Another one bites the dirt: a closer look into geophagy in tadpoles of Thoropa miliaris (Spix, 1824)

Another one bites the dirt: a closer look into geophagy in tadpoles of Thoropa miliaris (Spix, 1824) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Another one bites the dirt: a closer look into geophagy in tadpoles of Thoropa miliaris (Spix, 1824) Gustavo Colaço, Júlio Lopes, Gabriel Fidalgo, Mendel C. Fonseca, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7708182/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2026 Read the published version in The Science of Nature → Version 1 posted You are reading this latest preprint version Abstract Geophagy is present in different animal groups, including adult frogs and their larvae. In some species, the ingestion of sediments is an intrinsic aspect of the biology of these animals. During an investigation on aspects of the natural history and morphology of tadpoles of Thoropa miliaris (Cycloramphidae) we noticed that their intestines were filled with sediments. The first question that occurred to us involved whether ingestion was accidental or part of their behavioral repertoire. These tadpoles are found in rockfaces wetted by a slow running film of water, where besides a slime film of algae and moss, we do not observe significant accumulation of sediments. Another question involved knowing if the sediments ingested were already available in their habitat, or if they were biting off small pieces of the rocks where they live. To investigate that, we used a combination of light microscopy and synchrotron high-resolution x-ray microtomography techniques to study the morphology of the sediments in the habitat and intestines of these larvae and based on that, determine its source. Cycloramphidae Habitat Intestinal Content Synchrotron Microtomography Natural History Sediments Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Geophagy (sand or clay eating) is well documented in several groups of animals (vertebrates and invertebrates). The most prominent cases among terrestrial vertebrates, includes bats (Voigt et al. 2008 ), birds (Downs et al. 2019 ), elephants (Houston et al. 2001 ) and primates (Pebsworth et al. 2019 ). In addition to reporting observations indicative of geophagy, studies on the subject infer reasons for this feeding behavior, which varies from nutritional complementation to detoxification (Johns and Duquette 1991 ; Diamond et al. 1999 ; Voigt et al. 2008 ; Young et al. 2011 ). Aquatic vertebrates that feed on sand include several marine fish species, as exemplified by parrotfishes that ingest sand deliberately (Veeramani et al. 2010 ) or accidentally while capturing prey (Feitosa & Ferreira 2015 ). Similar feeding behavior is also present in freshwater bottom dweller siluriform fishes (Lolis and Andrian 1996 ). Among marine invertebrates, holothurians (Schneider et al. 2013 ) and some polychaeta (Jang et al. 2021 ) represent remarkable examples of feeding habits that include ingestion of sediments and accessing microorganisms at the sea floor. Among anuran amphibians, there are reports of geophagy based on the presence of sediments in the digestive tract of adults of Rhinella margaritifera (Laurenti 1768) considered intentional (McCracken and Forstner 2006 ). In the cases of the pipid Pipa carvalhoi (Miranda-Ribeiro 1937), the scaphiopodid Spea intermontana (Cope, 1883) and the bufonids Rhinella ornata (Spix 1824) and Rhinella marina (Linnaeus 1758), the presence of sediments in the gut content was interpreted as accidental, that is, sediments were ingested with prey by a few individuals (Canedo et al. 2006 ; Evans and Lampo 1996 ; Feledi et al. 2025 ; Weygoldt 1976 ; Zack and Johnson 2008 ). In adults dicroglossid Hoplobatrachus occipitalis (Günther, 1858) mineral grains were one of the most frequent contents of the examined stomachs (Ogoanah and Uchedike 2011 ), although it was not among the most abundant. Abbott ( 1884 ) reported that among organic items, there was a sand-grain mass “cemented” by a dark matter in intestines of young scaphiopodid Scaphiopus holbrookii (Harlan, 1835), that may represent remnants of sand ingested by the tadpoles prior to metamorphosis. In the intestines of some bottom-dweller tadpoles, as in the Brazilian hylid Ololygon angrensis (Lutz 1973), sand grains represent one of the most frequently items, appearing as 12 to 26.3% of the total content (Sousa-Filho et al. 2007 ). This behavior does not appear to be unusual, and there are similar reports for tadpoles of distinct species and localities as summarized on Table I. Table 1 Reports of additional tadpoles in different localities for which sediment was identified in the digestive tract. Family Species Locality Reference Microhylidae Otophryne pyburni Campbell & Clarke 1998 Northern South America Wassersug & Pyburn 1987 ; see also Campbell & Clarke 1998 Ranidae Aquarana clamitans (Latreille 1801) North America (USA) Jenssen 1967 Ascaphidae Ascaphus truei Stejneger, 1899 North America (USA) Metter, 1964 Mantellidae Boophis majori (Boulenger 1896) Southeastern Coast of Africa (Madagascar) Schmidt et al. 2008 ; Grosjean et al. 2011 Mantellidae B . picturatus Glaw, Vences, Andreone, and Vallan, 2001 Southeastern Coast of Africa (Madagascar) Altig and McDiarmid 2006 ; Grosjean et al. 2011 Microhylidae Scaphiophryne gottlebei Busse and Böhme, 1992 Southeastern Coast of Africa (Madagascar) Mercúrio & Andreone, 2006 Rhacophoridae Rhacophorus vampyrus (Rowley, Le, Thi, Stuart, & Hoang, 2010) Southeast Asia (Vietnam) Vassilieva et al. 2013 Micrixalidae Micrixalus herrei Myers 1942 South Asia (India) Senevirathne et al. 2016 Dicroglossidae Nannophrys ceylonensis Günther, 1869 South Asia (Sri Lanka) Wickramasinghe et al. 2007 Dicroglossidae Fejervarya limnocharis (Gravenhorst, 1829) Southeast Asia (Vietnam) Vassilieva et al. 2025 Dicroglossidae Fejervarya moodiei (Taylor, 1920), Southeast Asia (Vietnam) Vassilieva et al. 2025 Dicroglossidae Hoplobatrachus chinensis (Osbeck, 1765), Southeast Asia (Vietnam) Vassilieva et al. 2025 Dicroglossidae Limnonectes dabanus (Smith, 1922), Southeast Asia (Vietnam) Vassilieva et al. 2025 Dicroglossidae Occidozyga lima (Gravenhorst, 1829), Southeast Asia (Vietnam) Vassilieva et al. 2025 Dicroglossidae Occidozyga martensii (Peters, 1867) Southeast Asia (Vietnam) Vassilieva et al. 2025 Dicroglossidae Quasipaa verrucospinosa (Bourret, 1937) Southeast Asia (Vietnam) Vassilieva et al. 2025 Alytidae Discoglossus pictus Otth, 1837 Southwestern Europe (Spain) Diaz-Paniagua 1985 Pelobatidae Pelobates cultripes (Cuvier, 1829) Southwestern Europe (Spain) Diaz-Paniagua 1985 Bufonide Epidalea calamita (Laurenti, 1768) Southwestern Europe (Spain) Diaz-Paniagua 1985 Hylidae Hyla meridionalis Boettger, 1874 Southwestern Europe (Spain) Diaz-Paniagua 1985 Ranidae Pelophylax perezi (López-Seoane, 1885) Southwestern Europe (Spain) Diaz-Paniagua 1985 Two events relating to the rediscovery that tadpoles of Thoropa miliaris (Spix 1824) ingested sediments occurred during research dealing with natural history and morphological studies of these larvae. First, during a morphological study of these tadpoles at the Brazilian Synchrotron Light Laboratory (LNLS) , the physicists of our team while evaluating the use of this technology to survey anatomical structures (see Fidalgo et al. 2018 ) were surprised to realize that a considerable number of mineral grains was revealed in the acquired images of the digestive tract. Although we have even adjusted the setup so the incidence of X-rays on the sediments would not interfere with the images, we did not report this finding previously. The second surprise occurred during the investigation carried out by an undergraduate student and coauthor of this work (MCF) in the Laboratory of Natural History, Anatomy and Systematic of Amphibians of Universidade Federal Rural do Rio de Janeiro; sediments were discovered while dissecting intestines of these tadpoles to study their diet. We found earlier reports of similar findings for Thoropa tadpoles that appear in natural history studies by Barth ( 1956 ) for T . miliaris tadpoles and by Wassersug and Heyer ( 1983 ) for tadpoles of Thoropa petropolitana (Wandolleck 1907). In this latter study, the authors stated that these tadpoles “had mineral grains” in their guts. Since tadpoles of species of the Thoropa thrive in a hygropetric environment (Barth 1956 ), swimming in thin films of water that run over rock faces (Bokermann 1965 ; Colaço and Silva 2022 ; Dias et al. 2021 ) near waterfalls or in areas where water seeps and drips from the soil on higher grounds (Fig. 1). In this wet habitat we observe moss, small insects, and some slimy biofilm thriving during the rainy season. Smaller than one-millimeter grains of sediments are present, but in lesser amounts that are barely seen over the rocks were tadpoles are found. These observations drove us to further investigate the subject. Our goals for this study were twofold. First, to determine if the ingestion of grains was accidental or if these tadpoles bite off small and loose crystals from the rocky surface where they live. We would be able to determine that by analyzing the morphology of the grains in the sediment and by the relative amount of minerals (the habitat where tadpoles live is composed of granitic-gneissic rocky-outcrop, see below) that compose the rock (that would be indicative of some sort of selection of the easy grains tadpoles were able to bite off). On the other hand, sediment grains available in such habitat may have marks of weathering and transporting, indicated by rounded surfaces. In contrast if they were broken off, at least some angular geometry would be present. Herein, we report the results of a survey of the ingested grains content of T. miliaris tadpoles combining light microscopy and synchrotron microtomography (SR-µCT) analysis. These analyses were designed to better describe the geological aspects of the sediments ingested by the tadpoles (geometry and mineralogy of grains). We compared ingested to habitat sediments and rocks to determine if tadpoles ingest grains that are available in their habitats or not. Figure 1. Rockface in Mangaratiba, Rio de Janeiro, Brazil, where tadpoles were sampled. (A) example of a hygropetric habitat with some covering vegetation and a film of water running over the exposed rock; and close-up of (B) a tadpole at GS39; (C) a tadpole at GS42; and (D) a recent-metamorphosed (GS46) Thoropa miliaris, found