 gamma rays ranged from approximately 4% to 35% for micronuclei; for bridges, the bystander components ranged up to 6% for GM15510 cells and up to 23% for GM15036 cells  and 3. IRBE is the ratio of doses that achieve the same biological effect for two radiation types. When the biological responses for both radiation types are linearly related to dose, as seen here, the RBE is also the ratio of the biological effects at the same dose. Here, the neutron RBE was calculated relative to cobalt-60 gamma rays by dividing the frequencies of micronuclei and bridges obtained in cells irradiated directly with neutrons at each dose to the frequencies obtained in cells irradiated directly with photons at the same dose. The RBE of neutrons for all doses and both cell lines is 2.0±0.13 for micronuclei and 5.8±2.9 for bridges , indicatThe experiments described here provide no cytogenetic evidence that fast neutrons are capable of inducing a bystander effect through medium-borne factors. The neutron beam used in these experiments was contaminated with photons, necessitating parallel evaluations to determine whether there is a positive or an inhibitory effect of these photons on the results of the neutron experiments. Our observation of an absence of a bystander effect following doses of photons that are associated with exposure to neutrons confirmed that there is a lack of a bystander effect in response to neutrons, regardless of the presence of photons. Wang et al. Different responses to neutrons were observed in the two cell lines we used. GM15510 cells cultured in ICCM from neutron irradiated cells showed substantial variation in micronuclei frequencies but no consistent dose-related response. In contrast, GM15036 cells had micronuclei frequencies lower than the corresponding 0-dose control value, although the control value was well within the historical range for this cell line. Although we did not measure apoptosis or necrosis, these outcomes may influence the responses seen in these cell lines.There is clear evidence of a bystander effect in response to high doses of photons when we used the same serum and cell lines as for the neutron experiments. This result indicates that the methods used in our study are capable of detecting a bystander effect if such an effect exists. To the best of our knowledge there is no other factor that could have prevented neutrons from inducing a bystander effect in these cells, assuming a bystander effect even exists. Our findings are in agreement with previous studies that have reported the lack of a bystander effect on neutron exposure using clonogenic cell survival assay in a human skin cell line The inability of fast neutrons to induce a cytogenetic bystander effect as shown here may be due to different types of damage induced at the molecular level compared to photons. Cellular recognition of DNA damage and the subsequent repair processes may differ between neutrons and photons. Furthermore, due to the lower levels of oxidative damage and free radical production by neutrons compared to photons Neutrons, depending on their energy, might be more effective in controlling certain tumor types where conventional photon therapy is ineffective This study used cells that lack gap junctions. There is a possibility that a neutron-induced bystander effect requires physical contact between cells, which could be tested by performing experiments using cell lines such as fibroblasts and keratinocytes that have gap junctions. If no bystander effect is induced in these cell lines, then it may be likely that neutrons do not have any ability to induce a bystander effect. Another possible explanation for the lack of a bystander effect with neutrons observed in this study may be the presence of dimethyl sulfoxide (DMSO), a scavenger of reactive oxygen species For cells irradiated with high doses of photons, a considerable amount of damage was attributed to the bystander component. The percent contribution by the bystander exposure to the direct exposure was highest at the lowest dose delivered (0.5 Gy) and then it appears to saturate as dose increases, perhaps because there is saturation either of the bystander signals or the cellular responses to those signals We report two RBEs for neutron radiation, one for micronuclei and the other for nucleoplasmic bridges. These two genetic endpoints have different mechanisms of formation. Micronuclei are formed from lagging chromosomes and acentric fragments at anaphase, while nucleoplasmic bridges are formed when centromeres of dicentric chromosomes are pulled in opposite directions during mitosis In conclusion, we found no evidence for a bystander effect following exposure to fast neutrons (17 MeV average energy) or to doses of cobalt-60 photons equivalent to 5% of the neutron dose. As expected, a bystander effect was seen with high doses of photons, as evaluated by micronuclei frequencies and nucleoplasmic bridges. These results will facilitate refined estimates of the risk-benefit ratio of neutron therapy and may be valuable to those who are concerned about the health effects of exposure of space travel. We have also shown that these fast neutrons have a relative biological effectiveness of 2.0±0.13 for micronuclei and 5.8±2.9 for bridges compared to cobalt-60. Understanding the biological effects of neutrons may also enable more refined evaluations of the standards for radiation protection and safety.Normal human lymphoblastoid cell lines (GM15036 and GM15510) obtained from the Coriell Cell Repository were used for these experiments because they have previously been shown to exhibit a bystander effect in response to gamma radiation 2 incubator at 37°C. Approximately every 3 days the cells were counted and passaged by seeding at a concentration of 3×105 cells/ml. Cell culture for all the gamma radiation experiments was performed at Wayne State University (WSU), Detroit, Michigan. For the neutron radiation experiments, cells were grown at WSU up to a concentration of 1×106 cells/mL, then non-vented T-25 flasks were completely filled with media (60 ml) containing cells at this concentration. Two flasks per cell line were then shipped overnight to the University of Washington Medical Center (UWMC), Seattle. Upon arrival, the flasks were immediately placed upright in a fully humidified 5% CO2 incubator at 37°C, and their caps were loosened. The flasks were left undisturbed for 24 hours after which the cells were counted and checked for viability using a hemocytometer and trypan blue staining. Cells were then passaged once and cultured as described above.Cell culture was performed using the standardized protocol provided by Coriell. The serum used in this study was prescreened for its ability to support a bystander effect with the cytokinesis-block micronucleus assay in these same cell lines. Since serotonin has been reported to play a role in the bystander effect 2 field (SSD of 148 cm). The average energy and neutron mean free path varies with field size and water-equivalent depth . For experiments reported in this work, the average neutron energy is about 17 MeV, and the neutron mean free path in water is about 9 cm. For every dose, two flasks were irradiated per cell line: one flask was used to assess the damage induced by direct radiation exposure and the other flask was used for medium transfer for the bystander effect as described below. For the direct damage and the bystander effect experiments, three different controls were used: pre-radiation, post-radiation, and transportation control. Pre-radiation and post-radiation control flasks were sham-irradiated with exposure times corresponding to the lowest (0.5 Gy) and the highest (4 Gy) neutron dose, respectively. The transportation control involved flasks that were transported with the cells that were irradiated, but remained inside the insulated box; these control flasks were further insulated with bubble wrap to maintain their temperature close to 37°C. This box was the same as that used to carry the flasks to and from the laboratory and the radiation center . For the bystander effect an additional media-only control was included; these flasks contained fresh, complete, culture media without any cells and were irradiated at the highest dose, i.e. 4 Gy. Media from these flasks was transferred to non-irradiated cells in the same manner as described for media transfer from flasks that contained cells. Following exposure the flasks were returned immediately to the laboratory and incubated for 28 hours. The cells were then harvested as described below. For each cell line, the neutron bystander experiment was performed twice, once each on different days. Replicate 2 had all the controls as described for replicate 1 except the transportation control was not included.All neutron irradiations were carried out using the fast clinical neutron therapy system (CNTS) at the University of Washington . All gamma irradiations were performed in the Gershenson Radiation Oncology Center, WSU. For neutron irradiations, the cells in culture medium in T-25 flasks were irradiated at room temperature with doses 0 (sham), 0.5, 1, 1.5, 2, 3 and 4 Gy. The CNTS generates fast neutrons by targeting a near monoenergetic 50.5 MeV proton beam at a Be target (10.5 mm thick with a radius of 0.635 cm). The neutron beam is shaped by a primary collimator composed of iron and a secondary collimator made up of individually movable leaves composed of iron with cylindrical polyethylene inserts. Cells in T-25 flasks with culture medium were placed on top of 2 