Densitometry from digitized images of X-radiographs: Methodology for measurement of coral skeletal density

Skeletons of massive coral colonies contain annual density bands that are revealed by X-radiography of slices cut along growth axes. These bands allow measurement of skeletal growth parameters such as annual extension rate and annual calcification rate. Such measurements have been important in understanding coral growth, in assessing environmental impacts and in recovering proxy environmental information. Measurements of coral calcification rate from annual density banding require measurements of skeletal density along tracks across skeletal slices and, until now, such density measurements have depended upon specialized and expensive equipment. Here, we describe a straightforward, inexpensive and accurate technique for measuring skeletal density from digitized images of X-radiographs of coral skeletal slices. An aragonitic step-wedge was included in each X-radiograph of a coral slice together with two aluminium bars positioned along the anode-cathode axis. Optical density was measured along tracks across the X-ray images of these different objects. The aragonite step-wedge provided a standard for converting optical density to skeletal density. The aluminium bars were used to correct for the heel effect—a variation in the intensity of the X-ray beam along the anode-cathode axis that would, otherwise, introduce large errors into measurements of skeletal density. Exposure was found to vary from X-radiographs to X-radiograph, necessitating the inclusion of the calibration standards in each X-radiograph of a coral slice. Results obtained using this technique compared well with results obtained by direct gamma densitometry of skeletal slices.     

Inferred calcification rate of a temperate azooxanthellate caryophylliid coral along a wide latitudinal gradient

Correlations between environmental parameters (depth temperature and solar radiation) and growth parameters (bulk skeletal density, linear extension rate and net calcification rate) of the solitary azooxanthellate coral, Caryophyllia inornata, were investigated along an 8° latitudinal gradient on the western Italian coasts. Net calcification rate correlated positively with both bulk skeletal density and linear extension rate, showing that C. inornata allocates calcification resources evenly to thickening the skeleton and increasing linear growth. Overall, the three growth parameters did not follow gradients in the two environmental parameters, showing a different trend compared to most studies on zooxanthellate corals. However, the results are in agreement with the only previous analysis of an azooxanthellate coral, Leptopsammia pruvoti, studied along the same latitudinal gradient. In a comparison of the response to temperature of all Mediterranean species whose growth has been investigated to date, azooxanthellate corals were more tolerant to temperature increases than zooxanthellate corals.     

Declining coral calcification in massive Porites in two nearshore regions of the northern Great Barrier Reef

Temporal and spatial variation in the growth parameters skeletal density, linear extension and calcification rate in massive Porites from two nearshore regions of the northern Great Barrier Reef (GBR) were examined over a 16‐year study period. Calcification rates in massive Porites have declined by approximately 21% in two regions on the GBR ～450 km apart. This is a function primarily of a decrease in linear extension (～16%) with a smaller decline in skeletal density (～6%) and contrasts with previous studies on the environmental controls on growth of massive Porites on the GBR. Changes in the growth parameters were linear over time. Averaged across colonies, skeletal density declined over time from 1.32 g cm^?3 (SE = 0.017) in 1988 to 1.25 g cm^?3 (0.013) in 2003, equivalent to 0.36% yr^?1 (0.13). Annual extension declined from 1.52 cm yr^?1 (0.035) to 1.28 cm yr^?1 (0.026), equivalent to 1.02% yr^?1 (0.39). Calcification rates (the product of skeletal density and annual extension) declined from 1.96 g cm^?2 yr^?1 (0.049) to 1.59 g cm^?2 yr^?1 (0.041), equivalent to 1.29% yr^?1 (0.30). Mean annual seawater temperatures had no effect on skeletal density, but a modal effect on annual extension and calcification with maxima at ～26.7 °C. There were minor differences in the growth parameters between regions. A decline in coral calcification of this magnitude with increasing seawater temperatures is unprecedented in recent centuries based on analysis of growth records from long cores of massive Porites. We discuss the decline in calcification within the context of known environmental controls on coral growth. Although our findings are consistent with studies of the synergistic effect of elevated seawater temperatures and pCO₂ on coral calcification, we conclude that further data on seawater chemistry of the GBR are required to better understand the links between environmental change and effects on coral growth.     

