Woodlice efficiently sequester copper (Cu) in ‘cuprosomes’ within hepatopancreatic ‘S’ cells. Binuclear ‘B’ cells in the hepatopancreas form iron (Fe) deposits; these cells apparently undergo an apocrine secretory diurnal cycle linked to nocturnal feeding. Synchrotron-based m-focus X-ray spectroscopy undertaken on thin sections was used to characterize the ligands binding Cu and Fe in S and B cells of Oniscus asellus (Isopoda). Main findings were: (i) morphometry confirmed a diurnal B-cell apocrine cycle; (ii) X-ray fluorescence (XRF) mapping indicated that Cu was co-distributed with sulfur (mainly in S cells), and Fe was co-distributed with phosphate (mainly in B cells); (iii) XRF mapping revealed an intimate morphological relationship between the basal regions of adjacent S and B cells; (iv) molecular modelling and Fourier transform analyses indicated that Cu in the reduced Cuþ state is mainly coordinated to thiol-rich ligands (Cu–S bond length 2.3 A˚ ) in both cell types, while Fe in the oxidized Fe3þ state is predominantly oxygen coordinated (estimated Fe–O bond length of approx. 2 A˚ ), with an outer shell of Fe scatterers at approximately 3.05 A˚ ; and (v) no significant differences occur in Cu or Fe speciation at key nodes in the apocrine cycle. Findings imply that S and B cells form integrated unit-pairs; a functional role for secretions from these cellular units in the digestion of recalcitrant dietary components is hypothesized.
Fig2_RawData
Figure 2. µXRF maps of element distributions in unstained methacrylate-embedded thin mid-tubule sections of woodlouse hepatopancreas.
A. Light micrograph of a transverse section from a woodlouse sampled at 02:00 hours (i.e. the beginning of ‘B’-cell restitution during darkness). Note that the morphology of the sections used for ?XRF is relatively unclear; this is due to a lack of differential contrast in sections that were unstained and infiltrated with methacrylate resin.
S5_sp_light3x10.tif
B. Superimposed Cu, Fe and Zn µXRF maps of the section depicted in 2A; note the co-distribution of Cu and Zn in ‘S’-cells (arrow heads), and the distribution of diffuse Fe mainly in ‘B’-cell apical cytoplasm that might be undergoing blebbing (arrows), and some Fe signal within the tubule lumen (broken arrows).
File Prefix 6191
C. Superimposed Cu, P and S µXRF maps of the section depicted in 2A; note the co-distribution of Cu and S in ‘S’-cells (arrowheads), and the mainly apical distribution of P in ‘B’-cells (arrows).
File Prefix 6191
D. Light micrograph of a mid-tubule transverse section from a woodlouse at 12:00 hours (i.e. at the beginning of ‘B’-cell extrusion during the day).
S4_sp_Light4x10
E. Superimposed Cu, Fe and Zn µXRF maps of the section depicted in 2D; note that there is a partial section of a second tubule in the top-right of the micrograph, and that the transition metal distribution patterns are similar to those seen in 2B.
File prefix - 5983
F. Expanded view µXRF maps for the region delineated by a broken-lined rectangle in 2E; note that Fe is distributed above and below the nuclear plane (arrow) of the prominent ‘B’-cell, albeit mainly in the apical cytoplasm.
File prefix - 5984
G and H are schematic diagrams derived from Hames and Hopkin [36] illustrating the gross difference between ‘B’- and ‘S’-cell morphology (G), and a ‘clock’ summarizing the diurnal cycle of apocrine secretion in ‘B’-cells in relation to the light:dark regime with our two XAS sampling points superimposed upon it (H).
File types included:
tif - Image files (Light Micrographs)
.dat : map data showing the stage positions in mm and windowed fluorescence counts. Column format:
Horizontal position ; vertical position; time per point (ms); I0; It; Idrain; mca file.
.edf file ESRF format file for program PyMCA containing the summed MCA files for each point in a map row by row. Binary data format
.rgb file windowed detector counts for selected chemical elements of interest , column format:
y index; x index; time; i0 it idrain; then elements as listed in first row.
Fig3_RawData
Figure 3.