cm of water-equivalent material and irradiated at 0.6 Gy/min from below (gantry at 180 degree) using an open 28.8×28.8 cmThe Be target system used to generate fast neutrons also delivers a photon dose of about 5% of the neutron dose, which raises the possibility that any bystander effect observed in the experiments could be due to photons rather than to the neutrons. To rule out this possibility, cells were acutely irradiated with cobalt-60 gamma rays at doses equivalent to the 5% of the delivered neutron dose, i.e. 0 (sham), 0.025, 0.05, 0.075, 0.10, 0.15 and 0.20 Gy. We also included a media-only control containing fresh media without cells, which was irradiated at the highest dose, i.e. 0.2 Gy.As a positive control for the bystander effect, cells were exposed to 0 (sham), 0.5, 1, 2, 3 and 4 Gy cobalt-60 gamma rays. To ensure that the bystander effect observed was actually due to signals produced by the irradiated cells rather than an effect of exposure of the culture medium or an artifact of the media transfer process, a media-only control was included in which fresh media without cells was irradiated at the highest dose (4 Gy) prior to being transferred to non-irradiated cells. For the gamma radiation experiments, flasks were transported to and from the laboratory and the radiation center, a 5-minute car ride, in an insulated container as described above.To assess the effects of direct radiation damage on these cells, Cytochalasin B  dissolved in DMSO  was added four hours after irradiation to directly-irradiated cells to block cytokinesis. The final concentration of DMSO in each culture was 1.1%. The cell cultures were then incubated at 37°C for 28 hours.Following irradiation, the cells were left undisturbed until media transfer, which was performed four hours after irradiation as previously described 2 glass coverslips.Twenty-eight hours following media transfer, the cultures were swirled and pipetted gently to resuspend and break up the clumps of cells. Cells that were directly irradiated as well as those treated with conditioned media were centrifuged onto ethanol-cleaned microscope slides for 4 minutes at 93 x g using a cytocentrifuge . The slides were air-dried, fixed in 100% methanol  for 15 minutes, and then stained with 100 µl of acridine orange (0.5 mg/ml in 1X PBS)  for 1 minute in the dark. The excess stain was removed by washing in 1X PBS for 1 minute. The slides were then mounted with 1X PBS and 25×25 mmAll slides were coded prior to scoring to prevent observer bias. The Nuclear Division Index (NDI) for each dose and treatment was determined by evaluating at least 200 cells and determined according to the following formula: Cells were then evaluated simultaneously for micronuclei, nucleoplasmic bridges and buds according to our “relaxed” criteria Analysis of variance (ANOVA) was performed to evaluate radiation-induced dose responses in cells directly irradiated with neutrons or high doses of photons, and in cells treated with ICCM from these irradiated cells. For each experiment, the independent variables considered were cell line, dose, and the interaction between cell line and dose. The dependent variables were micronuclei and bridges. The same set of analyses were performed for the neutron bystander data which included replicate experiments as an additional variable. The Tukey HSD test was used for post-hoc evaluations. These analyses were performed using JMP software version 6.0, SAS Institute Inc. Chi squared analyses were used to evaluate changes in the frequencies of micronuclei and bridges in the irradiated cells as a pooled group compared to the unirradiated (0-dose control) cells for the high dose photon experiments, and for the low dose photon contamination experiments.


In the course of avian evolution, the λmax of the most shortwave-sensitive cone, SWS1, has switched between violet (λmax > 400 nm) and ultraviolet (λmax < 380 nm) multiple times. This shift of the SWS1 opsin is accompanied by a corresponding short-wavelength shift in the spectrally adjacent SWS2 cone. Here, we show that SWS2 cone spectral tuning is mediated by modulating the ratio of two apocarotenoids, galloxanthin and 11’,12’-dihydrogalloxanthin, which act as intracellular spectral filters in this cell type. We propose an enzymatic pathway that mediates the differential production of these apocarotenoids in the avian retina, and we use color vision modeling to demonstrate how correlated evolution of spectral tuning is necessary to achieve even sampling of the light spectrum and thereby maintain near-optimal color discrimination.Color vision in birds is mediated by four types of cone photoreceptors whose maximal sensitivities  light. This ability results from a change in the light-sensitive protein within the violet cone photoreceptor that shifts its sensitivity towards UV light. This expansion of vision into the UV is complemented by a shift in the sensitivity of the blue cone photoreceptor. However, it is not well understood exactly how the sensitivity of the blue cone is shifted and how this shift impacts color vision.To find answers to these questions, Toomey et al. characterized the light-filtering carotenoid pigments from bird species with violet or UV sensitivity, and used computational models of bird vision to predict how these pigments affect the number of colors a bird can see. This approach revealed that blue cone sensitivity is fine-tuned through a change in the chemical structure of the light-filtering carotenoid pigments within the photoreceptor. Computational models also indicated the sensitivity of the violet and blue cones must shift in a coordinated manner to maximize the number of colors a bird can see.These results suggest that both blue and violet cone cells have been fine-tuned during evolution to enhance color vision in birds. An important next step is to investigate the underlying molecular mechanisms that coordinate the modification of the carotenoid pigments and the tuning of light-sensitive proteins in a wide range of bird species.DOI:http://dx.doi.org/10.7554/eLife.15675.002 Color vision systems are based on visual pigments consisting of a retinoid chromophore bound to an opsin protein . When thmax > 400 nm), and ultraviolet-sensitive species (UVS), whose SWS1 cone is maximally sensitive to ultraviolet light (λmax < 380 nm) (All birds can be classified into two groups: violet-sensitive species (VS), whose most short-wavelength sensitive cone (SWS1) is maximally sensitive to violet light (λ 380 nm) . In the  380 nm) . Althoug 380 nm) . This sh 380 nm) . HoweverAccordingly, between VS and UVS species, the sensitivity of the short-wavelength sensitive 2 (SWS2) cone is tuned to maximize its spectral separation from the adjacent SWS1 and medium-wavelength sensitive (MWS) cones . Howeverde novo and must acquire them through their diet , the C-type droplet contains primarily galloxanthin with a peak absorbance at 402 nm as well as an apocarotenoid of undetermined structure (Apo2) with short-wavelength-shifted peak absorbance at 380 nm  analysis, we found that the molecular weight of Apo2 was two daltons greater than that of galloxanthin ; Table 1To predict the site of this saturation, we calculated the wavelength of maximum absorbance for all eight possible monosaturated forms of galloxanthin based on the Woodward-Fieser and Fieser-Kuhn rules , and com1H NMR, and then used MS/MS spectroscopy to compare this standard to the products of ozonolysis of derivatized Apo2. The critical product of Apo2 derivatization-ozonolysis had a parent ion and fragmentation pattern identical to that of the standard, confirming the presence of saturation at the 11’,12’ double bond ; however, its functional role in that tissue is unknown  . We founWhole retina measurements, however, may not be the best indicator of the specific composition of the C-type oil droplets. The relationship between whole retina apocarotenoid levels and the filtering of the C-type droplet is potentially confounded by the additional presence of apocarotenoids in other oil droplet types . In otheTo measure the apocarotenoid content of the C-type droplets directly, we made detailed microspectrophotometric measurements of expanded droplets from two UVS and two VS species . We founBirds acquire carotenoids through their diets and can metabolize these precursors in a variety of ways . RadioacBCO2 and expressed it in HEK293 cells. We then delivered a zeaxanthin substrate to the cells and characterized the resulting apocarotenoid products by HPLC. Consistent with our hypothesis, cells expressing BCO2 yielded a product consistent with , with a characteristic single absorbance peak at 449 nm  ; 2007. Wbination .F6,12 = 19.59, p=1.50 x 10−5, dihydrogalloxanthin: F6,12 = 21.00, p=1.03 x 10−5, BCO2, RDH12, and RETSAT increased significantly through the course of development, mirroring the developmental trends in apocarotenoid accumulation  in 6 nm intervals across the spectrum from 370 to 500 nm, while holding all other parameters constant  model of color discrimination to predict the maximum number of discriminable colors for a range of hypothetical visual systems with varying C-type oil droplet spectral filtering . For thiconstant , and preIt is important to note that in this analysis we evaluate all possible discriminable colors; however an organism is likely to only encounter a subset of these colors in nature . In the −2), the color discrimination thresholds of birds largely follow Weber’s law, indicating that receptor noise scales as a fraction of the stimulus