Coral reefs, renowned for their vibrant biodiversity and ecological significance, host a rich array of species that occupy various microhabitats within their calcium carbonate formations. The sustainability of coral structures relies on maintaining an equilibrium between reef accretion, primarily driven by hermatypic corals, and processes that break down calcium carbonate substrates (Connell, 1978; Silbiger et al., 2018). While corals are the primary reef builders, they are complemented by the contributions of other organisms such as crustose coralline algae, calcareous sponges, foraminifera, and certain mollusks, all enhancing reef framework formation. This equilibrium faces constant challenges from erosion, occurring in two primary forms: physical erosion from wave action and currents, and bioerosion (often considered the predominant form of erosion), involving organisms that feed on or bore into the reef framework (Connell, 1978; Silbiger et al., 2018). While some reef inhabitants, such as corallivores (dominated by parrotfish and urchin species), directly alter the reef framework through grazing, cryptic “boring” organisms also play important yet hidden roles. Within the calcium carbonate coral skeletons, cryptic communities of bioeroding organisms thrive by actively penetrating and removing the coral’s structural materials through bioerosion activities (Glynn and Manzello, 2015; Fordyce et al., 2021; Rice et al., 2020).

Bioeroders modify the architectural complexity of reefs across multiple spatial scales, ranging from the micron to meter level (Davidson et al., 2018; Fordyce et al., 2020a; Glynn and Manzello, 2015; Roff et al., 2019). These bioeroders are broadly categorized as either surface-dwelling or internal-boring depending on their location and mode of erosive activity. External bioeroders reside on exposed reef surfaces, whereas internal bioeroders reside hidden within the calcium carbonate skeleton (Glynn and Manzello, 2015). Although the majority of internal bioeroders remain cryptic, that is, concealed within the substrates they penetrate, evidence suggests that their abundance and biomass may equal or surpass those of external bioeroders (Ginsburg, 1983; Weinstein et al., 2016). Additionally, internal bioeroders exhibit the highest taxonomic diversity among bioeroding organisms (Glynn and Manzello, 2015). The key taxa among the internal bioeroders that penetrate coral skeletons, also termed macroendoliths, include Porifera, Polychaeta, Sipuncula, Bivalvia, and Cirripedia (MacGeachy and Stearn, 1976). At the microscopic scale, microendolithic communities of cyanobacteria, chlorophytes, rhodophytes, and fungi actively penetrate structural calcium carbonate substrates (Hutchings, 1986; Perry and Harborne, 2016).

Despite the high biomass, diversity, and prevalence of internal bioeroders observed in many scleractinian corals, a comprehensive understanding of these cryptic organisms remains elusive (Schönberg et al., 2017). The profound inaccessibility of internal bioeroders significantly impedes research efforts, contributing to this knowledge gap. Moreover, the complexity of bioerosion processes, which intricately shape various facets of reef ecological functions (Fordyce et al., 2020a), poses additional challenges to understanding this phenomena. For instance, in contrast to the apparent detrimental impact on reef substrata strength, cavities generated by internal bioeroders augment habitat complexity, thereby enhancing the diversity and biomass of organisms associated with reefs (Glynn and Manzello, 2015). There is a critical need to enhance our understanding of bioeroders’ abundance, burrowing activities, and potential impacts on coral skeletal properties, as these organisms play a pivotal role in influencing coral reef resilience and stability (Schönberg et al., 2017).

To date, various methodologies have been employed to assess the abundance of internal bioeroders, including the cross-sectioning of bioeroder cavities, coral skeletal decalcification, and in situ surveys (Maher et al., 2018; Rice et al., 2019; Risk et al., 1995; Scott, 1988). While these approaches enable the direct quantification of living bioeroders in their natural habitat, they are limited in identifying cavities blocked by coral overgrowth, which may result from the death of bioeroders during the coral lifespan or coverage by other settlements. These methods provide limited data on the abundance of living bioeroders and lack information on cavity volume or the impact of bioeroders on substrate properties such as skeleton density and porosity.

