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Session Schedule & Abstracts

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Saturday 2nd July, 2016

Symposium: Diffusible iodine-based contrast-enhanced computed tomography (diceCT) and related imaging techniques for evolutionary morphology 3

Room: Salon A   2:30 pm–4:00 pm

Moderator(s): P. M. Gignac, A. N. Herdina, N. J. Kley, A. Morhardt, J. A. Clarke, & M. Colbert
DCT3-1  2:30 pm  Contrast-enhanced micro-CT imaging of fish and frogs: digital dissections and biomechanical applications. Porro L*, Royal Veterinary College, University of London; Brocklehurst R, University of Manchester; Adriaens D, Ghent University; Herrel A, CNRS/Muséum National d'Histoire Naturelle; Rayfield E, University of Bristol
Abstract: Traditional dissection is destructive and may be unsuitable for very small specimens or anatomical structures. Micro-computed tomography (µCT) is excellent for visualizing three-dimensional, hard-tissue anatomy in high-resolution; however, it cannot differentiate between low-density soft tissues. Methodological advancements in the use of various contrast-enhancing agents has resulted in detailed, three-dimensional digital dissections of an increasingly wide range of vertebrates. Contrast-enhanced µCT was used to differentiate between soft tissues in the heads of two extant teleost fishes—the northern pike (Esox lucius) and European eel (Anguilla anguilla)—and throughout the entire body of an African clawed frog (Xenopus laevis). Specimens were stained using either iodine potassium iodide (pike and Xenopus) or phosphomolybdic acid (eel), with concentrations and staining times varying with staining agent and specimen size. Visualization of pike and eel data resulted in detailed models of cranial musculature, with different relative muscle sizes being correlated to different feeding modes (suction versus biting). Digital dissection of Xenopus provides the first comprehensive guide to the anatomy of this model organism in over 90 years, including visualization of the skeleton, muscles, organs and nervous system in phenomenal detail. In addition to digital dissections for understanding and quantifying morphology, information from these contrast-enhanced CT data sets were used in biomechanical analyses. Muscle information from the fish were used to load finite element models of the skulls simulating biting while data from frog hind limbs will be applied to dynamic models of jumping.

DCT3-2  2:45 pm  The curious case of the vomeronasal organ in bats: genetics asks questions only anatomy can answer. Yohe LR*, Ecology & Evolution, Stony Brook University; Curtis AA, Mammalogy, American Museum of Natural History; Rosenthal H, Smithtown High School West; Hoffmann S, Anatomical Sciences, Stony Brook University; Martin KR, Ecology & Evolution, Stony Brook University; Davalos-Alvarez LM, Ecology & Evolution, Stony Brook University
Abstract: In mammals, pheromone communication signals are detected and processed in the vomeronasal system in the nose. Nearly every terrestrial mammal uses vomerolfaction, although variation is present among Old World primates and bats. The neurons of the vomeronasal organ (VNO) are packed with receptors that bind directly to pheromone compounds and relay the signal to the accessory olfactory bulb (AOB) for interpretation. The ion channel protein Trpc2 is indispensable for vomeronasal function, as it depolarizes the cell to transduce the signal to the brain. Histological evidence suggests that most bats lack a VNO or AOB, and the Trpc2 gene is pseudogenized in many lineages. New World leaf-nosed bats (Phyllostomidae) are one dramatic exception, showing well-developed vomeronasal system morphology and a functional Trpc2 gene. Recent genetic evidence, however, shows Trpc2 pseudogenization of some nectarivorous lineages. Do these species truly lack a vomeronasal system while all other phyllostomids retain it? We investigated if phyllostomid species with a nonfunctional Trpc2 also reflected this degraded vomeronasal system pattern at the morphological level. Using diffusible iodine-based contrast-enhanced computed tomography (diceCT), we characterized and measured vomeronasal structures in a diversity of phyllostomids. Many species with a disrupted Trpc2 gene show non-detectable vomeronasal organ structures. We emphasize that scanning parameters, such as peak X-ray tube voltage and amperage are critical to obtaining clear resolution of vomeronasal organ structures. This powerful method allowed us to bridge a connection between genes and morphology.