in this habitat mainly during the rainy season. Note that the animals are barely covered by the film of water. Material and Methods Sampling We surveyed tadpoles on rock-outcrops along an abandoned railroad (Fig. 1), now Avenida Litorânea, near Praia do Junqueira, in the Municipality of Mangaratiba (22°58'56"S 44°02'19"W – Google Earth), localized in a region called "Costa Verde" of the Brazilian State of Rio de Janeiro. The samples are housed at the Herpetological collection of Laboratory of Natural History, Anatomy and Systematic of Amphibians of Universidade Federal Rural do Rio de Janeiro . After collecting, the sampled tadpoles were anesthetized in MS222 (Tricaine methanesulfonate) dissolved in water. Larvae were fixed in formalin 10% and preserved in formalin 5%. Developmental stages of the tadpoles were determined following Gosner ( 1960 ) as modified by Colaço and Silva ( 2022 ). We examined the digestive tract content 21 tadpoles from stages GS28 to recent metamorphosed specimens (three specimens for stage). Additionally, at the collection site, we used a small brush to sweep the rock and collect small samples of sediments on the same spot we collected and observed tadpoles. These samples of sediments were photographed and compared to those found in the intestines of the larvae. Synchrotron beamline setup, data and image acquisition The SR-µCT analysis was used in the whole tadpole following the protocol of tomographic scanning developed in Fidalgo et al. ( 2018 ). For proceeding SR-µCT scan, the tadpoles were taken out of the formalin 10%, washed in distilled water, and dehydrated by immersing it in a series of ethanol solutions that graduated following 20%, 50%, 70% and 100% (10 minutes each). Thereafter, the specimens were placed in a polypropylene pipet tip filled up with 100 GL ethanol with the lower end melted to avoid leaking and the top under light pressure of a plunger. This research used the LNLS facilities, operated by the Brazilian Center for Research in Energy and Materials (CNPEM – Campinas/São Paulo). The tadpoles were scanned on the µCT beamline IMX (proposal IMX-20160615), using white beam to reduce the acquisition time of each tomography. The energy range by white beam in IMX beamline varies from 4–25 keV and the association of physical filters causes the average energy of the X-rays to be around 8 keV (pink beam). The image detection system consisted of a LuAG: Ce scintillator (50 µm thickness) and a CCD camera, model PCO2000 and 2X magnification lenses that provided a pixel size of 4.11 µm. Features of the intestines and its sand content were imaged and analyzed by in-line phase-contrast with the 10 cm sample-detector distance (SDD). This technique is responsible for enhancing the edge of internal structures (Paganin et al. 2002 ) and is recommended for biological samples because of soft tissue presence (Momose et al. 1996 ). After acquiring the projections, the images were reconstructed using the PyRAFT fast reconstruction algorithm (Miqueles et al. 2014 a, b), developed by the IMX team. The 3D visualization, data segmentation and geometrical analysis were performed using the Avizo Fire 8 software. Dissections, microscopic analysis, and intestinal content The tadpoles were dissected with small ophthalmologic scissors and forceps, with which we removed the skin of the abdominal area. The intestines were then removed and, with the aid of a small syringe filled with water and a thin needle, its content emptied into a small vial for microscopic analyses. Each intestinal content was placed into a small tube and centrifuged to separate sediments from the other contents. After that, the supernatant portion of the content was inspected under a microscope in search of organic content. Geometrical features (sphericity and roundness) and other properties ( e.g. habit, cleavage/fracture and luster) were used to identify and describe the mineralogy and maturity (chemical and textural) of the sediments (Deer et al. 1992 ; Boggs 2006 ). The ingested sediments were compared to sediment and rocks samples collected in the tadpole’s habitat. The sediments were analyzed using a Leica M205C stereomicroscope with a digital camera and a Leica Suit V2.0 image software. Results Description of intestine content and Comparison of ingested and habitat sediments All the intestines examined contained sediments mixed with some mucus (Fig. 2 ). Besides sediments, we also observed algae, cyanophycean, fragments of moss and other plants pieces, parts of ants, collembola, and a few keratodonts (keratinized tooth like structures on the mouth of tadpoles). As it was not the focus of the present study, we did not quantify or identify these materials at lower levels. The grains of sediment ranged in size from a few micrometers to 200𝜇m (0,2 mm). Sediments were observed from the buccopharyngeal cavity and gills till the posterior end of the intestines (Fig. 3 ). Grains of feldspar, quartz and minor pieces of mica were characterized (Figs. 3 and 4 ). The feldspar is a principal sediment particle, show size of 125 to 30 µm and are angular to very angular with low sphericity (Figs. 3 and 4 ). The feldspar grains are colorless to light gray with tabular, pyramidal or irregular geometries and show diagnostic two cleavage directions and resinous luster (Fig. 3 ). We identified two types of quartz crystals in the sediments. One type is represented by angular to sub-angular colorless grains, with 30–65 µm, low to medium sphericity and diagnostic conchoidal fracture and vitreous luster grains (Fig. 4 ). Another type is composed by rounded grain with high to medium sphericity, 4–8 µm of size and without fracture or luster (Fig. 4 ). The mica grains are varied from 250 to 31 µm and show sub-angular to sub-rounded geometry with low to medium sphericity (Figs. 3 and 4 ). The mica grains are black to brown (biotite), diagnostic vitreous luster and perfect cleavage in one direction. The sediments collected over the rocky habitat (Fig. 4 ) resemble those found inside the intestines in terms of mineralogy, roundness, sphericity and size range. Synchrotron microtomographic images and data analyses The SR-µCT enabled both, quantitative analysis of the sediments volume, and qualitative, such as sphericity and roundness of the grains, in 3D (Fig. 3 ). It also allowed an evaluation of the maturity of sediment deposit. Furthermore, the SR-µCT indicated the relative density of the imaged minerals. The SR-µCT images also allowed us to visualize that the sediment fills up the entire length of the intestines, however, there are intestinal sections with more concentration of larger grains. Although the 3D reconstruction images represent quartz grains with several of them quite easily discernible, it presents some differences when compared to those obtained via microscopic photography. Most of the quartz grains in the 3D images are sub-angular to sub-rounded with medium sphericity (Fig. 3 ), while grains under microscope (bidimensional) analysis are angular (Fig. 4 ). Distinct to quartz, feldspar and mica grains in 3D images are angulous compared to those observed under light microscopy (Fig. 4 ) and show diagnostic cleavage pattern in 3D image (Fig. 3 ). Discussion The presence of mineral grains in the gut content like the ones found in the environment is clear indicative that T. miliaris tadpoles ingest sediments already present in their habitat. Although the volume of sediments ingested is considerable, we cannot discard the possibility that its ingestion is accidental and unavoidable. Instead, we infer that as they move and graze by scraping the wet rock and suck in whatever may fit into their mouths and can be swallowed as is described for other tadpoles (Wassersug 1972 ). In this process it seems improbable that these tadpoles could select what to swallow, and because of that, sediments are included. Another possibility is that the suction force they use to pump water for respiration and to suck the food may cause the ingestion of sediment (grains are found scattered in the gill basket). We envision that future work on the intestinal wall may indicate that some adaptation for such a diet that includes sediments may be present, and as such, that the intestinal wall may be thicker than that of other tadpoles that feed on living matter only. Another interesting question that these results might indicate by future studies is whether together with the sediment grains, the tadpoles are also ingesting bacteria and other microorganisms associated with the grains and perhaps using this resource as nutrients. Sediment nature, size and morphology In sedimentology, maturity (textural or compositional) describes how physicochemical processes at Earth’s surface modified clastic sediment from its source-rock during weathering, erosion, transport and deposition (Krumbein 1941 ). The angular sediment grains with low sphericity composed by heterogenous mineralogy and granulometry are related to the lower amount of energy associated with transport and deposition. In the case at hand, all the grains examined in the intestinal content of the T. miliaris tadpoles presented features indicative that they have passed through a process of erosion. As such, they were made available to the site where the tadpoles ingested them, from a nearby rock source. The size of the grains and amount of sediment in the intestines of the geophagous tadpoles are diverse. Altig and McDiarmid ( 2006 ) reported grains for the entire extent of the intestine of B. picturatus . The dry mass of inside the gut of these tadpoles (Gosner 36; TL = 40.8) was 30% of wet mass, and 54% of gut content weight was only 15 largest grains (4.1 ± 0.7 mm). Grosjean et al. ( 2011 ) also analyzed the gut content of sediment grains in B. picturatus tadpoles, reporting sizes between 0.1 to 1.4 mm, with rare occurrence of grains larger than 1 mm and the majority with sizes varying from 0.2 to 0.4 mm. They also reported grains in the gut content of B. majori tadpoles, with smaller grains (< 0.2 mm). Senevirathne et al. ( 2016 ) reported the presence of sediment grains ranging from 0.1 to 0.7 mm through the entire length of the tadpole’s gut of the fully fossorial tadpole Micrixalus herrei , from India. For other species the gut content analysis only indicated the presence of mineral grains, the size or diversity were not reported. It is important to note that in these reported cases of geophagy the tadpoles live in riverine