Nearshore coral growth declining on the Mesoamerican Barrier Reef System

Anthropogenic global change and local stressors are impacting coral growth and survival worldwide, altering the structure and function of coral reef ecosystems. Here, we show that skeletal extension rates of nearshore colonies of two abundant and widespread Caribbean corals (Siderastrea siderea, Pseudodiploria strigosa) declined across the Belize Mesoamerican Barrier Reef System (MBRS) over the past century, while offshore coral conspecifics exhibited relatively stable extension rates over the same temporal interval. This decline has caused nearshore coral extension rates to converge with those of their historically slower growing offshore coral counterparts. For both species, individual mass coral bleaching events were correlated with low rates of skeletal extension within specific reef environments, but no single bleaching event was correlated with low skeletal extension rates across all reef environments. We postulate that the decline in skeletal extension rates for nearshore corals is driven primarily by the combined effects of long‐term ocean warming and increasing exposure to higher levels of land‐based anthropogenic stressors, with acute thermally induced bleaching events playing a lesser role. If these declining trends in skeletal growth of nearshore S. siderea and P. strigosa continue into the future, the structure and function of these critical nearshore MBRS coral reef systems is likely to be severely impaired.     

Skeletal form and growth in Acropora aspera (Dana) from the Pulau Seribu,Indonesia

Linear extension and calcium carbonate accretion were measured in the branching coral Acropora aspera (Dana) from shallow-water sites around Pulau Pari, Pulau Seribu, Indonesia, during both wet and dry monsoon periods. Skeletal density and corallite form were also monitored in specimens collected from sites, variously affected by wave energy resulting from the monsoonal influence. Although the reversing monsoon appeared to exert the greatest effect on skeleton growth (by influencing temperature and possibly number of “sun-hours”) wave energy was also shown to affect skeletal extension, skeletal accretion, and skeletal density. The scale of differences between growth rate measurements was greatest for weight of skeleton accreted between monsoon period (8-fold), followed by between site differences (maximum 3-fold during west monsoon) and finally between station differences (maximum 3-fold during west monsoon at an outer reef flat and reef edge station). Skeletal extension did not appear to be as sensitive to the reversing monsoon influence as skeletal accretion.     

Radiographic studies of reef coral exoskeletons: Rates and patterns of coral growth

X-radiographs were made of vertical slices through the centers of 47 hermatypic coral colonies collected at Eniwetok Atoll, Marshall Islands. The image thus obtained are useful for the study of colony geometry, development, and response to damage.Comparison of radioactive inclusions of known age with previously reported cyclic skeletal density variations normal to the axis of growth confirms the annual nature of the density banding. Growth rates based on density bands and radioactivity inclusions are calculated for all 47 specimens, and measurements of the individual ‘growth bands’ are presented for 25 of them.Bulk densities measured by X-ray transmission ranged from 1.0 to 2.2 g/cm³, with an average range of 1.3–1.6 g/cm³. Intra-specimen skeletal densities typically vary by 10–30%; the period of high density skeletal deposition appears to coincide with the season of higher rainfall and warmer surface water at Eniwetok. Pigment residues left by boring algae are more commonly found in low density portions of the skeletons, but this distribution is believed to result from rather than cause the variations in the density of the deposited aragonite.Linear growth rates for the same specimen vary by factors of two or more from year to year, but the 25 specimens studied did not show a common pattern in the linear growth rate. Other than showing some general trends in growth as a function of species and depth, linear growth rates do not appear to be a particularly informative parameter.The density and growth rate variations are important factors in the measurement of coral growth and metabolism, and to the study of environmental controls of coral growth.     

Decline in skeletal growth of the coral Porites?lutea from the Andaman Sea, South Thailand between 1984 and 2005

Of the few studies that have examined in situ coral growth responses to recent climate change, none have done so in equatorial waters subject to relatively high sea temperatures (annual mean >27°C). This study compared the growth rate of Porites lutea from eight sites at Phuket, South Thailand between two time periods (December 1984–November 1986 and December 2003–November 2005). There was a significant decrease in coral calcification (23.5%) and linear extension rates (19.4–23.4%) between the two sampling periods at a number of sites, while skeletal bulk density remained unchanged. Over the last 46 years, sea temperatures (SST) in the area have risen at a rate of 0.161°C per decade (current seasonal temperature range 28–30°C) and regression analysis of coral growth data is consistent with a link between rising temperature and reduced linear extension in the order of 46–56% for every 1°C rise in SST. The apparent sensitivity of linear extension in P. lutea to increased SST suggests that corals in this part of the Andaman Sea may already be subjected to temperatures beyond their thermal optimum for skeletal growth. Communicated by Environment Editor Prof. Rob van Woesik     