magnitude with small deviations attributable to photon-shot noise (−2), Weber’s law no longer holds, and the color discrimination thresholds are limited primarily by photon shot noise (−2), the avian visual system nears the absolute limit of color discrimination which is likely set by the intrinsic dark noise within the receptors . The influence of these sources of noise on discrimination depends upon light levels. In bright light, approximately equivalent to sunset or brighter . The optimal positions of the UVS species were short-wavelength shifted  relative to the optima for the VS species , a pattern consistent with the actual C-type oil droplet spectral filtering observed within the respective visual systems  were significantly greater than the VS species  and UVS species . However the predicted optima of spectral filtering in the dim and very dim light conditions differed substantially from the observed values  accumulates a UV-absorbing apocarotenoid in its lens . The MWS cone, has a yellow zeaxanthin-pigmented oil droplet (11 conjugated double bonds), and, as we show here, the SWS2 cone has an oil droplet containing a mixture of dihydrogalloxanthin and galloxanthin  . All of Modulation of the number of conjugated double bonds in the filtering pigments of SWS2 cones represents one instance of a general mechanism of spectral tuning utilized by a diversity of organisms. Many cold-blooded aquatic organisms dynamically shift the sensitivity of their visual system by changing the degree of conjugation of the visual pigment chromophore, retinal . In contHere, we present evidence that galloxanthin and dihydrogalloxanthin are products of an enzymatic pathway that likely includes BCO2, RDH12, and RETSAT. Each of these enzymes has previously been identified as a carotenoid- or retinoid-metabolizing enzyme , but ourRDH12 is the most highly expressed member of the short-chain dehydrogenase/reductase (SDR) family in avian photoreceptors . In mammWe have proposed that the 11’,12’ saturation of galloxanthin is mediated by RETSAT, an enzyme known to carry out an analogous saturation of retinol to produce 13,14-dihydroretinol . In thisChanges in the filtering of the C-type oil droplet between VS and UVS bird species shift the peak sensitivity of the SWS2 cone photoreceptor to maintain even spectral separation from the adjacent SWS1 and MWS photoreceptors. Interestingly, perfect spectral separation among all of the single cone photoreceptor subtypes would also require a blue-shift in the sensitivity of the MWS cone in UVS species. However, for the species measured to date, there are no systematic differences between VS and UVS species in the positioning of the spectral sensitivity of the MWS cone. This suggests that the spectral tuning of MWS may be mechanistically constrained or its precise tuning has a relatively limited impact on visual function.cut values and actual measured values, and an overall decline in color discrimination performance. This is not necessarily surprising, because the species we examined are all primarily active in bright daylight conditions and unlikely to be adapted for low-light vision. Interestingly, nocturnal species (e.g. owls) have been reported to have pale oil droplets that lack carotenoids  (Waters Corp.). We used a mobile phase gradient beginning with 20:80 water:methanol (v:v) up to 100% methanol over the course of 5 min. Detection was performed using a quadrupole/time-of-flight mass spectrometer  equipped with an electrospray ionization (ESI) source operated in both positive and negative modes of polarity. ESI conditions included a capillary voltage of 3.2 kV for positive mode, 2.8 kV for negative, cone voltage of 35 V, ion guide at 1 V, source temperature of 100°C, and nitrogen desolvation gas temperature of 400°C flowing at 600 L/h. We compared the observed apocarotenoids to authentic standards of galloxanthin and 3-OH-β-apo-12’-carotenol purchased from CaroteNature GmbH .Whole retinas from chickens (21 days post-hatch) were extracted three times with 500 μl of hexane:tert-butyl methyl ether 1:1 (vol:vol). The extracts were combined, dried under a stream of nitrogen, and saponified with 0.2M sodium hydroxide in methanol at room temperature, in the dark for six hours, as detailed in . We re-emax) for all possible monosaturated forms of galloxanthin, we used the Woodward–Fieser rule for molecules with ≤4 conjugated double bonds and the Fieser-Kuhn rule for molecules containing >4 double bonds  of 4-nitrobenzoyl chloride , filtered and concentrated to give a residue that solidified under vacuum. A portion of this solid was purified by silica gel preparative thin-layer chromatography (5% CH3OH/CH2Cl2) to give a white solid with the expected properties for model compound 2  δ 1.98 , 2.07 , 2.65 , 4.32 , 8.22 , 8.32 ; UV (CH3OH) λmax 257 nm; HPLC tR = 6.8 min (0.8 mL/min of 70% CH3OH/H2O on a 250 x 4.6 mm Polaris C18 column). Negative ion APCI MS showed the expected molecular ion for C13H12NO5 at m/z 251.08102. The ozonolysis products were chromatographed using an Eclipse plus HD C18  with a 0.1%(v/v) formic acid in water versus 0.1%(v/v) formic acid in methanol mobile phase gradient at 0.3 mL/min and 40°C column temperature. From 0–3 min a gradient was applied from 80/20 A/B to 0/100, held for 1 min, and then re-equilibrated over 1 min to initial conditions. Eluent was interfaced via an APCI probe in negative mode with a 6550 QTOF (Agilent) with 40 µA corona, 3 kV vcap, 40 psi nebulizer, 15 L/min drying gas at 290°C, and 500°C vaporizer. MS and MS/MS data were acquired at 2 Hz from 100–1700 m/z, and MS/MS spectra were collected at 10 eV.Approximately 200 mg (1.96 mmol) of 5-hydroxy-2-pentanone  was dissolved in CHmpound 2 : 1H NMR Haemorhous mexicanus), a UVS species, containing approximately 2 µg of the unknown apocarotenoid  added and the solution was saturated with ozone (O3) generated from dry O2 using a Welsbach  laboratory ozonator, model T-816 (the apocarotenoid solution containing presumed compound 4 (3). An equal volume of 25 mM phosphate buffer, pH 6, was added, the two layers shaken together  filtered and concentrated. Analysis of the residue by HPLC as for the model compound 2 showed a band at the comparable retention time (see above). Further analysis of this sample by LC-MS showed the presence of a component identical to model compound 2 and, importantly, no evidence for the smaller fragment (C9H7NO5), which would have formed by apocarotenoid ozonolysis had there been an 11’,12’ double bond present to oxidatively cleave.The HPLC isolated fraction from the retina of the house finch  followed by 38 ×  and 10 min at 72°C. The PCR products were purified on a 2% agarose gel, extracted with QIAquick Gel Purification kit (Qiagen) and the resulting products were sequenced by the Protein Nucleic Acid Chemistry Laboratory (PNACL) at Washington University using the same primers as used in the initial PCR reactions. We translated the resulting DNA sequence and identified the tuning sites at AA86, AA90, and AA93 and inferred VS vs UVS tuning following published analyses  and the relative abundance of the different apocarotenoids in the retinas of these species. To analyze the data in a phylogenetic context, we generated 100 phylogenies for all sampled bird species using the resources at subsets/ , with thsubsets/  and adjusubsets/ . For eac picante .The apocarotenoid compositions of the C-type droplets in chicken (VS), mallard (VS), zebra finch (UVS), and house finch (UVS) retinas were determined by collecting detailed absorbance spectra from expanded individual C-type droplets with microspectrophotometry and fitting these with the pure spectra of the seven major carotenoids and apocarotenoids in the retina. These methods are detailed in BCO2 and RDH12 from 21 day-old chicken retina cDNA and RETSAT from adult zebra finch retina cDNA using the primers listed in NdeI and BsrGI restriction sites and a C-terminal c-Myc tagging sequence. We confirmed the sequence of each gene by Sanger sequencing (PNACL). We cultured HEK293 cells  following the distributor’s recommended protocols for up to 25 passages. For the enzyme assays, we cultured the cells in 6-well plates (each well 9.6 cm2) to 80% confluency and transfected the enzyme expression constructs along with a vector containing a CMV promoter driving the expression of the reverse tetracyclin-controlled transactivator using the TransIT reagent . Twenty four hours after transfection, we induced protein expression by adding doxycycline to the media at a concentration of 1 μg/ml. We purified zeaxanthin from a bacterial culture transformed with the zeaxanthin-producing plasmid pAC Zeax, a gift of Francis Cunningham   . To prodWe eluted the carotenoids with a gradient mobile phase consisting of acetonitrile:methanol:dichloromethane (44:44:12) (vol:vol:vol) from 1–11 min, then a ramp up to acetonitrile:methanol:dichloromethane (35:35:30) from 25 min, followed by isocratic conditions through 30 min. The column was held at 30°C and the flow rate was 1.2 ml/min throughout the run. We monitored the samples at 400 nm and identified galloxanthin by comparison to an authentic standard purchased from CaroteNature GmbH . We identified dihydrogalloxanthin by comparing absorbance spectra and retention times to measurements of this compound in retina samples. We putatively identified 3-OH-β-apo-10’-carotenal by comparison to published reports of this apocarotenoid .BCO2, RDH12, and RETSAT from the retinal cDNA of 21 day-old chicken with PCR primers that included