habitats, where sediments were transported to riverbeds, and that some process of sorting them is based on dimension and river flow. In the tadpoles of T. miliaris , the textural and chemical maturity of the ingested sediment indicates that their rock source is close to the habitat and that transport shows a lower energy. Tadpoles of T. miliaris , and possibly of all other cycloramphids with exotrophic tadpoles, differ in habitat usage from the generalist lotic/lentic tadpoles. Their hygropetric habitats (see Colaço and Silva 2022 ; Dias et al. 2021 ) i.e., films of water running on rocky outcrops that barely cover the tadpoles' body. This habitat shows low energy to transport sediments and possibly explains the sediment content ingested by these tadpoles. Morphology and Natural History of Geophagous Tadpoles Several morphological and behavioral features of tadpoles are evidence of a series of adaptations of this life phase to their habitats (McDiarmid and Altig 1999 ; Duellman and Trueb 1986 ), allowing the anurans to occupy and explore different habitats and niches (Roelants et al. 2011 ). The geophagous tadpoles include species that explore benthic, fossorial and hygropetric microenvironments (the species focus of this study). In each of these species show a specific set of characters (and behaviors) that reflect the diversity of ways to explore even the same type of habitat. As amply exemplified here, the presence of sediments in the intestines of distinct tadpoles seems to be more common than previously thought. Only two groups of reported geophagous tadpoles do not live in sediment-rich riverine or ponds environments where the grains are much more abundant, the exotrophic members of the family Cycloramphidae, especially species of genus Thoropa and the Dicroglossidae frog, N. ceylonensis (possibly all Nannophrys ). These tadpoles differ from most species discussed here, in both habitat and morphology. They inhabit water films that run on rocky-outcrops either near rivers and waterfalls or isolated from those on areas where water that infiltrated the soil above the outcrop run after heavy rains (semiterrestrial sensu Altig and Johnston 1989 ; quasi-terrestrial sensu Colaço and Silva 2022 ). In both habitats, it accumulates much less sediments than on the button of rivers and streams. The Sri Lankan Dicroglossidae frog, Nannophrys ceylonensis shows habitat usage and tadpole natural history extremely similar to that observed in the Cycloramphidae species of the genus Cycloramphus (the ones with exotrophic tadpoles) and Thoropa (Altig and Johnston 1989 ; Dias et al. 2021 ). There, as in the Brazilian Atlantic Rain Forest, these frogs reproduce in crevasses associated with waterfalls and tadpoles that roam around grazing on wet rocks that form a hygropetric habitat (Wickramasinghe et al. 2004 ). More recently, Wickramasinghe et al. ( 2007 ) described the diet of these tadpoles, reporting an increase in sediment content in their gut as they developed with latter stages with up to 9% of minerals versus other contents. The other tadpoles with sediments in the intestines exhibit diverse behaviors, morphological adaptations, and microhabitats usage, and have been grouped into distinct ecomorphological guilds ( sensu Altig and Johnston 1986). The benthic and fossorial (or nearly so) forms being the one more commonly associated with this feeding strategy. These tadpoles may not have an alternative but to ingest food mixed with sediment. Moreover, these tadpoles often show specialized feeding morphologies (Senevirathne et al. 2016 ; Dias et al. 2024 ). The tadpoles of the microhylid O. pyburni are considered psammonic, meaning that they live in sediment-rich shallow forest streams (see Altig and Johnston 1986). These tadpoles bury themselves into streambeds and feed and breathe in these conditions (Wassersug and Pyburn 1987 ; MacCulloch et al. 2008 ). Another group is represented by tadpoles called “psammonektonic,” exemplified by Scaphiophryne gottlebei Busse and Böhme 1992, which are also associated to streambeds and show both benthic and nektonic behaviors, feeding during the day with half body burrowed obliquely into the sediment grains and at night are found in the water column (Mercurio and Andreone 2006 ). The tadpoles of the “lotic clasping” B. majori and B. picturatus , which although resemble the latter larvae, was placed in its own guild (Grosjean et al. 2011 ) and are trivet in a similar habitat (Altig and McDiarmid 2006 ; Schmidt et al. 2008 ), that is streams running through well-forested areas with the sediment-rich bottom. In these rivers, however, the size of the grains ingested by these tadpoles vary, what may be explained by preferences for microhabitats. The ones living in a river flow can ingest larger grains than lower energetic microhabitats such as creeks, lakes or hygropetric habitats (Grosjean et al. 2011 ). The tadpole of M. herrei is fossorial, using this habitat from eggs until metamorphosis, living associated to streambed sand, gravel, and rocks in forested areas (Senevirathne et al. 2016 ). Other geophagous tadpoles are classified as having a more “generalist” lentic/lotic-benthic ecomorphologies such as Ololygon angrensis (Sousa-Filho et al. 2007 ) and Aquarana clamitans (Jenssen 1967 ). In a study of the diet of tadpoles from Huelva, Spain, Diaz-Paniagua ( 1985 ) reported finding sediment in the intestine of tadpoles of five species. In addition, she inferred that the differences in percentages of sediment consumption may relate to habits. Accordingly, while D. pictus, E. calamita and P. perezi are typically benthonic (sense Altig and Johnston 1989 ) and show a higher proportion of sediment in their intestinal contents (82.5%, 77.8% and 100% respectively), the more nektonic tadpoles of H. meridionalis and P. cultripes ingested less grains volume (44.6% and 54.3% respectively). Metter ( 1964 ) found between 30–40% of fine sand grains in his sample of intestines in the lotic-suctorial tadpoles of the tailed frog A. truei , which he inferred to be ingested while the tadpoles feed on rocks covered by diatoms, in fast-flowing rocky mountain streams. Recently, Dias et al. ( 2024 ) described the buccopharyngeal morphology of specialized “sand-eating” mantellid tadpoles of genus Mantidactylus. They have found that these tadpoles, besides having highly modified oral discs, a unique arrangement of the buccopharyngeal structures, characterized mainly by the presence of “ruffled ridges.” They propose that these structures would serve to separate food particles from the grains of sediments. Although not “too specialized,” the mantellid B. picturatus have a prelingual arena and buccal floor arena densely papillated and pustulated, whereas on the roof a bunch of pustules and long papillae are present postnarial and buccal roof arena (Grosjean et al. 2011 ), being also associated as an adaptation to foraging in the sand. Internally, tadpoles of T. miliaris do not exhibit any morphology suggestive of an adaptation to catch and separate mineral grains, in contrast, they show a relative overall reduction of these papillae systems and secretory surfaces (Dias et al. 2021 ), even when ontogenetic factors are considered (Colaço et al. 2024 ). Is geophagy an adaptation? Because of the feeding mechanism of tadpoles, that involves grazing and suction, we infer that in these cases swallowing sediments is unavoidable. In other words, different from what is documented for adult frogs, when sediment is found in their digestive tract content and infer that was accidentally ingested, in the case of tadpoles in such habitats, individuals have no way to avoid ingesting sediments. In these cases, geophagy may represent an adaptation to obtaining food in these habitats, being an intrinsic characteristic of tadpoles that have evolved in these habitats and/or lifestyles exemplified before. Once it is impossible to sort mineral grains out of the swallowed material, some hypotheses were raised to explain this behavior. First, the mineral particles could play the role as micro-gastroliths, acting as a “grinder” (Nathan and James 1972 ; Sousa-Filho et al. 2007 ; Wickramasinghe et al. 2007 ) and helping to macerate the organic matter within the gut and improving digestibility of, for example vegetable matter, as do gastroliths – a term developed by Mayne ( 1854 ) – in birds and other animals, living and fossil (Wings 2007 ). Another possibility is that mixed with the sediments there are biofilms of microorganisms, such as bacteria, microalgae, protozoa and even very small metazoans (Vences et al. 2016 ; Sousa-Filho et al. 2007 ), that once ingested serves as food source in nutrient-depleted environments (Senevirathne et al. 2016 ). We foresee a possible test if sand may act as micro-gastroliths by comparing the state of some of the large and harder ingested food, such as moss and larger plant material, in a more anterior versus a more posterior portion of the intestine. For Thoropa , in addition to sediments, Barth ( 1956 ) reported diatoms, fungal spores, pteridophytes, vegetal detritus, and rests of small arthropods in the digestive tract of T. miliaris (some of these items were also found by us). Wassersug and Heyer ( 1983 ) reported similar findings for T. petropolitana (Wandolleck 1907). Based on these findings, we also hypothesize that the great quantity of mineral grains may increase the tadpole’s body density, helping with the anchoring on the wet and slippery rock. Also, further investigations are necessary to determine what sort of microorganisms may be associated with the sediments, both in the environment and in the intestine content. In addition, it would be interesting to perform some histological studies on the intestines to investigate if its wall is somehow distinct from that of other tadpoles. Final remarks Our results have some important implications for feeding in tadpoles, both in the field of anuran biology and conservation, as well as research in natural history (Tschinkel and Wilson 2014 ). Several authors have called attention to the secondary role Natural History research now plays in biological investigation; there are even claims that the field is in danger of extinction (Nanglu et al. 2023 ). These alert calls to the danger of this field of research disappearing seem to result from the specialization biologists are demanded to engage in since the XIX Century (Farber 1982 ). Contrary to this trend, and most importantly, our results