Simultaneous extension of both basic microstructural components in scleractinian coral skeleton during night and daytime,visualized by in situ 86Sr pulse labeling

Using in situ (12 h) pulse-labeling of scleractinian coral aragonitic skeleton with stable ⁸⁶Sr isotope, the diel pattern of skeletal extension was investigated in the massive Porites lobata species, grown at 5 m depth in the Gulf of Eilat. Several microstructural aspects of coral biomineralization were elucidated, among which the most significant is simultaneous extension of the two basic microstructural components Rapid Accretion Deposits (RAD; also called Centers of Calcification) and Thickening Deposits (TD; also called fibers), both at night and during daytime. Increased thickness of the ⁸⁶Sr-labeled growth-front in the RADs compared to the adjacent TDs revealed that in this species RADs extend on average twice as fast as TDs. At the level of the individual corallite, skeletal extension is spatially highly heterogeneous, with sporadic slowing or cessation depending on growth directions and skeletal structure morphology. Daytime photosynthesis by symbiotic dinoflagellates is widely acknowledged to substantially increase calcification rates at the colony and the corallite level in reef-building corals. However, in our study, the average night-time extension rate (visualized in three successive 12 h pulses) was similar to the average daytime extension (visualized in the initial 12 h pulse), in all growth directions and skeletal structures. This research provides a platform for further investigations into the temporal calibration of coral skeletal extension via cyclic growth increment deposition, which is a hallmark of coral biomineralization.     

Extension growth rates in two coral species from high-latitude reefs of Bermuda

Mean annual growth rates (skeletal linear extension) in the hermatypic coralsPorites astreoides Lamarck andDiploria labyrinthiformis (L.) were investigated mainly by X-radiography from a variety of localities at various depths on the high-latitude coral reefs of Bermuda. Growth rates of both species show an inverse curvilinear relationship with depth, with highest growth rates in the shallow inshore waters of Castle Harbour and lowest at the edge of the Bermuda platform and on the adjacent fore-reef slope. Annual density bands form seasonal couplets, with narrow, high density bands appearing to form in the spring-summer months and wider, low density bands over the rest of the year in both species. Comparison of the growth rates ofP. astreoides from Bermuda with those from lower latitude West Indian localities, particularly Jamaica, indicates an inverse relationship with latitude and a similar inverse curvilinear relationship with depth at both geographic locations. Growth rate-locality differences in Bermuda for both species are suggested to be controlled mainly by local differences in wave energy and food supply and possibly seasonal water temperature fluctuations; growth rate-depth differences by decreasing illumination with depth; and growth rate-latitudinal differences by reduction in winter water temperatures and light levels with increasing latitude.     

Variation in coral growth rates with depth at Discovery Bay,Jamaica

Growth rates, determined by X-radiographic measurement of skeletal extension, decreased with depth for four of six species of coral examined at Discovery Bay, Jamaica. Growth of Porites astreoides, Montastrea annularis, Colpophyllia natans, and Siderastrea siderea decreased significantly with depth over a 1- to 30-m depth range. In Montastrea cavernosa, the highest growth rate occurred in the middle of the sampled depth range. Agaricia agaricites had no measurable change in growth rate with depth. A compilation of available growth data for Atlantic and Pacific corals shows a strong pattern of highest growth rates a short distance below the surface and a decrease with depth.Operated by Martin Marietta Energy Systems, Inc., under Contract No. DE-AC05-840R21400 with the U.S. Department of Energy. Publication No. 2467, Environmental Sciences Division, ORNL     

Porites growth characteristics in a changed environment: Misima Island, Papua New Guinea