EcoRI or Xho1 restriction sites (EcoRI or Xho1 restriction site and confirmed the sequences by Sanger sequencing (PNACL). We then generated probe templates by PCR with T3 or T7 primers and generated digoxigen-labeled probes from these templates following established methods , incubated eggs, and reared chicks to specific developmental stages. Chicks were fed a carotenoid-rich diet . We harvested retinas from the embryos (E) at days 16, 18, and 20 of incubation and chicks at days 0, 7, 14, and 21 post-hatch (P). The birds were euthanized by carbon dioxide asphyxiation following an approved protocol (Washington University ASC protocol #20140072). We collected three biological replicates of retina tissue at each time point for each analysis. We measured apocarotenoids levels by HPLC as described above and used multiple analysis of variance (MANOVA) to compare the change in apocarotenoid levels over time.To measure the accumulation of apocarotenoids and the transcript levels of BCO2, RDH12, RETSAT, and GAPDH (t) levels used for quantification in the time course analyses. We assayed each time point and biological replicate in triplicate using Sybr Green PCR master mix  with an Applied Biosystems StepOne real time PCR system . We normalized enzyme transcript expression relative to GAPDH expression. We then averaged the technical replicates for each biological replicate and compared expression levels (ΔCt) over time for each gene with analysis of variance (ANOVA).For qPCR analysis, we harvested retinas and stored them in Trizol reagent  for up to six months at -80°C. We then extracted RNA from the samples following manufacturer’s recommendations . We generated cDNA from ~1 μg of RNA using Superscript IV reverse transcriptase . We selected primers flanking the last exon/exon junction of nd GAPDH . To testn-dimensional color space, where dimensionality corresponds to the number of spectrally different photoreceptors encoding the colors. The coordinate axes represent the quantum catch, Q, of these photoreceptors and are given by:To evaluate the role of the spectral tuning of C-droplet, we used mathematical models of color discrimination. Colors can be represented as loci within i is receptor type , λmax and λmin are the upper and lower wavelength limits of the visible spectrum, which are set to 700 nm and 300 nm respectively, where Receptor sensitivity was determined as:where template  and an otemplate , and o ≤ 1. The boundaries of the color solid are formed by the color loci of optimal color stimuli that are imaginary reflectance spectra with only two possible values, 0 or 1, and with n-1 transitions . The solid defined by optimal color stimuli has two apices corresponding to an ideal black As a measure of performance, we used the number of discriminable colors within the object-color solid. The color solid is the volume within color space occupied by all possible object colors given by reflectance spectra, u, containing the loci of all colors that cannot be discriminated from the central locus. The distance between the central locus and the surface of the confusion-volume represents threshold difference, which is a property that varies with position and direction in color space. The total number of discriminable colors for an object color-solid of volume, V, is given by:Each color locus in color space is surrounded by a 'confusion-volume', and integration is carried out over the complete solid volume. We assume that thresholds are determined by photoreceptor noise, which has proven a successful approach in modeling of color discrimination . Relevanand photon-shot noise that originates in the stochastic nature of photon absorption:In very dim light, dark noise predominates. This noise does not depend on the light signal:We consider three cases corresponding to different levels of illumination. In very dim light, we assume that the dark noise limits discrimination . The absFrom the photoreceptor noise, we can estimate the confusion-volume by:c, depends on the relationship between the threshold and noise but is omitted from the calculations as we assume that the threshold criterion is invariant. j(λ), and a maximum intensity of T (assuming a green illumination spectrum given by vegetation gives very similar results to those reported here). We assumed levels of illumination equivalent to a quantum catch of the LWS cone of 100,000, 100, and 10 photons sec.−1 for the bright, dim, and very dim conditions, respectively. We describe the quantum efficiency of photoreceptor i for the illuminating spectrum by:The parameter, and we can reformulate quantum catch  as:(10)Qx is given by:where andkLWS = 1, and we fix the spectral sensitivity of the UVS, MWS and LWS cones so that both kUVS/VS and kMWS are kept invariant. For each species we repeat the calculations of the color solids using different C-droplet positions, which yield different spectral variants of the SWS cone and thereby different values for kSWS. This approach is valid because we determine the optimal C-droplet position for the maximal number of discriminable colors, while absolute numbers are irrelevant. The shape of the color solid can now be represented by a 'shape factor', G. The shape factor is calculated differently depending on the origin of noise. In the case of very dim light:We normalize the scaling factors describing photoreceptor quantum efficiencies to the LWS cone, i.e. and the total number of discriminable colors is given by:If the fluctuations of absorbed photons limit the color discriminability, thenAnd the total number of discriminable colors is given byIn bright light, when both the Weber noise and the shot noise play role, the shape factor depends on the light intensity;and the total number discriminable colors are calculated using t-tests. We compared the optimal predicted C-type droplet filtering and the predicted number of discriminable colors between bright and dim light conditions with paired sample t-tests.More details of the theory behind object-color solids and how they can be computed are found in . We compWe modeled the increment spectral sensitivity function using a commonly adopted receptor noise-limited model (RNL) of color discrimination . The RNLThe increment spectral sensitivity function describes detection of monochromatic light superimposed on a large adaptive background, where sensitivity is expressed as the inverse of the detection thresholds. For large and static stimuli, detection is mediated by color vision. We account for background adaption by defining photoreceptor quantum catch in relation to the quantum catch of the background. From Note that for small =Δln(Qi) . For bac=Δln(Qi) . The chrω is the Weber fractions and tI at S = 1 JND. We assume that receptor noise is independent of receptor type and inversely proportional to the relative frequency of the receptor types within the retina by  whether the model of cone noise is accurate; and (2) how the conclusions of the model depend on the spectra distribution of inputs. Because the modeling is an essential piece of the paper, and indeed one of the most novel aspects of the paper, these issues need to be dealt with directly. Specifically, it would be very helpful to know how the conclusions of the model depend on the assumptions upon which it is based. Detailed comments from each reviewer follow, including elaboration of the points above. We are including the full reviews so that you can see the convergence onto the essential points above.Reviewer #1:This paper uses an impressive array of techniques to investigate coordinated shifts in spectral tuning of two short-wavelength sensitive photoreceptors in birds. The central finding of the paper – that shifts in the short wavelength photopigment spectral sensitivity produce coordinated shifts in the absorption properties of the oil droplet controlling spectral tuning of the neighboring photoreceptor – are well supported by the data. I am not an expert on photopigment chemistry, so will leave detailed evaluation of that part of the paper for the other reviewers. I did have some concerns about the model for the impact of these changes in color coding:1) Cone noise model.Investigating chromatic discrimination requires a model for noise introduced in the cones. The cone noise is modeled in the paper as a constant Weber fraction. But this appears inconsistent with direct measures of cone noise. Generally, intrinsic noise dominates total cone noise at low light levels. As light levels increase, a point is reached at which quantal fluctuations begin to contribute and in some cases dominate. This behavior is seen in Lamb and Simon (1977), Rieke and Baylor (2000), Angueyra and Rieke (2013). I suspect a noise model consistent with these measurements would alter the findings in the paper about the dependence of optimal spectral sampling on light level. If there is a solid justification for using a constant Weber fraction noise model that should be given in the context of what is known about the cones. If not, a model more in line with the cone measurements should be used.2) Measurement of pigment mixtures in oil droplets . The data showing how the measured absorption spectra were fit to obtain a mixture of carotenoids is quite important but is not shown in any detail. I think that data needs to be included, both as absorption spectra and template fits and as summary data.Reviewer #2:This is an exceptionally comprehensive study of how in birds filtering of the light by the SWS2 ('blue') cone, which has a single spectral type of opsin, modifies the