show that the field still needs the investment of people and resources to uncover some basic knowledge gaps about several aspects of animals (and plants) life history that may have consequences in all the other fields in biology. What we learn from natural history may result in future research associated with a simple question of what animals eat. Another important side note of our research relates to the importance of partnerships between interdisciplinary specialists. To understand some biological aspects of these tadpoles, we assembled a group of different specialists, such as zoologists, geologists and physicists, each adding insights into understanding of the problem studied herein. Declarations Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Conceptualization: GC, JL, GF, MVC, RCB and HRS. Methodology: GC, JL, GF, MCF, KP, MB, GL, MVC, RCB and HRS. Formal analysis: GC, JL, GF, MCF and HRS. Project administration: GC, MVC, RCB and HRS. Visualization: GC, JL, GF, MCF, MB, GL, RCB, HRS. Writing – original draft: GC, JL, GF, HRS. Writing – review & editing: GC, JL, GF, KP, MB, GL, GMS, MVC, RCB, HRS. Acknowledgement This research was supported and used facilities of the Brazilian Synchrotron Light Laboratory (LNLS), part of the Brazilian Center for Research in Energy and Materials (CNPEM) a private nonprofit organization under the supervision of the Brazilian Ministry for Science, Technology, and Innovations (MCTI). GC is a postdoctoral fellow from National Council for Scientific and Technological Development (CNPq) and Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ). JL is a postdoctoral fellow funded by Foundation for Scientific and Technological Research Support at UFRRJ (FAPUR) and ExxonMobil. MB and GL are doctoral fellows from Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES). GMS received a scientific initiation fellowship from CNPq. References Abbott CC (1884) Recent studies of the spade-foot toad. 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West N Am Nat 68(2):241–244. https://doi.org/10.3398/1527-0904(2008)68[241:fbtgbs]2.0.co;2 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 05 Jan, 2026 Read the published version in The Science of Nature → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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01:51:39","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146703,"visible":true,"origin":"","legend":"","description":"","filename":"948139d2189d43189232eb27642bf0151structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/a8a2479fcc4f02e52713db4f.xml"},{"id":93083524,"identity":"6d4d2eb9-308f-4c2b-ab4d-843ffbabe797","added_by":"auto","created_at":"2025-10-09 01:52:30","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156906,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/f6fce87bf12017a0b4d0e03d.html"},{"id":93083426,"identity":"60ea7b85-ca87-4b46-a0dd-b12c1174a284","added_by":"auto","created_at":"2025-10-09 01:48:20","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1091811,"visible":true,"origin":"","legend":"\u003cp\u003eRockface in Mangaratiba, Rio de Janeiro, Brazil, where tadpoles were sampled. (A) example of a hygropetric habitat with some covering vegetation and a film of water running over the exposed rock; and close-up of (B) a tadpole at GS39; (C) a tadpole at GS42; and (D) a recent-metamorphosed (GS46) Thoropa miliaris, found in this habitat mainly during the rainy season. Note that the animals are barely covered by the film of water.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/64741ef55743c9efd6ca2b25.jpeg"},{"id":93083525,"identity":"8d554051-eec8-4e56-9035-85fb2476022e","added_by":"auto","created_at":"2025-10-09 01:54:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":325249,"visible":true,"origin":"","legend":"\u003cp\u003eSediments in the intestine of Thoropa miliaris tadpole. (A) Tadpole at GS39 with the abdominal skin and muscles removed and exposed intestine showing to be filled with fine grains of sediments inside. (B) Close-up of area of a tadpole intestine (diameter of circle 1 mm), showing sediments by transparency; (C) close-up of an intestine that was cut and spilled some of the content, including this piece of mica (80 µm); (D) rectum of a recent-metamorphosed (GS46) open showing great concentration of sediments\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/3e1829b2bf54ec1df6c9cb91.jpeg"},{"id":93083710,"identity":"cc2d11af-1e0e-44dc-8831-f0149f153603","added_by":"auto","created_at":"2025-10-09 02:12:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175035,"visible":true,"origin":"","legend":"\u003cp\u003eSR-μCT images showing the sediments inside the tadpole of Thoropa miliaris body in 3D. (A) sediments inside the tadpole, which highlighted field represents sediments inside the branchial chamber (anteriorly) and the sediments inside the intestines (posteriorly); (B) the same area of the intestine showed in (A), however with adjusted parameters to show only the mineral grains with a few representative grains colored; in yellow feldspar, in blue quarts, and in green mica crystals. In (C), (D), and (E) a close-up of the same crystals to highlight some of their morphological properties.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/60550180ac54399290c4a960.png"},{"id":93083588,"identity":"f38ee841-eba7-43c8-a3c9-eba38fad46c6","added_by":"auto","created_at":"2025-10-09 01:55:05","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":582087,"visible":true,"origin":"","legend":"\u003cp\u003ePhotographs from light microscopy of mineral grains as found in the hygropetric habitat (A, B, and C) where tadpoles are found in 3 samples. In C we present a single large crystal of mica that better; And sediments sampled in the intestines of the tadpoles of Thoropa miliaris (D, E, and F). Blue arrow quarts, green arrow mica, and red arrow feldspar.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/93ba45604796ed8dd234afe3.jpeg"},{"id":100069916,"identity":"810bb1b1-b96f-4bb2-9bd3-1a43a41769a1","added_by":"auto","created_at":"2026-01-12 16:15:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3040280,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7708182/v1/80d0922a-40f2-4d9c-b368-c2285dd10223.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Another one bites the dirt: a closer look into geophagy in tadpoles of Thoropa miliaris (Spix, 1824)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGeophagy (sand or clay eating) is well documented in several groups of animals (vertebrates and invertebrates). The most prominent cases among terrestrial vertebrates, includes bats (Voigt et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), birds (Downs et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), elephants (Houston et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and primates (Pebsworth et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition to reporting observations indicative of geophagy, studies on the subject infer reasons for this feeding behavior, which varies from nutritional complementation to detoxification (Johns and Duquette \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Diamond et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Voigt et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Young et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Aquatic vertebrates that feed on sand include several marine fish species, as exemplified by parrotfishes that ingest sand deliberately (Veeramani et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) or accidentally while capturing prey (Feitosa \u0026amp; Ferreira \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similar feeding behavior is also present in freshwater bottom dweller siluriform fishes (Lolis and Andrian \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Among marine invertebrates, holothurians (Schneider et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and some polychaeta (Jang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) represent remarkable examples of feeding habits that include ingestion of sediments and accessing microorganisms at the sea floor.\u003c/p\u003e\u003cp\u003eAmong anuran amphibians, there are reports of geophagy based on the presence of sediments in the digestive tract of adults of \u003cem\u003eRhinella margaritifera\u003c/em\u003e (Laurenti 1768) considered intentional (McCracken and Forstner \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In the cases of the pipid \u003cem\u003ePipa carvalhoi\u003c/em\u003e (Miranda-Ribeiro 1937), the scaphiopodid \u003cem\u003eSpea intermontana\u003c/em\u003e (Cope, 1883) and the bufonids \u003cem\u003eRhinella ornata\u003c/em\u003e (Spix 1824) and \u003cem\u003eRhinella marina\u003c/em\u003e (Linnaeus 1758), the presence of sediments in the gut content was interpreted as accidental, that is, sediments were ingested with prey by a few individuals (Canedo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Evans and Lampo \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Feledi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Weygoldt \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Zack and Johnson \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In adults dicroglossid \u003cem\u003eHoplobatrachus occipitalis\u003c/em\u003e (G\u0026uuml;nther, 1858) mineral grains were one of the most frequent contents of the examined stomachs (Ogoanah and Uchedike \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), although it was not among the most abundant. Abbott (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1884\u003c/span\u003e) reported that among organic items, there was a sand-grain mass \u0026ldquo;cemented\u0026rdquo; by a dark matter in intestines of young scaphiopodid \u003cem\u003eScaphiopus holbrookii\u003c/em\u003e (Harlan, 1835), that may represent remnants of sand ingested by the tadpoles prior to metamorphosis. In the intestines of some bottom-dweller tadpoles, as in the Brazilian hylid \u003cem\u003eOlolygon angrensis\u003c/em\u003e (Lutz 1973), sand grains represent one of the most frequently items, appearing as 12 to 26.3% of the total content (Sousa-Filho et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This behavior does not appear to be unusual, and there are similar reports for tadpoles of distinct species and localities as summarized on Table I.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eReports of additional tadpoles in different localities for which sediment was identified in the digestive tract.