Another technique utilized to gain insights into the extent and characteristic patterns of skeletal excavation by diverse organisms is the use of X-ray scans (Hein and Risk, 1975; MacGeachy and Stearn, 1976; Highsmith et al., 1983; Risk and Sammarco, 1991). However, simple X-ray radiography offers limited insights, as it is a two-dimensional imaging technique and is typically characterized by lower resolution. Computed Tomography (CT), on the other hand, offers exceptional three-dimensional (3D) high-resolution imaging, facilitating the non-destructive analysis of intricate microstructural details within coral skeletons without physically altering or destroying the original skeleton (DeCarlo et al., 2015; Fordyce et al., 2020b; Kramer et al., 2022). Previous studies utilizing CT for non-destructive analysis have quantified carbonate production, bioeroding activities, and net accretion on coral substrates (Silbiger et al., 2014, 2016, 2017; DeCarlo et al., 2015; Enochs et al., 2016, 2021; Newman et al., 2023). However, detailed methodological evaluations of CT for quantitative analysis of cryptic internal coral bioeroders, such as calcareous bivalves, are insufficient.

Here, we present a case study that evaluates the effectiveness of µCT scanning in conducting high-resolution morphometric analyses of common coral-boring species in the northern Red Sea, and present evidence of the advantages of this technique over traditional methods. Furthermore, we address potential concerns regarding the use of preserved coral samples for µCT scanning. Our complementary approach provides precise quantification of internal calcareous bioeroders that can be applied to advance our understanding of the interactions between these organisms and their calcifying hosts.

Modern 3D imaging techniques, such as computed tomography, are gaining popularity in the study of calcifying organisms as this tool facilitates ecologically relevant examinations of internal structural changes (Silbiger et al., 2016; Gutiérrez et al., 2018; Fordyce et al., 2020b; Kramer et al., 2023). However, coral reef research has not yet fully harnessed its potential to provide novel ecological insights. Here, we demonstrate that µCT serves as a powerful, precise, and non-destructive tool to study quantitative demographical and morphological aspects of bioeroders in their coral hosts, and provide spatial visualization of the of the internal bioeroder population within the host skeleton.

While prior studies share the use of volumetric µCT analysis for quantifying overall bioerosion patterns and processes in coral skeletons, including that of macro bioeroders (Silbiger et al., 2014, 2016, 2017; DeCarlo et al., 2015; Enochs et al., 2016, 2021; Newman et al., 2023), a key distinction lies in our focus on specific cryptic calcareous bivalves species rather than general bioerosion measures or broader taxonomical level. By targeting these major yet understudied bioeroders, we evaluate μCT’s ability to precisely quantify their abundance, size structure, spatial distribution, and the geometry of their individual boring cavities - aspects not explicitly examined in previous studies. Moreover, our direct comparative analysis between μCT and decalcification techniques highlights μCT’s advantages as a rapid, non-destructive method for assessing bioeroder population demographics and impacts on skeletal porosity. Overall, our work provides a complementary, fine-scale perspective on coral-bioeroder interactions, demonstrating μCT as a practical tool for advanced ecological investigations in this area.

µCT was advantageous for locating lithophagid bivalves and their boreholes ( Figure 1 ). This group plays a significant role in internal bioerosion and contributes to the destruction of diverse calcareous substrates (Glynn and Manzello, 2015; Morton, 1983; Rice et al., 2019). Specifically, genera such as Leiosolenus and Lithophaga engage in boring activities within live and dead coral structures, respectively. These cryptic molluscs achieve peak abundances in subtidal reef zones, with certain species extending their bioerosive activities to the lower depth limits of reef-building corals (Glynn and Manzello, 2015). Our 3D analyses of coral specimens provide detailed insights into the impacts of boring bivalves on coral skeletons, enabling accurate assessments of bioerosion processes by locating the precise location, distribution, growth angle, and abundance of bioeroders within corals ( Figures 1 ,  2 ), and to measure their body size with microscale precision ( Figure 3 . We were able to detect bivalves ranging in size from 0.5mm to 28mm, with varying sizes among the Leiosolenus species ( Figure 1 ; Table 1 ). This size variation aligns with the skeletal polyp dimensions of the different coral morphologies examined. In contrast to small-polyp corals (<1.2mm in diameter for S. pistillata), large-polyp corals (5mm and 2.5mm in diameter for E. forskaliana and A. myriophthalma, respectively) are typically associated with larger skeletal features, thus providing more space for larger bivalves to settle and bore.

Assessing the body size of boring bivalve assemblagescould provide unprecedented information regarding their demographic characteristics, including recruitment events, reproductive maturity, and longevity (Dietzel et al., 2020; Kramer et al., 2020). However, reports of the size-frequency distribution of boring organisms, let alone of lithophagid bivalves, are lacking and non-existent in many species. Scott (1988) sought to assess the abundance of the internal bioeroder Leiosolenus bisulcatus within live and dead coral hosts. Coral host fragments were collected, and counts were made of both living and deceased Leiosolenus individuals. However, aside from the clear destructive and irreversible drawbacks associated with this method, it lacks the capacity to furnish details regarding burrow volume and its effect on coral porosity.

