Fig 1.
Schematic illustrations of a cut through the medial plane of a Nautilus and full thickness shell wall at the aperture and CLFM image of Nautilus shell showing inclined banding visible by CLFM in the outer prismatic layer relative to actively growing surface of the shell.
SIMS measurements in this study were only from the outer prismatic layer of shell and no analyses are reported from the nacreous layer because growth banding in the nacreous layer is too thin for depth migration to be subsampled by 10-μm SIMS spots. The CLFM image shows only the outer prismatic layer of the Nautilus shell. Growth bands were formed in the layer parallel to the growth surface and are likely expressed due to differences in the proportion of aragonite to intercrystalline organic matter [22]. Growth bands extend from the exterior surface of the shell to the nacreous-prismatic boundary and are inclined by ~11° to the exterior surface. Growth rates in literature (averaging 158 μm/day as represented by the scale bar) are typically reported as apertural growth per day, which corresponds to the distance along the exterior surface in the figure [21]. There are three major patterns within growth bands: 1) There is less contrast between the light and dark portions of bands near the exterior surface. 2) Near the exterior surface spots of brightly luminescing aragonite are small and surrounded by small spots of less luminescing aragonite (less graininess) (A). 3) Near the nacreous-prismatic boundary there are larger spots of brightly luminescing aragonite surrounded by continuous areas that do not luminesce as brightly (B). The intensity of fluorescence was measured along the distance perpendicular to banding by averaging lines of pixels parallel to growth banding.
Fig 2.
Map of the collection location of Nautilus macromphalus (AMNH 105621) near New Caledonia with δ18OArg predicted for the temperatures observed in the water depths Nautilus inhabits.
A) Map of the southern end of New Caledonia showing the collection site (Amédée) of the Nautilus macromphalus used in this study. The 750 m bathymetry is the approximate lower limit of Nautilus habitat in the area defined by implosion depth. B) Water temperature for the map area [15] varies between the surface to 840 m depth. Gray bars indicate the approximate seasonal range in temperatures in the map area at each depth. The black line shows the average annual temperature with depth. A higher amount of seasonal variability is present in the upper 100 meters. C) The expected oxygen isotope ratio calculated using Eq 1 for an aragonite precipitated in isotope equilibrium with seawater at the temperatures shown in B assuming a constant δ18OSW (0.5‰ VSMOW) with depth. The δ18OArg will vary by up to 4‰ over the range of 20°C for the depths that Nautilus can inhabit. The gray bars show expected seasonal variability in δ18OArg and the black line is the average expected δ18OArg with depth in the map area.
Fig 3.
The outer prismatic layer of a portion of the polished surface from Nautilus macromphalus (AMNH 105621) imaged by four different techniques: A) SE, B) CLFM, C) Plain light dissecting microscope, D) UV.
The oldest shell precipitated is in the upper right corner of all images. Growth proceeds to the lower left. All images are of the same portion of the shell. Five bands are highlighted with white lines. A) SE image showing the polished shell surface. Lines of pores in the SE image (A) correlate to low fluorescence in CLFM (B), bright portions of the band in the plain light correlate to dissecting microscope image (C) and dark portions in the long wavelength UV (~360 nm) fluorescence image (D). These cavities are the same as those intersected by some SIMS analysis pits (Fig 4). B) CLFM (true color) image showing growth bands. Growth bands extend from the exterior surface of the outer prismatic layer (top of the image) to the interior boundary with the nacreous layer. Note that the banding closer to the nacreous boundary is more pronounced than the banding toward the exterior of the shell. C) Plain-light dissecting microscope image (true color) of polished shell surface. Banding is apparent but discontinuous. D) UV fluorescence (true color) image of the shell. Faint banding is present with an orientation equal to that found in other imaging techniques. CLFM images on the uncoated sample mount were made using a Bio-Rad MRC-1024 scanning confocal microscope at the W. M. Keck Laboratory for Biological Imaging at UW-Madison operated with a 40 mW laser at a wavelength of 488 nm. Images of banding within the outer prismatic layer were best expressed through an emission filter that detects visible green light (λ = 505 to 539 nm). Growth-band orientation in the prismatic layer was used to place SIMS transects.
Fig 4.
Representative SE images illustrating the criteria used for qualitative SIMS pit categorization for both the wild-caught and aquarium-reared Nautilus and beanplots showing the distribution of δ18OArg and O16H1/O16 across pit categories from Nautilus macromphalus (AMNH 105621).
Cavities that the SIMS analysis pits intersected are likely left from the removal of organic matter during roasting or from plucking during polishing. Organic matter in the outer prismatic layer of mollusks has banding but also irregular clumps [22]. In the images above, cavities are outlined by a red dashed line. All pits are approximately 1-μm deep. Category 1 (C1) This pit is classified as regular in appearance because it has a uniform texture throughout the walls and bottom of the pit (wild-caught, n = 134; aquarium-reared, n = 25). Category 2 (C2) This pit minimally intersects two small cavities and therefore is also classified as regular in appearance (wild-caught, n = 85; aquarium-reared, n = 5). Category 3 (C3) A more significant intersection with a cavity at the bottom of this analysis pit makes it irregular (wild-caught, n = 52; aquarium-reared, n = 16). Category 4 (C4) A larger intersection with a cavity, but this cavity intersection is on the wall of the pit rather than on the bottom. This pit is also considered to be irregular and 5% of analyses had cavity intersections similar to this pit (wild-caught, n = 14; aquarium-reared, n = 7). Category 5 (C5) This pit is an example of some of the most extreme pit-cavity intersections and is a highly irregular pit (wild-caught, n = 8; aquarium-reared, n = 0). Beanplots [71] below show the estimated density distribution of δ18OArg (top) or O16H1/O16 (bottom) for the wild-caught Nautilus macromphalus where we have paired analyses (2/14/2014-2/15/2014). The thin black line extending across the beanplots indicates the mean for all analyses, short lines extending from each category indicate each category mean, and short white lines within each category indicate individual observations.