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLocality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMicrohylidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eOtophryne pyburni\u003c/em\u003e Campbell \u0026amp; Clarke \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNorthern South America\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWassersug \u0026amp; Pyburn \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; see also Campbell \u0026amp; Clarke \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRanidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAquarana clamitans\u003c/em\u003e (Latreille 1801)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNorth America (USA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eJenssen \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1967\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAscaphidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAscaphus truei\u003c/em\u003e Stejneger, 1899\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNorth America (USA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMetter, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1964\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMantellidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eBoophis majori\u003c/em\u003e (Boulenger 1896)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheastern Coast of\u0026nbsp;Africa (Madagascar)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSchmidt et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Grosjean et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMantellidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eB\u003c/em\u003e. \u003cem\u003epicturatus\u003c/em\u003e Glaw, Vences, Andreone, and Vallan, 2001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheastern Coast of\u0026nbsp;Africa (Madagascar)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAltig and McDiarmid \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Grosjean et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMicrohylidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eScaphiophryne gottlebei\u003c/em\u003e Busse and B\u0026ouml;hme, 1992\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheastern Coast of\u0026nbsp;Africa (Madagascar)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMerc\u0026uacute;rio \u0026amp; Andreone, 2006\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRhacophoridae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eRhacophorus vampyrus\u003c/em\u003e (Rowley, Le, Thi, Stuart, \u0026amp; Hoang, 2010)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMicrixalidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMicrixalus herrei\u003c/em\u003e Myers 1942\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouth Asia (India)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSenevirathne et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eNannophrys ceylonensis\u003c/em\u003e G\u0026uuml;nther, 1869\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouth Asia (Sri Lanka)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWickramasinghe et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eFejervarya limnocharis\u003c/em\u003e (Gravenhorst, 1829)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFejervarya moodiei (Taylor, 1920),\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eHoplobatrachus chinensis\u003c/em\u003e (Osbeck, 1765),\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLimnonectes dabanus\u003c/em\u003e (Smith, 1922),\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eOccidozyga lima\u003c/em\u003e (Gravenhorst, 1829),\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOccidozyga \u003cem\u003emartensii\u003c/em\u003e (Peters, 1867)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicroglossidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eQuasipaa verrucospinosa\u003c/em\u003e (Bourret, 1937)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoutheast Asia (Vietnam)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVassilieva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlytidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eDiscoglossus pictus\u003c/em\u003e Otth, 1837\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouthwestern Europe (Spain)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiaz-Paniagua \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePelobatidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePelobates cultripes\u003c/em\u003e (Cuvier, 1829)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouthwestern Europe (Spain)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiaz-Paniagua \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBufonide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEpidalea calamita\u003c/em\u003e (Laurenti, 1768)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouthwestern Europe (Spain)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiaz-Paniagua \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHylidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eHyla meridionalis\u003c/em\u003e Boettger, 1874\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouthwestern Europe (Spain)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiaz-Paniagua \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRanidae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePelophylax perezi\u003c/em\u003e (L\u0026oacute;pez-Seoane, 1885)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSouthwestern Europe (Spain)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDiaz-Paniagua \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTwo events relating to the rediscovery that tadpoles of \u003cem\u003eThoropa miliaris\u003c/em\u003e (Spix 1824) ingested sediments occurred during research dealing with natural history and morphological studies of these larvae. First, during a morphological study of these tadpoles at the \u003cem\u003eBrazilian Synchrotron Light Laboratory (LNLS)\u003c/em\u003e, the physicists of our team while evaluating the use of this technology to survey anatomical structures (see Fidalgo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) were surprised to realize that a considerable number of mineral grains was revealed in the acquired images of the digestive tract. Although we have even adjusted the setup so the incidence of X-rays on the sediments would not interfere with the images, we did not report this finding previously. The second surprise occurred during the investigation carried out by an undergraduate student and coauthor of this work (MCF) in the \u003cem\u003eLaboratory of Natural History, Anatomy and Systematic of Amphibians\u003c/em\u003e of \u003cem\u003eUniversidade Federal Rural do Rio de Janeiro;\u003c/em\u003e sediments were discovered while dissecting intestines of these tadpoles to study their diet.\u003c/p\u003e\u003cp\u003eWe found earlier reports of similar findings for \u003cem\u003eThoropa\u003c/em\u003e tadpoles that appear in natural history studies by Barth (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1956\u003c/span\u003e) for \u003cem\u003eT\u003c/em\u003e. \u003cem\u003emiliaris\u003c/em\u003e tadpoles and by Wassersug and Heyer (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) for tadpoles of \u003cem\u003eThoropa petropolitana\u003c/em\u003e (Wandolleck 1907). In this latter study, the authors stated that these tadpoles \u0026ldquo;had mineral grains\u0026rdquo; in their guts. Since tadpoles of species of the \u003cem\u003eThoropa\u003c/em\u003e thrive in a hygropetric environment (Barth \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1956\u003c/span\u003e), swimming in thin films of water that run over rock faces (Bokermann \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Cola\u0026ccedil;o and Silva \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) near waterfalls or in areas where water seeps and drips from the soil on higher grounds (Fig.\u0026nbsp;1). In this wet habitat we observe moss, small insects, and some slimy biofilm thriving during the rainy season. Smaller than one-millimeter grains of sediments are present, but in lesser amounts that are barely seen over the rocks were tadpoles are found. These observations drove us to further investigate the subject.\u003c/p\u003e\u003cp\u003eOur goals for this study were twofold. First, to determine if the ingestion of grains was accidental or if these tadpoles bite off small and loose crystals from the rocky surface where they live. We would be able to determine that by analyzing the morphology of the grains in the sediment and by the relative amount of minerals (the habitat where tadpoles live is composed of granitic-gneissic rocky-outcrop, see below) that compose the rock (that would be indicative of some sort of selection of the easy grains tadpoles were able to bite off). On the other hand, sediment grains available in such habitat may have marks of weathering and transporting, indicated by rounded surfaces. In contrast if they were broken off, at least some angular geometry would be present. Herein, we report the results of a survey of the ingested grains content of \u003cem\u003eT. miliaris\u003c/em\u003e tadpoles combining light microscopy and synchrotron microtomography (SR-\u0026micro;CT) analysis. These analyses were designed to better describe the geological aspects of the sediments ingested by the tadpoles (geometry and mineralogy of grains). We compared ingested to habitat sediments and rocks to determine if tadpoles ingest grains that are available in their habitats or not.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFigure 1. Rockface in Mangaratiba, Rio de Janeiro, Brazil, where tadpoles were sampled. (A) example of a hygropetric habitat with some covering vegetation and a film of water running over the exposed rock; and close-up of (B) a tadpole at GS39; (C) a tadpole at GS42; and (D) a recent-metamorphosed (GS46) Thoropa miliaris, found in this habitat mainly during the rainy season. Note that the animals are barely covered by the film of water.\u003c/em\u003e\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSampling\u003c/h2\u003e\u003cp\u003eWe surveyed tadpoles on rock-outcrops along an abandoned railroad (Fig.