 The construction in 1988 of an open-cut gold mine and ore-processing facility at Misima Island, Papua New Guinea, resulted in disturbance of the adjacent fringing coral reef, mostly because of large increases in sedimentation. This provided an opportunity to examine whether growth characteristics of the major reef-building coral, Porites, changed in response to this sudden and sustained increase in sedimentation. Annual variation in skeletal density was measured in 93 colonies variously affected by sedimentation. The colonies provided data for average annual density, annual extension and annual calcification covering the periods 5 y before and 5 y after mining operations began. The average depth of skeleton occupied by tissue (tissue layer thickness) was also measured for most colonies. There was high mortality of Porites in regions strongly affected by increased sedimentation. In colonies that survived, density, extension and calcification tended to be less (in some cases significantly) in the period after mining operations began compared with pre-construction levels. However, these decreases were not linked with proximity to the mine site and probably reflect a regional-scale response of Porites growth to some other environmental change. This suggests that periods of high sedimentation may not be recorded by the growth characteristics of massive Porites. Average growth characteristics of surviving Porites from Misima Island were similar to those from inshore reefs of the northern Great Barrier Reef (GBR). Tissue layer thickness in Porites from the control areas at Misima Island were also similar to colonies from the northern inshore GBR reefs. However, tissue layer thickness significantly decreased with increased proximity to the mine site at Misima Island. Accepted: 15 May 1999     

Coral Transplantation: Regeneration and Growth of Acropora Fragments in a Nursery

Abstract Coral reef degradation has been widely reported for the past 20 years. Because the recovery rate is usually low, various methods of restoration have been explored in different regions of the world. Among the effective and commonly used methods to restore coral communities is the transplantation of coral colonies or fragments. In this investigation fragments of Acropora pulchra were used in a semiprotected nursery in southern Taiwan between 1996 and 1998 to test, in situ, the possible effects of different factors on the generation of new branches and the initial skeletal extension rates of transplants. The variables under study here were the origin and length of the fragments, their new orientation, presence of tissue injury, and position in the fragment. All these factors were found to make a difference in either one or both aspects of coral growth (i.e., branching frequency and skeletal extension rate). These two factors clearly determine the success rate of a small fragment developing into a large colony that has a much higher probability to survive and grow on its own. It is now obvious that the efficiency of coral generation through fragment culture can be enhanced if the variables examined here are taken into consideration. Once coral colonies are formed, they can be fragmented again to generate more corals or can be transplanted to a suitable site.     

Linear extension rate is independent of colony size in the coral Pocillopora damicornis

Measurements of the skeletal extension rate of branches of the reef coral Pocillopora damicornis showed that the linear extension rate is independent of colony size for colonies from 1.9 to 19 cm in diameter. Analysis of existing data from Western Australia, Samoa, the Great Barrier Reef and Hawaii supports the finding that linear extension is not related to colony size in this species.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Coral density and predation affect growth of a reef-building coral

The influence of predation on the growth of stony corals has gained increased attention, although the degree to which coral conspecific density can modify the effects of corallivores remains poorly studied. Here, a field experiment was used to quantify the independent and combined effects of coral colony density and coral predators on the skeletal growth of massive Porites. Predator exclusion increased coral growth by 20%. Increasing coral density increased growth by 30%. However, the effect of predators was independent of coral density. Possible alternative mechanisms for increased skeletal growth at higher colony density include changes in near-field flow, resulting in increases in photosynthetic activity, nutrient uptake, or the increased accessibility of coral mutualists.     

Regional decline in growth rates of massive Porites corals in Southeast Asia

This study reports the first well‐replicated analysis of continuous coral growth records from warmer water reefs (mean annual sea surface temperatures (SST) >28.5 °C) around the Thai–Malay Peninsula in Southeast Asia. Based on analyses of 70 colonies sampled from 15 reefs within six locations, region‐wide declines in coral calcification rate (ca. 18.6%), linear extension rate (ca. 15.4%) and skeletal bulk density (ca. 3.9%) were observed over a 31‐year period from 1980 to 2010. Decreases in calcification and linear extension rates were observed at five of the six locations and ranged from ca. 17.2–21.6% and ca. 11.4–19.6%, respectively, whereas decline in skeletal bulk density was a consequence of significant reductions at only two locations (ca. 6.9% and 10.7%). A significant link between region‐wide growth rates and average annual SST was found, and Porites spp. demonstrated a high thermal threshold of ca. 29.4 °C before calcification rates declined. Responses at individual locations within the region were more variable with links between SST and calcification rates being significant at only four locations. Rates of sea temperature warming at locations in the Andaman Sea (Indian Ocean) (ca. 1.3 °C per decade) were almost twice those in the South China Sea (Pacific Ocean) (ca. 0.7 °C per decade), but this was not reflected in the magnitude of calcification declines at corresponding locations. Considering that massive Porites spp. are major reef builders around Southeast Asia, this region‐wide growth decline is a cause for concern for future reef accretion rates and resilience. However, this study suggests that the future rates and patterns of change within the region are unlikely to be uniform or dependent solely on the rates of change in the thermal environment.     