Table 1.
Summary of SIMS analyses of oxygen isotope ratios measured at WiscSIMS on the Nautilus belauensis (AMNH 102555) and confocal microscope brightness.
These are ion microprobe analyses of oxygen isotope ratios, grey scale values from confocal laser fluorescence images, and distance perpendicular to banding for analyses that were shown to be regular based on pit morphology. See supplementary material for a complete list of measurements.
Table 2.
Summary of SIMS analyses of oxygen isotope ratios measured at WiscSIMS on the Nautilus macromphalus (AMNH 105621) and confocal microscope brightness.
These are ion microprobe analyses of oxygen isotope ratios, grey scale values from confocal laser fluorescence images, and distance perpendicular to banding for analyses that were shown to be regular based on pit morphology. See supplementary material for a complete list of measurements.
Fig 5.
SIMS results and CLFM brightness for transects perpendicular to banding in the outer prismatic layer of the wild-caught Nautilus macromphalus (AMNH 105621).
SIMS analysis and CLFM imaging was done on this 8 mm long portion of vacuum roasted and polished Nautilus macromphalus shell. The background grayscale is based on the residual CLFM brightness after lowess regression to correct for differences in brightness within and between images. Oxygen isotope ratios are plotted against hours of growth, assuming that each dark-light-dark cycle is 24 hours. The most recently precipitated shell is at time 0. Overlapping transects are color coded to show where correlation across the shell was carried out to produce the composite record. Transect locations are highlighted on the CLFM image of the outer prismatic layer. Correlation shows agreement within instrumental precision between transects correlated across the shell. There is considerable oxygen isotope variability within daily individual growth bands and across multiple bands. A higher resolution map of the shell surface is available in the supplementary information (S1 Fig).
Fig 6.
SIMS results and CLFM brightness for transects perpendicular to banding in the outer prismatic layer of the aquarium-reared Nautilus belauensis (AMNH 102555).
SIMS analysis and CLFM imaging was done on this 3-mm long portion of vacuum roasted and polished Nautilus belauensis shell. The background grayscale is based on the residual CLFM brightness after lowess regression to correct for differences in brightness across and between images. Oxygen isotope ratios are plotted against hours of growth, assuming that each dark-light-dark cycle is 24 hours and the scales of the x and y axes match those of Fig 5. The most recently precipitated shell is at time 0. Overlapping transects are color coded to show where correlation across the shell was done to produce the composite record. Transect locations are highlighted on the CLFM image of the outer prismatic layer. Correlation shows general agreement within instrumental precision between transects correlated across the shell. Finding variability in this sample is particularly interesting because it is one of three individuals that is often cited as an example of Nautilus precipitating oxygen in isotopic equilibrium with seawater [12]. The positive shift in δ18OArg that takes place over several days is attributed to evaporation of ~8 liters of water from the 80 L incubation tank in which this individual was living [45,47,76]. A higher resolution map of the shell surface is available in the supplementary information (S2 Fig).
Fig 7.
A comparison of SIMS measured δ18OArg to telemetry results that have been converted to δ18OArg.
Telemetry is done using ultrasonic transmitters that are attached to the dorsal shell of Nautilus and are monitored from boats or stationary underwater receivers [17–19]. SIMS results can be compared to telemetry records if water temperatures and δ18O are known, growth is continuous, and time averaging is calculated. Green lines in A and B are telemetry data converted to δ18OArg assuming a thermal gradient of 0.3°C/m, with a constant δ18Osw of 0.5‰ (VSMOW) and using Eq 1. Overlying red lines are hypothetical SIMS data approximate time averaging from a 10-μm spot diameter SIMS spot assuming 35 μm/day growth. The gray background is the 2SD instrumental precision envelope around the SIMS analyses. A) Regular shallow-deep migrations observed in Nautilus belauensis off Palau [17] would likely leave a SIMS result with regular variability. B) Rapid excursions to depths at dawn and dusk, like those observed in N. pompilius off Osprey Reef, Australia [19] would leave less variability in the SIMS results due to the effects of time averaging. C) Actual SIMS results for the wild-caught Nautilus macromphalus from New Caledonia suggest that depth migration can be detected; however, there is not a regular migratory pattern like that observed at Palau. A different style of depth migration pattern, influenced by the local bathymetry or cover availability, could explain the differences between the telemetry data and the SIMS measurements. The δ18Oarg variation measured by SIMS in this study shows that the wild-caught sample spent significant amounts of time in both shallow and deep water, a behavior more similar to that observed by Ward et al. [17] and Carlson et al. [18] near Palau (fringing reef) than that observed by Dunstan et al. [19] near Osprey Reef (submerged atoll), Australia. Dunstan et al. [19] suggest that the behavioral difference between Osprey Reef and Palau is due to available hiding locations, and therefore it is possible that hiding locations on the fore-reef of the fringing reef near New Caledonia are less abundant than those on Osprey Reef. Differences in behavior could also be due to other factors of location or differences between species.