DCT3-3  3:00 pm  Comparative morphology of bat cranial muscles using contrast-enhanced micro-CT imaging. Vander Linden A*, University of Massachusetts Amherst; Santana S E , University of Washington
Abstract: By harnessing the power of micro-CT imaging, functional morphologists have been able to describe, quantify and compare many aspects of the diverse skeletal anatomy of bats, and to relate this diversity to functional, ecological, and lineage diversity. However, similar progress has not been achieved for soft tissues, and many aspects of the morphological and functional diversity of bats remain unknown. Here we describe, for the first time, how soft-tissue components of the cranial anatomy of bats can be fully visualized in adult individuals using diffusible iodine-based contrast-enhanced computed tomography (diceCT) protocols DiceCT methods have proven successful in the study of soft-tissue morphology in invertebrates, vertebrate embryos, archosaurs, rodents, lagomorphs and carnivorans. This research capitalizes on these previous studies and compares tissue contrast levels resulting from treatments of Lugol's iodine on intact bat heads. Using a taxonomically and ecologically diverse sample of bat species, we are able to visualize the precise anatomy of jaw adductor muscle groups that are difficult to access via traditional dissection. We further demonstrate the value of diceCT methods for generating realistic computer renditions of the three-dimensional anatomy of structures, including the brain and cranial musculature, allowing for accurate documentation, quantification and modeling of their function.

DCT3-4  3:15 pm  Advantages and difficulties of alcoholic iodine staining for correlative 2D and 3D microCT imaging and histomorphology in bat developmental studies. Herdina* AN*, Department of Theoretical Biology, University of Vienna, Vienna, Austria; Nugraha TP, Research Center for Biology, Indonesian Institute of Sciences, Cibinong, Indonesia; Semiadi G, Research Center for Biology, Indonesian Institute of Sciences, Cibinong, Indonesia; Gro?schmidt K, Bone and Biomaterials Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria; Haase A, Bone and Biomaterials Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria; Lina PHC, Department of Terrestrial Zoology, Naturalis Biodiversity Center, Leiden, Netherlands; Godlevska L, Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, Kiev, Ukraine; Vlaschenko A, Feldman Ecopark Bat Rehabilitation Center, Lisne, Ukraine; Metscher BD, Department of Theoretical Biology, University of Vienna, Vienna, Austria; *present address: , Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
Abstract: Iodine contrast staining has successfully been used in a number of studies on fetal and adult vertebrates, because soft tissues can thus be differentiated in microCT images. In this ongoing study, we use 1% (w/v) elemental iodine in 100% ethanol (I2E) to study the development of reproductive organs in a sample of 14 Cynopterus brachyotis (Chiroptera: Pteropodidae) from the collection of the Museum Zoologicum Bogoriense (Indonesia) and compare it to the postnatal development of three other bat species. Bats occupy a large variety of different ecological niches and have evolved diverse morphological specializations. The prenatal development of different bat organ systems has mainly been studied using histological methods, dissection, or clearing and staining. The skeletal system has also been studied with x-ray techniques including microCT (X-ray microtomography). In a previous study on the postnatal development of the baculum (os penis) in the bat species Pipistrellus pipistrellus we found that the distal part of the baculum reaches its adult shape before the proximal part. The different states of medullary cavity development we found suggest the medullary cavity first forms from the ventral side of the baculum, where the branches of the base meet the shaft and that it is later replaced by a secondary medullary cavity. In the long term, we will compare the pre- and postnatal development of the reproductive organs of different bat species, to test if different baculum shapes develop similarly or if they start to calcify at different ossification centers. Correlating 3D microCT imaging with serial, surface-stained, undecalcified ground sections of the material enables us to get a precise histomorphological evaluation of a larger number of samples and even of other species.