\u0026nbsp;1), now Avenida Litor\u0026acirc;nea, near Praia do Junqueira, in the Municipality of Mangaratiba (22\u0026deg;58'56\"S 44\u0026deg;02'19\"W \u0026ndash; Google Earth), localized in a region called \"Costa Verde\" of the Brazilian State of Rio de Janeiro. The samples are housed at the Herpetological collection of \u003cem\u003eLaboratory of Natural History, Anatomy and Systematic of Amphibians\u003c/em\u003e of \u003cem\u003eUniversidade Federal Rural do Rio de Janeiro\u003c/em\u003e. After collecting, the sampled tadpoles were anesthetized in MS222 (Tricaine methanesulfonate) dissolved in water. Larvae were fixed in formalin 10% and preserved in formalin 5%. Developmental stages of the tadpoles were determined following Gosner (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1960\u003c/span\u003e) as modified by Cola\u0026ccedil;o and Silva (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We examined the digestive tract content 21 tadpoles from stages GS28 to recent metamorphosed specimens (three specimens for stage). Additionally, at the collection site, we used a small brush to sweep the rock and collect small samples of sediments on the same spot we collected and observed tadpoles. These samples of sediments were photographed and compared to those found in the intestines of the larvae.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSynchrotron beamline setup, data and image acquisition\u003c/h3\u003e\n\u003cp\u003eThe SR-\u0026micro;CT analysis was used in the whole tadpole following the protocol of tomographic scanning developed in Fidalgo et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For proceeding SR-\u0026micro;CT scan, the tadpoles were taken out of the formalin 10%, washed in distilled water, and dehydrated by immersing it in a series of ethanol solutions that graduated following 20%, 50%, 70% and 100% (10 minutes each). Thereafter, the specimens were placed in a polypropylene pipet tip filled up with 100 GL ethanol with the lower end melted to avoid leaking and the top under light pressure of a plunger.\u003c/p\u003e\u003cp\u003eThis research used the LNLS facilities, operated by the Brazilian Center for Research in Energy and Materials (CNPEM \u0026ndash; Campinas/S\u0026atilde;o Paulo). The tadpoles were scanned on the \u0026micro;CT beamline IMX (proposal IMX-20160615), using white beam to reduce the acquisition time of each tomography. The energy range by white beam in IMX beamline varies from 4\u0026ndash;25 keV and the association of physical filters causes the average energy of the X-rays to be around 8 keV (pink beam). The image detection system consisted of a LuAG: Ce scintillator (50 \u0026micro;m thickness) and a CCD camera, model PCO2000 and 2X magnification lenses that provided a pixel size of 4.11 \u0026micro;m.\u003c/p\u003e\u003cp\u003eFeatures of the intestines and its sand content were imaged and analyzed by in-line phase-contrast with the 10 cm sample-detector distance (SDD). This technique is responsible for enhancing the edge of internal structures (Paganin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and is recommended for biological samples because of soft tissue presence (Momose et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). After acquiring the projections, the images were reconstructed using the PyRAFT fast reconstruction algorithm (Miqueles et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003ea, b), developed by the IMX team. The 3D visualization, data segmentation and geometrical analysis were performed using the Avizo Fire 8 software.\u003c/p\u003e\n\u003ch3\u003eDissections, microscopic analysis, and intestinal content\u003c/h3\u003e\n\u003cp\u003eThe tadpoles were dissected with small ophthalmologic scissors and forceps, with which we removed the skin of the abdominal area. The intestines were then removed and, with the aid of a small syringe filled with water and a thin needle, its content emptied into a small vial for microscopic analyses. Each intestinal content was placed into a small tube and centrifuged to separate sediments from the other contents. After that, the supernatant portion of the content was inspected under a microscope in search of organic content.\u003c/p\u003e\u003cp\u003eGeometrical features (sphericity and roundness) and other properties (\u003cem\u003ee.g.\u003c/em\u003e habit, cleavage/fracture and luster) were used to identify and describe the mineralogy and maturity (chemical and textural) of the sediments (Deer et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Boggs \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The ingested sediments were compared to sediment and rocks samples collected in the tadpole\u0026rsquo;s habitat. The sediments were analyzed using a Leica M205C stereomicroscope with a digital camera and a Leica Suit V2.0 image software.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eDescription of intestine content and Comparison of ingested and habitat sediments\u003c/h2\u003e\u003cp\u003eAll the intestines examined contained sediments mixed with some mucus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Besides sediments, we also observed algae, cyanophycean, fragments of moss and other plants pieces, parts of ants, collembola, and a few keratodonts (keratinized tooth like structures on the mouth of tadpoles). As it was not the focus of the present study, we did not quantify or identify these materials at lower levels.\u003c/p\u003e\u003cp\u003eThe grains of sediment ranged in size from a few micrometers to 200\u0026#120583;m (0,2 mm). Sediments were observed from the buccopharyngeal cavity and gills till the posterior end of the intestines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Grains of feldspar, quartz and minor pieces of mica were characterized (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The feldspar is a principal sediment particle, show size of 125 to 30 \u0026micro;m and are angular to very angular with low sphericity (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The feldspar grains are colorless to light gray with tabular, pyramidal or irregular geometries and show diagnostic two cleavage directions and resinous luster (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We identified two types of quartz crystals in the sediments. One type is represented by angular to sub-angular colorless grains, with 30\u0026ndash;65 \u0026micro;m, low to medium sphericity and diagnostic conchoidal fracture and vitreous luster grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Another type is composed by rounded grain with high to medium sphericity, 4\u0026ndash;8 \u0026micro;m of size and without fracture or luster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The mica grains are varied from 250 to 31 \u0026micro;m and show sub-angular to sub-rounded geometry with low to medium sphericity (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The mica grains are black to brown (biotite), diagnostic vitreous luster and perfect cleavage in one direction. The sediments collected over the rocky habitat (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) resemble those found inside the intestines in terms of mineralogy, roundness, sphericity and size range.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSynchrotron microtomographic images and data analyses\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe SR-\u0026micro;CT enabled both, quantitative analysis of the sediments volume, and qualitative, such as sphericity and roundness of the grains, in 3D (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It also allowed an evaluation of the maturity of sediment deposit. Furthermore, the SR-\u0026micro;CT indicated the relative density of the imaged minerals. The SR-\u0026micro;CT images also allowed us to visualize that the sediment fills up the entire length of the intestines, however, there are intestinal sections with more concentration of larger grains.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough the 3D reconstruction images represent quartz grains with several of them quite easily discernible, it presents some differences when compared to those obtained via microscopic photography. Most of the quartz grains in the 3D images are sub-angular to sub-rounded with medium sphericity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), while grains under microscope (bidimensional) analysis are angular (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Distinct to quartz, feldspar and mica grains in 3D images are angulous compared to those observed under light microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and show diagnostic cleavage pattern in 3D image (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe presence of mineral grains in the gut content like the ones found in the environment is clear indicative that \u003cem\u003eT. miliaris\u003c/em\u003e tadpoles ingest sediments already present in their habitat. Although the volume of sediments ingested is considerable, we cannot discard the possibility that its ingestion is accidental and unavoidable. Instead, we infer that as they move and graze by scraping the wet rock and suck in whatever may fit into their mouths and can be swallowed as is described for other tadpoles (Wassersug \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). In this process it seems improbable that these tadpoles could select what to swallow, and because of that, sediments are included. Another possibility is that the suction force they use to pump water for respiration and to suck the food may cause the ingestion of sediment (grains are found scattered in the gill basket). We envision that future work on the intestinal wall may indicate that some adaptation for such a diet that includes sediments may be present, and as such, that the intestinal wall may be thicker than that of other tadpoles that feed on living matter only. Another interesting question that these results might indicate by future studies is whether together with the sediment grains, the tadpoles are also ingesting bacteria and other microorganisms associated with the grains and perhaps using this resource as nutrients.