Skeletal growth, ultrastructure and composition of the azooxanthellate scleractinian coral Balanophyllia regia

The biomineralization process and skeletal growth dynamics of azooxanthellate corals are poorly known. Here, the growth rate of the shallow-water dendrophyllid scleractinian coral Balanophyllia regia was evaluated with calcein-labeling experiments that showed higher lateral than vertical extension. The structure, mineralogy and trace element composition of the skeleton were characterized at high spatial resolution. The epitheca and basal floor had the same ultrastructural organization as septa, indicating a common biological control over their formation. In all of these aragonitic skeletal structures, two main ultrastructural components were present: “centers of calcification” (COC) also called rapid accretion deposits (RAD) and “fibers” (thickening deposits, TD). Heterogeneity in the trace element composition, i.e., the Sr/Ca and Mg/Ca ratios, was correlated with the ultrastructural organization: magnesium was enriched by a factor three in the rapid accretion deposits compared with the thickening deposits. At the interface with the skeleton, the skeletogenic tissue (calicoblastic epithelium) was characterized by heterogeneity of cell types, with chromophile cells distributed in clusters regularly spaced between calicoblasts. Cytoplasmic extensions at the apical surface of the calicoblastic epithelium created a three-dimensional organization that could be related to the skeletal surface microarchitecture. Combined measurements of growth rate and skeletal ultrastructural increments suggest that azooxanthellate shallow-water corals produce well-defined daily growth steps.     

A precise and non-destructive method to calculate the surface area in living scleractinian corals using X-ray computed tomography and 3D modeling

The surface area of corals represents a major reference parameter for the standardization of flux rates, for coral growth investigations, and for investigations of coral metabolism. The methods currently used to determine the surface area of corals are rather approximate approaches lacking accuracy, or are invasive and often destructive methods that are inapplicable for experiments involving living corals. This study introduces a novel precise and non-destructive technique to quantify surface area in living coral colonies by applying computed tomography (CT) and subsequent 3D reconstruction. Living coral colonies of different taxa were scanned by conventional medical CT either in air or in sea water. Resulting data volumes were processed by 3D modeling software providing realistic 3D coral skeleton surface reconstructions, thus enabling surface area measurements. Comparisons of CT datasets obtained from calibration bodies and coral colonies proved the accuracy of the surface area determination. Surface area quantifications derived from two different surface rendering techniques applied for scanning living coral colonies showed congruent results (mean deviation ranging from 1.32 to 2.03%). The validity of surface area measurement was verified by repeated measurements of the same coral colonies by three test persons. No significant differences between all test persons in all coral genera and in both surface rendering techniques were found (independent sample t-test: all n.s.). Data analysis of a single coral colony required approximately 15 to 30 min for a trained user using the isosurface technique regardless of the complexity and growth form of the latter, rendering the method presented in this study as a time-saving and accurate method to quantify surface areas in both living coral colonies and bare coral skeletons. Communicated by Biology Editor Dr Michael Lesser     

Heterotrophy mitigates the response of the temperate coral Oculina arbuscula to temperature stress

Anthropogenic increases in atmospheric carbon dioxide concentration have caused global average sea surface temperature (SST) to increase by approximately 0.11°C per decade between 1971 and 2010 – a trend that is projected to continue through the 21st century. A multitude of research studies have demonstrated that increased SSTs compromise the coral holobiont (cnidarian host and its symbiotic algae) by reducing both host calcification and symbiont density, among other variables. However, we still do not fully understand the role of heterotrophy in the response of the coral holobiont to elevated temperature, particularly for temperate corals. Here, we conducted a pair of independent experiments to investigate the influence of heterotrophy on the response of the temperate scleractinian coral Oculina arbuscula to thermal stress. Colonies of O. arbuscula from Radio Island, North Carolina, were exposed to four feeding treatments (zero, low, moderate, and high concentrations of newly hatched Artemia sp. nauplii) across two independent temperature experiments (average annual SST (20°C) and average summer temperature (28°C) for the interval 2005–2012) to quantify the effects of heterotrophy on coral skeletal growth and symbiont density. Results suggest that heterotrophy mitigated both reduced skeletal growth and decreased symbiont density observed for unfed corals reared at 28°C. This study highlights the importance of heterotrophy in maintaining coral holobiont fitness under thermal stress and has important implications for the interpretation of coral response to climate change.