DCT3-5  3:30 pm  DiceCT and the staining of old museum specimens, exemplified by the analysis of venom glands in viperid snakes. Mahlow K.*, Museum für Naturkunde Berlin; Mueller J., Museum für Naturkunde Berlin
Abstract: Thanks to advances in computed tomography and histological staining methods, the comparative study of soft-tissue anatomy across wider taxonomic scales is becoming increasingly popular, and by now also involves non-freshly sampled specimens from natural history collections. However, staining is an invasive approach and in many cases it is not allowed to alter valuable collection specimens, either in structure or in color. In addition, the specimens have often been preserved using diverse fixations, some of which prevent the tissues from being successfully stained. Here we report on our experiences with the most common fixations like formalin and ethanol, as well as with specialized cases such as gasoline, and compare their effect on the staining of soft tissues by considering also body mass and the individual and historical age of the specimens. Furthermore, we present a case study on the comparative morphology of venom glands and associated muscles in viperid snakes, which represent a great challenge for staining due to the often old and large-sized material characterized by high muscle density, strong fixation with formalin and/or gasoline, and the additional storage in ethanol. Careful development of protocols and comparative tests of different approaches has now made it possible to even visualize the thin fibers of the pit organ of crotalid vipers. Also, we were able to calculate approximate staining periods based on the estimated fixation and storage times, the body mass of the specimen, and the fixation chemicals. To destain the material we discovered it is best to use watery solutions for staining, which results in a slow dissolving of the stain after return into storage ethanol, as more traditional destaining solutions such as sodium-thiosulfate are not appropriate for most museums specimens because of their chemical reactions with the inorganic components of the bone tissue.

DCT3-6  3:45 pm  Diffusible iodine-based contrast enhancement of large, post-embryonic, intact vertebrates for CT scanning: staining, destaining, and long-term storage . Morhardt AC*, Department of Biological Sciences, Ohio University; Ridgely RC, Department of Biomedical Sciences, Ohio University; Witmer LM, Department of Biomedical Sciences, Ohio University
Abstract: Diffusible, iodine-based contrast enhancement of computed tomography (diceCT) is a successful technique for studying in situ soft tissues in fixed specimens, but studies have identified soft-tissue shrinkage as an artifact of staining with physiologically hypertonic solutions of Lugol's iodine (aqueous iodine potassium iodide; I2KI). Shrinkage introduces error into analyses of quantitative data collected from stained tissues. Our study seeks to clarify the effects of physiologically hypertonic I2KI on large, post-embryonic, intact vertebrate specimens. In our study, results from post-mortem, fixed lab mouse (Mus musculus) specimens show a significant difference in mass loss between specimens stained with hypertonic I2KI and those pretreated with 20% sucrose and stained with isotonic I2KI. Additionally, an age-matched set of seven Virginia opossum (Didelphis virginiana) pouch young was used to test the effects of different physiological pretreatment solutions and concentrations of I2KI. Of protocols tested, results indicate that, when combined, PBS pre-treatment and hypertonic (15%) I2KI staining resulted in 41.6% mass loss and equivalent volume loss. Pretreating with a 20% sucrose solution and staining with physiologically-isotonic (1.25%) I2KI greatly reduced mass and volume losses. Results are supported by data from a wide range of vertebrate taxa (i.e., African-clawed frogs, alligators, several mammals and birds). Therefore, to mitigate the shrinkage effects of hypertonic I2KI, we recommend pretreatment with sucrose and staining with isotonic I2KI, although this protocol comes at the cost of added time for staining and the need to refresh the I2KI solution. Finally, destaining using sodium thiosulfate allows stained specimens to regain pre-stain coloration and remain stable for future re-staining and/or gross dissection. Together, these practices limit shrinkage and make diceCT more of a reversible, non-destructive technique.

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