\u003c/p\u003e\n\u003ch3\u003eSediment nature, size and morphology\u003c/h3\u003e\n\u003cp\u003eIn sedimentology, maturity (textural or compositional) describes how physicochemical processes at Earth\u0026rsquo;s surface modified clastic sediment from its source-rock during weathering, erosion, transport and deposition (Krumbein \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1941\u003c/span\u003e). The angular sediment grains with low sphericity composed by heterogenous mineralogy and granulometry are related to the lower amount of energy associated with transport and deposition. In the case at hand, all the grains examined in the intestinal content of the \u003cem\u003eT. miliaris\u003c/em\u003e tadpoles presented features indicative that they have passed through a process of erosion. As such, they were made available to the site where the tadpoles ingested them, from a nearby rock source.\u003c/p\u003e\u003cp\u003eThe size of the grains and amount of sediment in the intestines of the geophagous tadpoles are diverse. Altig and McDiarmid (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported grains for the entire extent of the intestine of \u003cem\u003eB. picturatus\u003c/em\u003e. The dry mass of inside the gut of these tadpoles (Gosner 36; TL\u0026thinsp;=\u0026thinsp;40.8) was 30% of wet mass, and 54% of gut content weight was only 15 largest grains (4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm). Grosjean et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) also analyzed the gut content of sediment grains in \u003cem\u003eB. picturatus\u003c/em\u003e tadpoles, reporting sizes between 0.1 to 1.4 mm, with rare occurrence of grains larger than 1 mm and the majority with sizes varying from 0.2 to 0.4 mm. They also reported grains in the gut content of \u003cem\u003eB. majori\u003c/em\u003e tadpoles, with smaller grains (\u0026lt;\u0026thinsp;0.2 mm). Senevirathne et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported the presence of sediment grains ranging from 0.1 to 0.7 mm through the entire length of the tadpole\u0026rsquo;s gut of the fully fossorial tadpole \u003cem\u003eMicrixalus herrei\u003c/em\u003e, from India. For other species the gut content analysis only indicated the presence of mineral grains, the size or diversity were not reported. It is important to note that in these reported cases of geophagy the tadpoles live in riverine habitats, where sediments were transported to riverbeds, and that some process of sorting them is based on dimension and river flow.\u003c/p\u003e\u003cp\u003eIn the tadpoles of \u003cem\u003eT. miliaris\u003c/em\u003e, the textural and chemical maturity of the ingested sediment indicates that their rock source is close to the habitat and that transport shows a lower energy. Tadpoles of \u003cem\u003eT. miliaris\u003c/em\u003e, and possibly of all other cycloramphids with exotrophic tadpoles, differ in habitat usage from the generalist lotic/lentic tadpoles. Their hygropetric habitats (see Cola\u0026ccedil;o and Silva \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) i.e., films of water running on rocky outcrops that barely cover the tadpoles' body. This habitat shows low energy to transport sediments and possibly explains the sediment content ingested by these tadpoles.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMorphology and Natural History of Geophagous Tadpoles\u003c/h2\u003e\u003cp\u003eSeveral morphological and behavioral features of tadpoles are evidence of a series of adaptations of this life phase to their habitats (McDiarmid and Altig \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Duellman and Trueb \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), allowing the anurans to occupy and explore different habitats and niches (Roelants et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The geophagous tadpoles include species that explore benthic, fossorial and hygropetric microenvironments (the species focus of this study). In each of these species show a specific set of characters (and behaviors) that reflect the diversity of ways to explore even the same type of habitat. As amply exemplified here, the presence of sediments in the intestines of distinct tadpoles seems to be more common than previously thought.\u003c/p\u003e\u003cp\u003eOnly two groups of reported geophagous tadpoles do not live in sediment-rich riverine or ponds environments where the grains are much more abundant, the exotrophic members of the family Cycloramphidae, especially species of genus \u003cem\u003eThoropa\u003c/em\u003e and the Dicroglossidae frog, \u003cem\u003eN. ceylonensis\u003c/em\u003e (possibly all \u003cem\u003eNannophrys\u003c/em\u003e). These tadpoles differ from most species discussed here, in both habitat and morphology. They inhabit water films that run on rocky-outcrops either near rivers and waterfalls or isolated from those on areas where water that infiltrated the soil above the outcrop run after heavy rains (semiterrestrial \u003cem\u003esensu\u003c/em\u003e Altig and Johnston \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; quasi-terrestrial \u003cem\u003esensu\u003c/em\u003e Cola\u0026ccedil;o and Silva \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In both habitats, it accumulates much less sediments than on the button of rivers and streams.\u003c/p\u003e\u003cp\u003eThe Sri Lankan Dicroglossidae frog, \u003cem\u003eNannophrys ceylonensis\u003c/em\u003e shows habitat usage and tadpole natural history extremely similar to that observed in the Cycloramphidae species of the genus \u003cem\u003eCycloramphus\u003c/em\u003e (the ones with exotrophic tadpoles) and \u003cem\u003eThoropa\u003c/em\u003e (Altig and Johnston \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There, as in the Brazilian Atlantic Rain Forest, these frogs reproduce in crevasses associated with waterfalls and tadpoles that roam around grazing on wet rocks that form a hygropetric habitat (Wickramasinghe et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). More recently, Wickramasinghe et al. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) described the diet of these tadpoles, reporting an increase in sediment content in their gut as they developed with latter stages with up to 9% of minerals versus other contents.\u003c/p\u003e\u003cp\u003eThe other tadpoles with sediments in the intestines exhibit diverse behaviors, morphological adaptations, and microhabitats usage, and have been grouped into distinct ecomorphological guilds (\u003cem\u003esensu\u003c/em\u003e Altig and Johnston 1986). The benthic and fossorial (or nearly so) forms being the one more commonly associated with this feeding strategy. These tadpoles may not have an alternative but to ingest food mixed with sediment. Moreover, these tadpoles often show specialized feeding morphologies (Senevirathne et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The tadpoles of the microhylid \u003cem\u003eO. pyburni\u003c/em\u003e are considered psammonic, meaning that they live in sediment-rich shallow forest streams (see Altig and Johnston 1986). These tadpoles bury themselves into streambeds and feed and breathe in these conditions (Wassersug and Pyburn \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; MacCulloch et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Another group is represented by tadpoles called \u0026ldquo;psammonektonic,\u0026rdquo; exemplified by \u003cem\u003eScaphiophryne gottlebei\u003c/em\u003e Busse and B\u0026ouml;hme 1992, which are also associated to streambeds and show both benthic and nektonic behaviors, feeding during the day with half body burrowed obliquely into the sediment grains and at night are found in the water column (Mercurio and Andreone \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The tadpoles of the \u0026ldquo;lotic clasping\u0026rdquo; \u003cem\u003eB. majori\u003c/em\u003e and \u003cem\u003eB. picturatus\u003c/em\u003e, which although resemble the latter larvae, was placed in its own guild (Grosjean et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and are trivet in a similar habitat (Altig and McDiarmid \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Schmidt et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), that is streams running through well-forested areas with the sediment-rich bottom. In these rivers, however, the size of the grains ingested by these tadpoles vary, what may be explained by preferences for microhabitats. The ones living in a river flow can ingest larger grains than lower energetic microhabitats such as creeks, lakes or hygropetric habitats (Grosjean et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The tadpole of \u003cem\u003eM. herrei\u003c/em\u003e is fossorial, using this habitat from eggs until metamorphosis, living associated to streambed sand, gravel, and rocks in forested areas (Senevirathne et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOther geophagous tadpoles are classified as having a more \u0026ldquo;generalist\u0026rdquo; lentic/lotic-benthic ecomorphologies such as \u003cem\u003eOlolygon angrensis\u003c/em\u003e (Sousa-Filho et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and \u003cem\u003eAquarana clamitans\u003c/em\u003e (Jenssen \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). In a study of the diet of tadpoles from Huelva, Spain, Diaz-Paniagua (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) reported finding sediment in the intestine of tadpoles of five species. In addition, she inferred that the differences in percentages of sediment consumption may relate to habits. Accordingly, while \u003cem\u003eD. pictus, E. calamita\u003c/em\u003e and \u003cem\u003eP. perezi\u003c/em\u003e are typically benthonic (sense Altig and Johnston \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and show a higher proportion of sediment in their intestinal contents (82.5%, 77.8% and 100% respectively), the more nektonic tadpoles of \u003cem\u003eH. meridionalis\u003c/em\u003e and \u003cem\u003eP. cultripes\u003c/em\u003e ingested less grains volume (44.6% and 54.3% respectively). Metter (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1964\u003c/span\u003e) found between 30\u0026ndash;40% of fine sand grains in his sample of intestines in the lotic-suctorial tadpoles of the tailed frog \u003cem\u003eA. truei\u003c/em\u003e, which he inferred to be ingested while the tadpoles feed on rocks covered by diatoms, in fast-flowing rocky mountain streams.\u003c/p\u003e\u003cp\u003eRecently, Dias et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) described the buccopharyngeal morphology of specialized \u0026ldquo;sand-eating\u0026rdquo; mantellid tadpoles of genus \u003cem\u003eMantidactylus.\u003c/em\u003e They have found that these tadpoles, besides having highly modified oral discs, a unique arrangement of the buccopharyngeal structures, characterized mainly by the presence of \u0026ldquo;ruffled ridges.