Panel A. Cu K-edge XANES spectra from:
O. asellus ‘S’-cells at 02:00 hours (red; i-iv)
Spectral range 5960-65 (n=5)
Spectral range 6085-89 (n=5)
Spectral range 6099-103 (n=5)
Spectral range 6123-27 (n=5)
O. asellus 12:00 hours (green; v-vi);
Spectral range 5993-98 (n=4)
Spectral range 6174 (n=1)
O. asellus ‘B’-cells at 02:00 hours (blue; vii-xi)
Spectral range 5975-79 (n=5)
Spectral range 6090-94 (n=5)
Spectral range 6110-14 (n=5)
Spectral range 6133-37 (n=5)
Spectral range 6148-49 (n=2)
O. asellus 12:00 hours (brown; xii) clock time
Spectral range 6175 (n=1)
see Table 2 for the fit data
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
Fig4_RawData
Figure 4.
A. Cu K-edge EXAFS data (solid lines) and fits (broken lines) from:
O. asellus ‘S’-cell at 02:00 hours (red; i)
Spectral Range 5960-65 (n=5)
O. asellus ‘B’-cells at 02:00 hours (blue; ii & iii)
Spectral Range 5975-79 (n=5)
Spectral Range 6148-49 (n=2)
O. asellus ‘B’-cells 12:00 hours (brown; iv)
Spectral Range 6175 (n=2)
(see Table 3 for corresponding quantitative data).
B. Fourier transforms of the Cu EXAFS data (solid lines) and fits (broken lines) depicted in Fig. 4A.
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
Fig5_RawData
Figure 5.
A. Fe K-edge XANES spectra from:
O. asellus ‘S’-cell at 12:00 hours (green; i)
Spectral Range 6172 (n=1)
O. asellus ‘B’-cells at 02:00 hours (blue; ii - iv)
Spectral Range 5967-74 (n=8)
Spectral Range 6104-08 (n=5)
Spectral Range 6128-32 (n=5)
O. asellus ‘B’-cells 12:00 hours (brown; v - vi)
Spectral Range 5985-91 (n=7)
Spectral Range 6170-71 (n=2)
(see Table 2 for the fit data)
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
Fig6_RawData
Figure 6.
A. Fe K-edge EXAFS data (solid lines) and fits (broken lines) from:
O. asellus ‘S’-cell at 12:00 hours (green; i)
Spectral Range 6172 (n=1)
O. asellus ‘B’-cells 02:00 hours (blue; ii)
Spectral Range 5967-74 (n=7)
O. asellus ‘B’-cells 12:00 hours (brown; iii & iv)
Spectral Range 5985-91 (n=7)
Spectral Range 6170-71 (n=2)
(see Table 3 for corresponding quantitative data).
B. Fourier transforms of the Fe EXAFS data (solid lines) and fits (broken lines) depicted in Fig. 6A.
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
Fig7_RawData
Figure 7. Radiation sensitivity of Cu assessed by collecting a sequential series of 5 XANES spectra from given regions in 3 different ‘B’-cells (panels A, B, C).
The thin methacrylate-embedded sections were obtained from a woodlouse sampled at 02:00 hours. In each panel, the large down-pointing vertical arrow indicates that the top spectrum was collected first and the lowest spectrum last. Note that the shoulder on the absorption edge at 8982.5 eV (broken arrows), whilst remaining visible, is progressively eroded from the third acquired spectrum onwards.
Panel A
Spectral Range 6110-14 (n=5)
Panel B
Spectral Range 6090-94 (n=5)
Panel C
Spectral Range 6133-37 (n=5)
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
Fig8_RawData
Figure 8.
Radiation sensitivity of Fe assessed by collecting a sequential series of 5 XANES spectra from given regions in a
‘B’-cell (panel A)
Spectral Range 6104-08 (n=5)
‘S’-cell (panel B)
Spectral Range 6128-32 (n=5)
The thin methacrylate-embedded sections were obtained from a woodlouse sampled at 02:00 hours.
In both panels, the large down-pointing vertical arrow indicates that the top spectrum was collected first and the lowest spectrum last. Note that successive spectra remain very similar.
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
FigS2_RawData
Figure S2. µXRF maps of element distributions in unstained methacrylate-embedded thin mid-tubule sections of woodlouse hepatopancreas sampled at 02:00 hours (i.e. the beginning of ‘B’-cell restitution during darkness).