spectral sensitivity of this photoreceptor in concert with shifts in the shorter wavelength sensitive SWS1 (Violet/UV) cone opsin spectral sensitivity. This relationship between the spectral sensitivities of the two types of photoreceptor has been known since around 2000, but has received little attention.Similarly, the role on carotenoid metabolism in tuning the coloured oil droplets in avian cones has been studied, but to my knowledge this is far the most comprehensive investigation of carotenoid metabolism in this context.It is therefore the link between the biochemistry and visual function that gives this work its main significance beyond the specialised fields of carotenoid metabolism and avian colour vision. One can quantitatively specify performance, here in terms of the number of discriminable colours, and hence link the trait (carotenoid metabolism) to its function (colour discrimination) in a way that is seldom possible in other domains.Thus this is a highly novel contribution to visual evolution and ecology, which has hitherto looked at phenomena such as photoreceptor noise and spectral tuning, and the optical design of eyes, but not spectral filtering by photostable pigments (carotenoid or other). As far as I can tell the work is done impeccably, and insofar as my competence allows, I have no concerns about the findings.I do however have two questions:1) At no point does the paper address the question of why shifts between UVS and VS type opsin occur so often in avian evolution, or why intermediate variants do not evolve. This is, of course, not the main subject, but a natural question to ask, so it would at least be good to know that we have no idea!2) More substantively, the model of performance evaluates the number of discriminable colours available to the different types of eye taking account of photoreceptor noise. This measure is potentially misleading:It is a priori obvious (and certainly doesn't need the modelling done here) that reducing spectral overlap increases the volume of the colour solid so that if the Weber fractions of the different cone types are fixed  then is obviously beneficial to reduce – or ideally eliminate – spectral overlap (see Eq 12).It is mainly when the effects of photon noise (Eq 13) are taken into account that the modelling of the kind is useful. The model estimates a 'confusion volume', and hence the number of discriminable colours under a natural illuminant. These volumes depend upon photon noise, and hence quantum catch. This raises two questions: i) what are the spectral compositions of the stimuli that are being discriminated, and ii) how do they relate to the reflectances of real or even hypothetical objects?For example, if spectra are generated at random – say  with a reflectance of either 0 or 1 at each 1-nm interval – then it is easy to see that the density of spectra falling in the central part of the colour space will greatly exceed that in the periphery potentially reducing the benefit of minimising spectral overlap.In reality most potentially discriminable colours, perhaps 90% – 99% of the 8-million or so, will hardly ever occur, whereas most natural spectra will reside in a very small proportion of these colour spaces. It should therefore be explained how the estimates of the number of discriminable colours might be relevant to any natural task.Therefore, although it is intuitively reasonable that photoreceptor spectral sensitivities should overlap 'by a certain amount but not too much' I do wonder if the number of colours is a misleading characterisation of the utility of the birds' colour vision.Reviewer #3:Toomey and colleagues present here in this study a thorough investigation of the tuning mechanisms behind avian vision in the shorter wavelength spectrum. Birds have been shown to have their shortwave-sensitive pigments (SWS1) tuned either to the violet (VS) or the ultraviolet (UVS) spectrum and this SWS1 shift is accompanied by a same direction shift in the tuning of SWS2 pigments. Colour vision in birds is achieved by the presence of 4 types of cone opsins but is also regulated by the presence of oil droplets in cone photoreceptors. In this study, the authors demonstrate that the SWS2 tuning shift is mediated by modulating the ratio of 2 carotenoids  present in the oil droplet found in SWS2 cones. They elucidate the molecular structure and identity of one of these carotenoids which was previously unknown and go on to report the ratio of these two carotenoids in 21 and 24 species of VS and UVS birds, respectively, showing a correlation between the ratios, SWS2 tuning and VS/UVS pigments. They show that shorter shifted SWS2 pigments are found in UVS species and have a higher amount of dihydrogalloxanthin while VS species have more galloxanthin. They then further investigate the possible enzymatic pathway from where dietary zeaxanthin is converted to dihydrogalloxanthin and galloxanthin through a 3-step enzymatic process. They show in vitro evidence that their proposed pathway can generate these 2 carotenoids and further confirm expression of the enzymes in the avian retina during development. Finally, creating a modelling system of the avian UVS and VS visual system, Toomey and colleagues were able to investigate the role of the complementary SWS1 tuning and SWS2 oil-droplets in colour discrimination in both VS and UVS species.I believe this is a thorough study of the tuning mechanisms used by avian species to modulate their visual system and consequently colour discrimination. The authors offer a complete hypothesis of how this process works and what are the direct effect on the visual system of these species.eLife. Your revised article has been favorably evaluated by Eve Marder as the Senior editor and Fred Rieke as the Reviewing editor.Thank you for resubmitting your work entitled "Complementary shifts in photoreceptor spectral tuning unlock the full adaptive potential of ultraviolet vision in birds" for further consideration at The manuscript has been improved but there are a few remaining issues that need to be addressed before acceptance, as outlined below:1) All figure supplements should be referred to in the main text. One place this comes up in particular is in describing the spectral fits in 2) Subsection “Complementary SWS1 opsin tuning and C-type oil droplet spectral filtering facilitate color discrimination”: Reviewer #2 raised a concern about the actual color space relevant for vision, and whether the analysis of the number of discriminable colors was relevant. Other readers may share this concern. Hence some of the response to this concern could make its way into the paper, and the introduction to this section seems like a good place. This could entail describing in a few sentences the 'ideal' analysis, based on known input spectra for each species natural environment, and why this is not feasible currently.3) In the first paragraph of the subsection “C-type oil droplet spectral filtering is determined by the accumulation of two apocarotenoids, galloxanthin and dihydrogalloxanthin”: define phylogenetic inertia.4) In the second paragraph of the subsection “A proposed enzymatic pathway for apocarotenoid metabolism in the SWS2 cone”: LWS is a bit distracting here – perhaps mention that part of your hypothesis is that these observations about expression also hold for SWS cones. All three reviewers commented on the comprehensive nature of the study and the importance of the research topic. A specific area of concern was the modeling of color discrimination. Two specific issues came up in this regard: (1) whether the model of cone noise is accurate; and (2) how the conclusions of the model depend on the spectra distribution of inputs. Because the modeling is an essential piece of the paper, and indeed one of the most novel aspects of the paper, these issues need to be dealt with directly. Specifically, it would be very helpful to know how the conclusions of the model depend on the assumptions upon which it is based. Detailed comments from each reviewer follow, including elaboration of the points above. We are including the full reviews so that you can see the convergence onto the essential points above.Reviewer #1:This paper uses an impressive array of techniques to investigate coordinated shifts in spectral tuning of two short-wavelength sensitive photoreceptors in birds. The central finding of the paper – that shifts in the short wavelength photopigment spectral sensitivity produce coordinated shifts in the absorption properties of the oil droplet controlling spectral tuning of the neighboring photoreceptor – are well supported by the data. I am not an expert on photopigment chemistry, so will leave detailed evaluation of that part of the paper for the other reviewers. I did have some concerns about the model for the impact of these changes in color coding:1) Cone noise model.Investigating chromatic discrimination requires a model for noise introduced in the cones. The cone noise is modeled in the paper as a constant Weber fraction. But this appears inconsistent with direct measures of cone noise. Generally, intrinsic noise dominates total cone noise at low light levels. As light levels increase, a point is reached at which quantal fluctuations begin to contribute and in some cases dominate. This behavior is seen in Lamb and Simon (1977), Rieke and Baylor (2000), Angueyra and Rieke (2013). I suspect a noise model consistent with these measurements would alter the findings in the paper about the dependence of optimal spectral sampling on light level. If there is a solid justification for using a constant Weber fraction noise model that should be given in the context of what is known about the cones. If not, a model more in line with the cone measurements should be used.