\u0026rdquo; They propose that these structures would serve to separate food particles from the grains of sediments. Although not \u0026ldquo;too specialized,\u0026rdquo; the mantellid \u003cem\u003eB. picturatus\u003c/em\u003e have a prelingual arena and buccal floor arena densely papillated and pustulated, whereas on the roof a bunch of pustules and long papillae are present postnarial and buccal roof arena (Grosjean et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), being also associated as an adaptation to foraging in the sand. Internally, tadpoles of \u003cem\u003eT. miliaris\u003c/em\u003e do not exhibit any morphology suggestive of an adaptation to catch and separate mineral grains, in contrast, they show a relative overall reduction of these papillae systems and secretory surfaces (Dias et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), even when ontogenetic factors are considered (Cola\u0026ccedil;o et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIs geophagy an adaptation?\u003c/h2\u003e\u003cp\u003eBecause of the feeding mechanism of tadpoles, that involves grazing and suction, we infer that in these cases swallowing sediments is unavoidable. In other words, different from what is documented for adult frogs, when sediment is found in their digestive tract content and infer that was accidentally ingested, in the case of tadpoles in such habitats, individuals have no way to avoid ingesting sediments. In these cases, geophagy may represent an adaptation to obtaining food in these habitats, being an intrinsic characteristic of tadpoles that have evolved in these habitats and/or lifestyles exemplified before. Once it is impossible to sort mineral grains out of the swallowed material, some hypotheses were raised to explain this behavior. First, the mineral particles could play the role as micro-gastroliths, acting as a \u0026ldquo;grinder\u0026rdquo; (Nathan and James \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Sousa-Filho et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wickramasinghe et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and helping to macerate the organic matter within the gut and improving digestibility of, for example vegetable matter, as do gastroliths \u0026ndash; a term developed by Mayne (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1854\u003c/span\u003e) \u0026ndash; in birds and other animals, living and fossil (Wings \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Another possibility is that mixed with the sediments there are biofilms of microorganisms, such as bacteria, microalgae, protozoa and even very small metazoans (Vences et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sousa-Filho et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), that once ingested serves as food source in nutrient-depleted environments (Senevirathne et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We foresee a possible test if sand may act as micro-gastroliths by comparing the state of some of the large and harder ingested food, such as moss and larger plant material, in a more anterior versus a more posterior portion of the intestine.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003eThoropa\u003c/em\u003e, in addition to sediments, Barth (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1956\u003c/span\u003e) reported diatoms, fungal spores, pteridophytes, vegetal detritus, and rests of small arthropods in the digestive tract of \u003cem\u003eT. miliaris\u003c/em\u003e (some of these items were also found by us). Wassersug and Heyer (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) reported similar findings for \u003cem\u003eT. petropolitana\u003c/em\u003e (Wandolleck 1907). Based on these findings, we also hypothesize that the great quantity of mineral grains may increase the tadpole\u0026rsquo;s body density, helping with the anchoring on the wet and slippery rock. Also, further investigations are necessary to determine what sort of microorganisms may be associated with the sediments, both in the environment and in the intestine content. In addition, it would be interesting to perform some histological studies on the intestines to investigate if its wall is somehow distinct from that of other tadpoles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFinal remarks\u003c/h2\u003e\u003cp\u003eOur results have some important implications for feeding in tadpoles, both in the field of anuran biology and conservation, as well as research in natural history (Tschinkel and Wilson \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Several authors have called attention to the secondary role Natural History research now plays in biological investigation; there are even claims that the field is in danger of extinction (Nanglu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These alert calls to the danger of this field of research disappearing seem to result from the specialization biologists are demanded to engage in since the XIX Century (Farber \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Contrary to this trend, and most importantly, our results show that the field still needs the investment of people and resources to uncover some basic knowledge gaps about several aspects of animals (and plants) life history that may have consequences in all the other fields in biology. What we learn from natural history may result in future research associated with a simple question of what animals eat. Another important side note of our research relates to the importance of partnerships between interdisciplinary specialists. To understand some biological aspects of these tadpoles, we assembled a group of different specialists, such as zoologists, geologists and physicists, each adding insights into understanding of the problem studied herein.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: GC, JL, GF, MVC, RCB and HRS. Methodology: GC, JL, GF, MCF, KP, MB, GL, MVC, RCB and HRS. Formal analysis: GC, JL, GF, MCF and HRS. Project administration: GC, MVC, RCB and HRS. Visualization: GC, JL, GF, MCF, MB, GL, RCB, HRS. Writing \u0026ndash; original draft: GC, JL, GF, HRS. Writing \u0026ndash; review \u0026amp; editing: GC, JL, GF, KP, MB, GL, GMS, MVC, RCB, HRS.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported and used facilities of the Brazilian Synchrotron Light Laboratory (LNLS), part of the Brazilian Center for Research in Energy and Materials (CNPEM) a private nonprofit organization under the supervision of the Brazilian Ministry for Science, Technology, and Innovations (MCTI). GC is a postdoctoral fellow from National Council for Scientific and Technological Development (CNPq) and Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ). JL is a postdoctoral fellow funded by Foundation for Scientific and Technological Research Support at UFRRJ (FAPUR) and ExxonMobil. MB and GL are doctoral fellows from Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES). GMS received a scientific initiation fellowship from CNPq.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbott CC (1884) Recent studies of the spade-foot toad. Am Nat 18(11):1075\u0026ndash;1080\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAltig R, Johnston GF (1989) Guilds of anuran larvae: relationships among developmental modes, morphologies, and habitats. Herpetol monogr 81\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/1466987\u003c/span\u003e\u003cspan address=\"10.2307/1466987\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAltig R, McDiarmid RW (2006) Descriptions and biological notes on three unusual mantellid tadpoles (Amphibia: Anura: Mantellidae) from southeastern Madagascar. 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West N Am Nat 68(2):241\u0026ndash;244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3398/1527-0904(2008)68[241:fbtgbs]2.0.co;2\u003c/span\u003e\u003cspan address=\"10.3398/1527-0904(2008)68[241:fbtgbs]2.0.co;2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cycloramphidae, Habitat, Intestinal Content, Synchrotron Microtomography, Natural History, Sediments","lastPublishedDoi":"10.21203/rs.3.rs-7708182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7708182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGeophagy is present in different animal groups, including adult frogs and their larvae. In some species, the ingestion of sediments is an intrinsic aspect of the biology of these animals. During an investigation on aspects of the natural history and morphology of tadpoles of \u003cem\u003eThoropa miliaris\u003c/em\u003e (Cycloramphidae) we noticed that their intestines were filled with sediments. The first question that occurred to us involved whether ingestion was accidental or part of their behavioral repertoire. These tadpoles are found in rockfaces wetted by a slow running film of water, where besides a slime film of algae and moss, we do not observe significant accumulation of sediments. Another question involved knowing if the sediments ingested were already available in their habitat, or if they were biting off small pieces of the rocks where they live. To investigate that, we used a combination of light microscopy and synchrotron high-resolution x-ray microtomography techniques to study the morphology of the sediments in the habitat and intestines of these larvae and based on that, determine its source.\u003c/p\u003e","manuscriptTitle":"Another one bites the dirt: a closer look into geophagy in tadpoles of Thoropa miliaris (Spix, 1824)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 21:54:16","doi":"10.21203/rs.3.rs-7708182/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d67c0c5-3746-471d-b78e-52f16bd3cfea","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:08:40+00:00","versionOfRecord":{"articleIdentity":"rs-7708182","link":"https://doi.org/10.1007/s00114-025-02061-4","journal":{"identity":"the-science-of-nature","isVorOnly":false,"title":"The Science of Nature"},"publishedOn":"2026-01-05 15:58:12","publishedOnDateReadable":"January 5th, 2026"},"versionCreatedAt":"2025-10-08 21:54:16","video":"","vorDoi":"10.1007/s00114-025-02061-4","vorDoiUrl":"https://doi.org/10.1007/s00114-025-02061-4","workflowStages":[]},"version":"v1","identity":"rs-7708182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7708182","identity":"rs-7708182","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}