A. Light micrograph of a transverse section.
S5_sp_light1x10.tif
B. Superimposed Cu, Fe and Zn µXRF maps of the section depicted in S1A; note the regular 1:1 distribution of ‘S’- (*) and ‘B’-cells (•) in the tubule wall.
Prefix 5955
C. Expanded view µXRF maps for the region delineated by a broken-lined rectangle in 2E; note the hallmarks of Cu/Zn-containing ‘S’-cell cytoplasm ‘under’ the basal aspect of the ‘B’-cell (dotted line), the co-distribution of Cu and Zn (light blue) as well as partitioning of Cu (green) and Zn (dark blue) in the ‘S’-cell, and possibly some Zn-containing focal regions in the ‘B’-cell (broken arrows).
Prefix 5956
File types included:
tif - Image files (Light Micrographs)
.dat : map data showing the stage positions in mm and windowed fluorescence counts. Column format:
Horizontal position ; vertical position; time per point (ms); I0; It; Idrain; mca file.
.edf file ESRF format file for program PyMCA containing the summed MCA files for each point in a map row by row. Binary data format
.rgb file windowed detector counts for selected chemical elements of interest , column format:
y index; x index; time; i0 it idrain; then elements as listed in first row.
FigS3_RawData
Figure S3. µXRF maps of element distributions in unstained methacrylate-embedded thin mid-tubule sections of woodlouse hepatopancreas sampled at 02:00 hours (i.e. the beginning of ‘B’-cell restitution during darkness).
A. Light micrograph of a transverse section; note two spherical structures (arrows) in the lumen that might represent the newly shed apical cytoplasm (‘blebbing’) of two adjacent ‘B’-cells.
S5_sp_light2.tif
B. Superimposed Cu, Fe and Zn µXRF maps of the section depicted in S2A; note the Fe-rich ‘blebs’ in the lumen (solid arrows) corresponding with the structures labelled in S2A, as well as two more diffuse entities (broken arrows) possibly representing ‘older’ blebs that were not discernible in the micrograph.
Sample Prefix 6076
File types included:
tif - Image files (Light Micrographs)
.dat : map data showing the stage positions in mm and windowed fluorescence counts. Column format:
Horizontal position ; vertical position; time per point (ms); I0; It; Idrain; mca file.
.edf file ESRF format file for program PyMCA containing the summed MCA files for each point in a map row by row. Binary data format
.rgb file windowed detector counts for selected chemical elements of interest , column format:
y index; x index; time; i0 it idrain; then elements as listed in first row.
FigS4_RawData
Figure S4. Radiation sensitivity of Cu assessed by collecting a sequential series of 5 XANES spectra from given regions in 3 different ‘S’-cells (panels A, B, C).
Panel A
Spectral Range 6099-6103 (n=5)
Panel B
Spectral Range 6085-89 (n=5)
Panel C
Spectral Range 6123-27 (n=5)
The thin methacrylate-embedded sections were obtained from a woodlouse sampled at 02:00 hours. In each panel, the large down-pointing vertical arrow indicates that the top spectrum was collected first and the lowest spectrum last. Note that the small shoulder on the absorption edge at 8982.5 eV (broken arrows) is slightly eroded from the third acquired spectrum onwards (cf. Fig. 7).
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
FigS5_RawData
Figure S5. Radiation sensitivity of Zn assessed by collecting a sequential series of 5 XANES spectra from given regions in 2 different ‘S’-cells (panels A and B).
Panel A
Spectral Range 6142-46
Panel B
Spectral Range 6116-21
The thin methacrylate-embedded sections were obtained from a woodlouse sampled at 0200 hours. In each panel, the large down-pointing vertical arrow indicates that the top spectrum was collected first and the lowest spectrum last. Note that the absorption edge peak at ~9670 eV (broken arrows) is slightly eroded from the 2nd acquisition onwards (A) and from the 4th onwards (B).
File type provided: XAS
_dtc.dat - deadtime corrected (DTC) XAS files; column format:
Monochromator angle : energy : time per point : I0 : It; Idrain : raw fluorescence counts channels 1-9: summed raw fluorescence counts: DTC flu counts channels 1-9: summed DTC fluorescence counts