-2 for chicken and 1 cd m-2 for budgerigar, Lind and Kelber 2009, J. Exp Biol. 212:3693 Olsson et al. 2015, J. Exp Biol. 218:184). Therefore, for the major part of the day when the birds are active, cone noise is likely to display Weber behavior.The bird species we model in this study are primarily diurnal . In diurnal chickens and budgerigars, behavioral measures of spectral sensitivity thresholds can be explained by a constant Weber fraction at light levels greater than or equal to sunset conditions  and budgerigars .A) Bright light: we assume that intrinsic noise  and photon shot noise are important. In chickens, behavioral thresholds consistent with Weber-like noise are seen at light levels ≥ 10 cd m-2 .B) Dim light: we assume photon-shot noise dominates and depends upon light intensity in a linear fashion. In chickens, behavioral thresholds consistent with photo-shot noise are observed at light levels of 0.1-10 cd m-2 . Estimates of dark noise in avian cone photoreceptors are not currently available, and we have therefore chosen an arbitrary level for these calculation. The specific value of the noise parameter does not change our prediction of the relative performance of the various cone/oil droplet configuration. However, the estimates of absolute number of discriminable colors cannot be validated without direct measurement of dark noise.C) Very dim light: we assume dark noise dominates and is invariant with respect to light intensity. In chickens, behavioral thresholds consistent with dark noise are observed at light levels of 0.025-0.1 cd mThese three conditions yield different predictions as to the positioning of the C-type droplet spectral filtering that will maximize the number of discriminable colors. In the bright condition, where we assume Weber-like scaling of the noise, the model predictions are consistent with the observed droplet filtering in VS and UVS species. In both dim and very dim conditions the model predictions are substantially short-wavelength shifted relative to the observed values. A short-wavelength shift in C-type droplet filtering increases spectral overlap between the SWS2 and SWS1 cones, but also increases the quantum catch of the receptors. As we have modeled the dim and very dim conditions, the benefit of increased quantum catch outweighs the cost of increased spectral overlap because the discrimination thresholds are directly dependent on intensity. Together these results indicate that in the diurnal species we examine, spectral filtering of the C-type oil droplets is fine-tuned for color discrimination in bright light conditions where discrimination thresholds display Weber-like behavior.However, this may not be the case for all bird species. Nocturnal birds such as owls are reported to have pale, carotenoid-deficient oil droplets with much reduced spectral filtering . Across a broader taxonomic scale, oil droplets are often depigmented or completely lost in nocturnal avian taxa . This suggests that oil droplet spectral filtering is not adaptive for nocturnal vision.2) Measurement of pigment mixtures in oil droplets . The data showing how the measured absorption spectra were fit to obtain a mixture of carotenoids is quite important but is not shown in any detail. I think that data needs to be included, both as absorption spectra and template fits and as summary data.We have added detailed plots of the absorbance spectra and fitted curves for each individual droplet measured from each of the species in Reviewer #2:. As far as I can tell the work is done impeccably, and insofar as my competence allows, I have no concerns about the findings.I do however have two questions:1) At no point does the paper address the question of why shifts between UVS and VS type opsin occur so often in avian evolution, or why intermediate variants do not evolve. This is, of course, not the main subject, but a natural question to ask, so it would at least be good to know that we have no idea!max of SWS1 opsin from VS to UVS can be affected by as little as a single amino acid change in the opsin (S90C). In fact, this amino acid substitution can be brought about by a single nucleotide change at the DNA level. For this reason, the VS to UVS shift is easily ‘accessible’ in an evolutionary sense. The S90C substitution is predicted to cause deprotonation of the chromophore Schiff-base linkage in the opsin . This deprotonation results in a large blue-shift in the peak spectral sensitivity that dwarfs more subtle shifts brought about by other changes in the opsin. Therefore, the evolution of intermediate tunings may be constrained by the photochemistry of the visual pigment.The molecular mechanism of SWS1 opsin spectral tuning offers an explanation for why these transitions occur so frequently and why intermediate tunings are not typically observed. The shift of the λAt the level of ultimate causation, currently relatively little is understood about the selective pressures that might be driving the VS to UVS shift. This is, of course, a very interesting question. However, much of this discussion is speculative and to include it here would require an extensive Introduction and Discussion that is beyond the scope of the paper. Nevertheless, we have edited lines  to acknowledge this question.2) More substantively, the model of performance evaluates the number of discriminable colours available to the different types of eye taking account of photoreceptor noise. This measure is potentially misleading:It is a priori obvious (and certainly doesn't need the modelling done here) that reducing spectral overlap increases the volume of the colour solid so that if the Weber fractions of the different cone types are fixed  then is obviously beneficial to reduce – or ideally eliminate – spectral overlap (see Eq 12).It is mainly when the effects of photon noise (Eq 13) are taken into account that the modelling of the kind is useful. The model estimates a 'confusion volume', and hence the number of discriminable colours under a natural illuminant. These volumes depend upon photon noise, and hence quantum catch. This raises two questions: i) what are the spectral compositions of the stimuli that are being discriminated, and ii) how do they relate to the reflectances of real or even hypothetical objects?For example, if spectra are generated at random – say  with a reflectance of either 0 or 1 at each 1-nm interval – then it is easy to see that the density of spectra falling in the central part of the colour space will greatly exceed that in the periphery potentially reducing the benefit of minimising spectral overlap.In reality most potentially discriminable colours, perhaps 90% – 99% of the 8-million or so, will hardly ever occur, whereas most natural spectra will reside in a very small proportion of these colour spaces. It should therefore be explained how the estimates of the number of discriminable colours might be relevant to any natural task.Therefore, although it is intuitively reasonable that photoreceptor spectral sensitivities should overlap 'by a certain amount but not too much' I do wonder if the number of colours is a misleading characterisation of the utility of the birds' colour vision.This is a good point and a challenge we have discussed extensively. We agree that it is unlikely that the spectral world of any given bird species completely fills color space. However, we have a very limited understanding of the spectra that are available and important for specific bird species. Restricting our analyses to specific regions of color space would largely be arbitrary and could also be misleading. Therefore, we chose to limit our assumptions and model the entirety of color space.Our calculations of the spectral sensitivity function indicate that C-type spectral shifts have their most pronounced effect in the 'blue' region (400-500 nm). If we were to restrict our analysis to this region, our results would possibly be somewhat clearer, but less general.The manuscript has been improved but there are a few remaining issues that need to be addressed before acceptance, as outlined below:1) All figure supplements should be referred to in the main text. One place this comes up in particular is in describing the spectral fits in We have added references to We have added descriptive labels to each subsection of We have revised 2) Subsection “Complementary SWS1 opsin tuning and C-type oil droplet spectral filtering facilitate color discrimination”: Reviewer #2 raised a concern about the actual color space relevant for vision, and whether the analysis of the number of discriminable colors was relevant. Other readers may share this concern. Hence some of the response to this concern could make its way into the paper, and the introduction to this section seems like a good place. This could entail describing in a few sentences the 'ideal' analysis, based on known input spectra for each species natural environment, and why this is not feasible currently.We have added a discussion of the relevance of the discriminable color analysis . We acknowledge that ideally we would limit the analysis to the colors that are ecologically relevant for a given species. However, to date, such a “chromatic ecosystem” has not been defined for any species and we chose to analyze all of color space in order to limit the assumptions we make.3) In the first paragraph of the subsection “C-type oil droplet spectral filtering is determined by the accumulation of two apocarotenoids, galloxanthin and dihydrogalloxanthin”: define phylogenetic inertia.We have clarified the meaning of phylogenetic inertia in the first paragraph of the subsection “C-type oil droplet spectral filtering is determined by the accumulation of two apocarotenoids, galloxanthin and dihydrogalloxanthin”.4) In the second paragraph of the subsection “A proposed enzymatic pathway for apocarotenoid metabolism in the SWS2 cone”: LWS is a bit distracting here – perhaps mention that part of your hypothesis is that these observations about expression also hold for SWS cones.We have revised this statement to clarify the logic of our hypothesis .