Bioerosion by bivalves is caused by the secretion of acid, which dissolves the substrate and allows them to create a burrow, typically occupying polyp spaces (Lazar and Loya, 1991). Utilizing µCT scans, we segmented and measured individual burrow volumes created by the bioeroders within the host skeleton ( Figure 2 ). Measuring these volumes provides insights into the impact of internal bioerosion on skeletal integrity and porosity, which varies among coral species owing to factors such as morphological groups and environmental conditions (Foster et al., 2016; Hughes, 1987; Kramer et al., 2023). We showed that boring cavities in the encrusting E. forskaliana accounted for over 60% of the total porosity, boring cavities in the massive A. myriophthalma contributed nearly one-third of the coral porosity, and boring cavities in the branching S. pistillata reached up to 25% of the coral porosity ( Table 1 ). Associating these findings with bivalve size and abundance suggests that the extent of internal bioerosion is influenced by bivalve size and coral morphology, namely, larger bivalve species create expansive boring cavities that contribute substantially to skeletal porosity.

Additionally, examining the internal coral skeleton in 3D provided flexibility in identifying bivalves that were “blocked” ( Table 1 ) by the coral skeletal overgrowth, obstructing their burrowing progress towards the coral surface. Such information can easily be overlooked during field observations of live boring bivalves or when examining a predetermined 2D slice angle of the skeleton since these approaches offer inadequate estimation for macroborers (Silbiger et al., 2016). A comparison between µCT scan examination and bivalve counting after skeleton decalcification revealed that µCT scans yielded more accurate bivalve abundance estimates in 9 of 12 S. pistillata samples ( Table 1 ). This superiority is attributed to the ability of µCT to capture very small bivalves (< 1 mm) effectively, which is especially beneficial in corals inhabited by mostly small-sized bioeroders, such as S. pistillata. In contrast, using the skeleton decalcification method proved insufficient as it can be prone to human error and oversight in identifying and locating bivalves hidden within the tissue, and there is a risk of losing very small bivalves during the process ( Figure 4 ). Furthermore, skeletal decalcification is significantly more time-consuming than X-ray scanning, taking up to 24h (depending on skeletal density) compared to just 1h in µCT. However, while reducing processing time, µCT scanning may potentially increase costs. Therefore, to balance accuracy and affordability, it is important to clearly state the research objective. If µCT is required, it is necessary to predetermine the optimal scan parameters required to identify and measure the structures of interest. Lastly, we demonstrated the ability to scan corals with fixed tissues as a powerful tool for advancing comprehensive research, eliminating the need for additional sampling efforts. Our investigation revealed that X-ray tomography had no adverse effects on tissue integrity compared to samples that had not undergone scanning, ensuring the suitability of the samples for subsequent histological analysis. This finding carries significant implications, offering valuable opportunities for utilizing existing sample collections and museum specimens more effectively. Traditionally, formalin-fixed coral specimens have been primarily utilized to investigate cellular composition and other histological characteristics from the preserved tissues. However, we highlight that the non-destructive nature of μCT allows to visualize and analyze their internal calcareous structures without compromising on the integrity of the tissue, thereby allowing for subsequent analyses.

Our approach demonstrated that utilizing µCT for bioeroder analysis in corals surpasses the traditional 2D X-ray radiography technique, sectioning, and/or skeletal decalcification owing to its non-destructive nature, time efficiency, precision, and provision of high-resolution 3D reconstructions of internal and external coral-bivalve features. Our study highlights the advantages of µCT in quantifying bore-holes down to the sub-millimeter scale and offering high-resolution 3D visualization of their distribution within coral skeletons. Additionally, we demonstrated that with proper sample preparation, µCT can be used with fixed coral tissues, which is important for studying delicate structures or samples that need to be preserved.

As we navigate an era of climate change, investigating the impact of shifting oceanic conditions on marine calcifiers has become increasingly imperative (Fordyce et al., 2020b). Incorporating this powerful tool into our toolkit fills essential gaps in our understanding of the interaction between internal bioeroders and their coral hosts, which is necessary for unraveling the factors influencing coral health and resilience. Lastly, µCT generates digital image data that can be readily shared among researchers, fostering collaboration and advancing our collective understanding of coral ecosystems.