Depressive symptoms and reduced quality of life (QOL) are parts of the chronic stress syndrome and predictive of adverse outcome in patients with ischemic heart disease (IHD). Chronic stress is associated with increased sensitivity for pain, which can be measured by algometry as Pressure Pain Sensitivity (PPS) on the sternum.To evaluate if stress focus by self-measurement of PPS, followed by stress reducing actions including acupressure, can decrease depressive symptoms and increase psychological well-being in people with stable IHD.Observer blinded randomized clinical trial over 3 months of either intervention or treatment as usual (TAU). Statistical analysis: Intention to treat.Primary endpoint: change in depressive symptoms as measured by Major depression inventory (MDI). Other endpoints: changes in PPS, Well-being (WHO-5) and mental and physical QOL (SF-36).Two hundred and thirteen participants with IHD were included: 106 to active treatment and 107 to TAU. Drop-out: 20 and 12, respectively. The active intervention included self-measurement of PPS twice daily followed by acupressure as mandatory action, aiming at a reduction in PPS. At 3 months PPS decreased 28%, to 58, in active and 11%, to 72, in TAU, p<0.001. MDI decreased 22%, to 6.5, in active group vs. 12%, to 8.3 in TAU, p = 0.040. WHO-5 increased to 71.0 and 64.8, active group and TAU, p = 0.015. SF-36 mental score sum increased to 55.3 and 53.3, active and TAU, p = 0.08.PPS measurements followed by acupressure reduce PPS, depressive symptoms and increase QOL in patients with stable IHD.NCT01513824ClinicalTrials.gov  The bidirectional interaction between depression and ischemic heart disease (IHD) has been documented numerous times and is generally accepted Depression, quality of life and general well-being is all part of the chronic stress concept Chronic stress and depression is associated with widespread increased pain sensitivity, leading to both hyperalgesia (pain induced by noxious stimuli) and allodynia (pain induced by non-noxious stimuli) The gold standard in measuring pain sensitivity is by algometry. Recently a simple and handheld algometric device has been designed to asses pressure pain sensitivity (PPS) Several studies have evaluated the effect of various stress-reducing interventions in patients with IHD and some have shown to improve the prognosis and to reduce the risk of new cardiovascular events Acupressure, i.e. applying a continuous pressure for approximately one minute at specific hyperalgesic points at the body, has been shown to reduce both local and spreading pain in chronic low back pain and neck pain syndromes Thus we hypothesized that the combination of daily self-measurements of PPS aiming at increased empowerment followed by acupressure aiming to restore DNIC, together would resolve in a reduction of the following elements of chronic stress: Depressive symptoms measured by MDI, general well-being measured by WHO-5, and physically and mentally QOL measured by SF-36 QOL-score.Aiming to test this hypothesis we performed an observer blinded randomized clinical trial with blinded outcome assessment over 3 month in which the active group measured PPS twice daily followed by acupressure as mandatory action. Our primary end point was changes in MDI.www.regionh.dk/vek, identifier: H-4-2010-135, and amendment 31962) and the Danish Data Protection Agency (identifier: 2011-41-7022), and was registered on www.clinicaltrials.gov (identifier: NCT01513824). All participants gave their written informed consent after oral and written information about the study. The study was performed according to the declaration of Helsinki.The original protocols and a CONSORT checklist are available as supporting information see . The stuInclusion criteria: 1) documented IHD defined as having had a MI, percutaneous coronary intervention (PCI) or coronary artery bypass graft surgery (CABG), 2) completed cardiac rehabilitation more than six months prior to inclusion, 3) age 75 years or younger at inclusion; exclusion criteria: 1) hospitalization due to psychiatric disease prior to IHD, 2) scheduled cardiac surgery, 3) changes in heart medication within the last month prior to inclusion, 4) a chronic competing disorder clearly impairing the patients QOL, 5) chronic pain syndromes such as fibromyalgia or severe arthritis 361 patients with IHD participated in a cross-sectional study on the associations between PPS and stress in patients with IHD After the baseline visit the participants were allocated randomly with a 1∶1 ratio to either the intervention arm or to treatment as usual (TAU). Randomization was stratified according to age, sex, MDI, diabetes, and chronic heart failure. The randomization was performed and carried out by an external statistician using a computerized randomization sequence with a block size of 16, which was unknown to the investigators. The participants received the allocation result by a confidential e-mail generated automatically. One hundred and six participants were allocated to active intervention and 107 to TAU.A single center, two-armed, parallel-group, observer-blinded randomized clinical superiority trial. The participants were enrolled at the trial cite in Copenhagen (Denmark).All subjects were instructed by a professional instructor  to perform PPS measurements twice daily, in the morning (before breakfast) and in the evening (before going to bed). After the measurement, if the PPS level and thus the stress-level was high, the participant should perform acupressure as a mandatory stress-reducing procedure, as well as reflection on both the PPS level and on ones general feeling of need for stress handling on a voluntary basis.All subjects received a personal PPS measurement instrument, together with an instruction manual and were individually instructed during a 45 minutes period at their private homes on how to use the PPS device. This included education in performing acupressure. The acupressure procedure has previously been described in “Magnusson et al,” The participants were offered a new appointment and/or 1–2 phone contacts with the instructor if they had problems with self-measurements of PPS after the visit, or had other questions.www.songheart.org. On the website each participant was able to track results and changes in PPS during the intervention period.The participants were instructed to report their PPS measurements each day on their personal login on the website Both the active and the TAU group was informed at baseline, that their stress level was elevated and received a booklet on general stress handling principles Change in MDI from baseline to follow up after three months.Changes in stress-level as measured by PPS as well as changes in well being as measured by WHO-5.Changes in quality of life as measured by SF-36 physical (PCS) and mental (MCS) QOL scale.The stress-reducing effects in participants with the highest depression/stress level measured as the subgroup of participants with MDI≥15 at baseline and the subgroup of participants having the highest 50% of both PPS and Clinical stress signs score (CSS) at baseline.Before and after three months all participants came to the metabolic ward in the morning. The PPS level was measured by a professional after 5–10 min of resting in a supine position. The PPS measurement procedure has previously been described in details www.songheart.org) and each participant received a personal profile with login. The day prior to the visit at the metabolic ward, the participants answered the following questionnaires on their www.songheart.org profile: 1) MDI, assessing depressive symptoms on a score form 0–50, zero being equivalent to no signs of depressive symptoms, 2) WHO-5, assessing psychological well-being with 100 equivalent to best psychological well-being, 3) SF-36, assessing PCS and MCS QOL with 100 equivalent to best QOL. At baseline a fourth questionnaire was included, measuring CSS as well as a demographic questionnaire. The CSS score is a newly developed score of 56 clinical stress symptoms experienced during the last four weeks A website was established for the study . Both the observed and values are reported. For testing of statistical significance we included all randomized patients in our analysis regardless of subsequent adherence to treatment. We imputed missing values treating the outcomes as dependent variables using multiple imputations. For this we used a linear regression model with the following predictor variables: Allocation, sex, and age. The imputation was conducted using 100 imputations and 20 iterations. The pooled estimates from these imputations were subsequently used for analysis Cohens effect size was used to compare active to TAU group using ITT data. The effect size was evaluated according to Hedgen and Olkin as the difference in mean change score from baseline to follow up between active group and TAU divided by the pooled standard deviation Analyses were performed using the statistical package SPSS version 19. All statistical tests were two-sided and p-values below 0.05 were considered statistically significant.Twenty dropouts (19%) were reported in the active group and 12 (11%) in the TAU group, p = 0.18.  Drop outNinety four in the active group reported repeated PPS measurements at home, in mean 90 measurements over three months (range 6–192).MDI was significantly reduced by 22% after three months in the active group and by 12% in the TAU group. The reduction was significantly greater in the active group as compared with the TAU group, which resulted in a difference between the two groups at three months, p = 0.040 .WHO-5, increased by 6.4% in the active group and by 3.7% in the TAU group from baseline to follow up , which resulted in significantly different levels between active group and TAU at 3 months (p = 0.016) .PPS was significantly reduced by 28% after three months in the active group and 11% in the TAU group (both p<0.001), and at 3 months the PPS values had become different between the two groups (p<0.001).SF-36 PCS was improved in both groups although not reaching statistically difference at three months, p = 0.14. SF-36 MCS improved in the active group (p = 0.004), however, the difference after three month compared with TAU values only tended to be significant (p = 0.09) .Two subgroup analyses were performed: One on subjects with MDI≥15 at baseline and another on subjects having both the PPS and CSS within the upper half at baseline .Fourty-two participants had MDI≥15 at baseline and 21 were randomized to active and 21 to TAU group, and 18 subjects in each group completed the study . Fifty-nThe effect of active vs. TAU for the whole group was 0.12 for MDI, 0.11 for WHO-5 and 0.63 for PPS. Effect size for the subgroup with MDI≥15 was 0.35 for MDI, 0.21 for WHO-5 and 0.43 for PPS and for the subgroup with both the highest PPS and CSS, the effect size was 0.52 for MDI, 0.62 for WHO-5 and 0.46 for PPS.In the present randomized interventional trial we found, that in patients with stable IHD the combination of daily self-measurements of PPS followed by acupressure and reflection on PPS as a surrogate for current stress-level resulted in a modest but statistically significant improvement in PPS, MDI and WHO-5.This beneficial effect of the intervention program was more pronounced in subgroups of patients with higher baseline levels of components of the chronic stress syndrome, such as elevated MDI, as well as the combination of an elevated PPS and CSS. These subgroup analyses, however, were hampered by a limited sample size.The primary endpoint was a reduction in MDI. MDI was chosen as a well validated often used psychometric questionnaire on depressive symptoms. It is well known that patients with IHD as well as other chronic diseases have increased levels of depressive symptoms which leads to a poor outcome Similar to a positive effect on MDI we found a beneficial effect of the intervention on general well-being as measured by WHO-5′s well-being index. The intervention resulted in an increase in WHO-5 with a small effect size, however subgroup analysis demonstrated a substantially increase in well-being, although the difference at three months between the active and the TAU group, did not reach statistically significance probably due to a limited number of subjects in the subgroup analyses.The intervention was based on the concept that self-measurement of pain sensation threshold measured by PPS resulted in an awareness of ones current stress-level, leading to reflection as well as to action i.e. increased empowerment, aiming at improving overall well-being.Acupressure was the only mandatory action for reducing pain sensation in the intervention program. General advice about stress reduction was presented for both the active and the TAU group in the form of a booklet on stress-coping Parts of the intervention program have been used with success previously in patients with advanced angina pectoris in a non-randomized fashion PPS measures pain sensitivity threshold like other algometers within pain research. We have recently demonstrated a close correlation between PPS and another pressure pain algometer An inherent weakness of pressure pain algometry is the fact that there is no objective measure of pain threshold since it is the subject itself that reacts to discomfort and ends the measurement. Therefore a potential bias to repeated measurements is habituation both mentally and physically. If present this could result in a progressive reduction in PPS over time. We have performed validation studies in which we have found a very close association between two measurements performed within 5 seconds as well as with one day between measurements In the present study the PPS measure was reduced with 22 units in the active group. The PPS device is constructed such that a reduction of 30 units equals a doubling of the pressure applied to the sternum. This quite robust change also speaks against a drift in PPS sensation due to habituation.The instructors measuring PPS at baseline as well as after three months were blinded to the randomization results, the answers of the questionnaires, the results of the PPS home measurements and to the concrete PPS result while measuring at baseline and follow up visits. The latter is due to the construction of the PPS device, which first shows the result of the pressure when ending the pressure, thus revealing only the final result.Chronic illness in general is associated with both stress and depressive symptoms which includes IHD Whether the reduction in pain sensitivity is the primary event leading to reduced feeling of depressive symptoms and increased well-being as found by an improvement in MDI and WHO-5 results, or the intervention works primarily by changing depressive symptoms and well-being and thereafter leads to a reduction in pain sensitivity is not clear. Concerning the mandatory intervention, acupressure, it is a well known clinical observation, that acupressure on a distinct sore point of the body leads to pain reduction locally with a spreading effect into the surroundings, which also has been demonstrated in females with chronic neck pain We aimed at 300 and included 213 participants. The power analysis was based on an effect size of 0.40, but we only obtained an effect size of 0.12. This means that the study ended up underpowered. However looking at both primary endpoint (MDI) and the other endpoints we found a rather clear tendency towards a beneficial effect on all parameters studied during intervention. This indicates that all bights the effect size was rather small, it seems real and is not a result of a type 1 error. Although the study was underpowered we did find significant changes probably avoiding a rather large risk of a type 2 error.The randomization procedure included DM and chronic heart failure yes/no, however, the randomization ended up with significantly more diabetics in the active group and in contrast less patients with chronic heart failure compared with the TAU group. Given the small number of diabetics and patients with heart failure the consequence of this randomization failure is probably mild or absent.The study was single blinded. This was intentionally since the intervention was of an open nature i.e. the participants should reflect on the PPS measurements and also because sham PPS measurement giving false values did not seem ethically acceptable. However, the single-blinded design may have caused bias to the results of the intervention and may have affected the responses from the participants regarding the questionnaires. With regard to the PPS measurements they were performed by professionals who did not have knowledge about the allocation to active or control group. Further the PPS device was designed in a way making the measure non-visible before the end of each measurement for both instructor and patient. Thus the results from PPS measurements can be regarded as double blinded.Rather few in- and exclusion criteria make our study representative to most patients with IHD, further the intervention technique used is easy to adapt and use, and without side effects.Statistical evaluation was performed by an ITT analysis using all randomized subjects (n = 106). This is the most correct, however not always used in RCT Self-measurements of PPS followed by acupressure as a mandatory action and otherwise individual reflection, in subjects with stable IHD resulted in a reduction in PPS, reduced amounts of depressive symptoms and increased well-being. This effect seemed more pronounced among the most psychologically vulnerable subjects. Due to the study ending up being underpowered further studies which are properly powered should be performed in order to establish the impact of this new type of intervention.The concept of biofeedback guided stress handling, i.e. increased patient empowerment, based on frequent self-measurement of PPS might be broadened out to other chronic illnesses like diabetes, as well as to otherwise healthy subjects on work places where the employees demonstrate signs of increased stress due to a high psychological stress demand.Protocol S1Trial Protocol (Danish).(PDF)Click here for additional data file.Protocol S2Trial Protocol (English).(PDF)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.