The Development of the Chimaeroid Pelvic Skeleton and the Evolution of Chondrichthyan Pelvic Fins.

Pelvic girdles, fins and claspers are evolutionary novelties first recorded in jawed vertebrates. Over the course of the evolution of chondrichthyans (cartilaginous fish) two trends in the morphology of the pelvic skeleton have been suggested to have occurred. These evolutionary shifts involved both an enlargement of the metapterygium (basipterygium) and a transition of fin radial articulation from the pelvic girdle to the metapterygium. To determine how these changes in morphology have occurred it is essential to understand the development of extant taxa as this can indicate potential developmental mechanisms that may have been responsible for these changes. The study of the morphology of the appendicular skeleton across development in chondrichthyans is almost entirely restricted to the historical literature with little contemporary research. Here, we have examined the morphology and development of the pelvic skeleton of a holocephalan chondrichthyan, the elephant shark (Callorhinchus milii), through a combination of dissections, histology, and nanoCT imaging and redescribed the pelvic skeleton of Cladoselache kepleri (NHMUK PV P 9269), a stem holocephalan. To put our findings in their evolutionary context we compare them with the fossil record of chondrichthyans and the literature on pelvic development in elasmobranchs from the late 19th century. Our findings demonstrate that the pelvic skeleton of C. milii initially forms as a single mesenchymal condensation, consisting of the pelvic girdle and a series of fin rays, which fuse to form the basipterygium. The girdle and fin skeleton subsequently segment into distinct components whilst chondrifying. This confirms descriptions of the early pelvic development in Scyliorhinid sharks from the historical literature and suggests that chimaeras and elasmobranchs share common developmental patterns in their pelvic anatomy. Alterations in the location and degree of radial fusion during early development may be the mechanism responsible for changes in pelvic fin morphology over the course of the evolution of both elasmobranchs and holocephalans, which appears to be an example of parallel evolution.

Paratympanic sinuses in juvenile Alligator.

Crocodylia has an extensive epithelial pneumatic space in the middle ear, paratympanic sinus system. Although fossil and extant crocodylian paratympanic sinus systems have been studied recently using the computed tomography (CT) and three-dimensional (3D) reconstruction data, due to the soft tissue nature of the pneumatic system and presence of its surrounding soft tissue structures, some boundaries, and definitions of each extension remain ambiguous. We describe the comprehensive paratympanic sinus system in posthatched alligator using soft tissue enhanced CT data with 3D reconstructions. The data are compared to the available data to discuss the ontogenetic pattern in alligator. We introduce further divided entities of the pneumatic system based on their associated bony and soft tissue structures and epithelial membrane and clarify the pneumatic terminologies. We then re-visit the potential homology of the paratympanic sinus in Archosauria. Epithelial boundaries of the ventral portion of the pneumatic system from the histological data suggest that the dual origin of the basioccipital diverticulum derived from the tympanic sinus and basicranial diverticulum medially. The presence of the epithelial boundary and pneumatic changes in ontogeny suggests that the middle ear may function differently in developmental stages. Lastly, a morphogenetic tree is constructed to help future work of comparative developmental studies of the paratympanic sinus system between crocodiles and birds.

Nano-CT imaging of larvae in the ant Pheidole hyatti reveals coordinated growth of a rudimentary organ necessary for soldier development.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.

Artificial turf infill associated with systematic toxicity in an amniote vertebrate.

Artificial athletic turf containing crumb rubber (CR) from shredded tires is a growing environmental and public health concern. However, the associated health risk is unknown due to the lack of toxicity data for higher vertebrates. We evaluated the toxic effects of CR in a developing amniote vertebrate embryo. CR water leachate was administered to fertilized chicken eggs via different exposure routes, i.e., coating by dropping CR leachate on the eggshell; dipping the eggs into CR leachate; microinjecting CR leachate into the air cell or yolk. After 3 or 7 d of incubation, embryonic morphology, organ development, physiology, and molecular pathways were measured. The results showed that CR leachate injected into the yolk caused mild to severe developmental malformations, reduced growth, and specifically impaired the development of the brain and cardiovascular system, which were associated with gene dysregulation in aryl hydrocarbon receptor, stress-response, and thyroid hormone pathways. The observed systematic effects were probably due to a complex mixture of toxic chemicals leaching from CR, such as metals (e.g., Zn, Cr, Pb) and amines (e.g., benzothiazole). This study points to a need to closely examine the potential regulation of the use of CR on playgrounds and artificial fields.

Plastic Teabags Release Billions of Microparticles and Nanoparticles into Tea.

The increasing presence of micro- and nano-sized plastics in the environment and food chain is of growing concern. Although mindful consumers are promoting the reduction of single-use plastics, some manufacturers are creating new plastic packaging to replace traditional paper uses, such as plastic teabags. The objective of this study was to determine whether plastic teabags could release microplastics and/or nanoplastics during a typical steeping process. We show that steeping a single plastic teabag at brewing temperature (95 °C) releases approximately 11.6 billion microplastics and 3.1 billion nanoplastics into a single cup of the beverage. The composition of the released particles is matched to the original teabags (nylon and polyethylene terephthalate) using Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The levels of nylon and polyethylene terephthalate particles released from the teabag packaging are several orders of magnitude higher than plastic loads previously reported in other foods. An initial acute invertebrate toxicity assessment shows that exposure to only the particles released from the teabags caused dose-dependent behavioral and developmental effects.

Development of the paratympanic pneumatic system of Japanese quail.

Avian heads are characterized as having two extensive air-filled systems lined with epithelia; the paranasal and paratympanic sinuses. Many diverticula derived from the paratympanic sinus system are known to reticulate with each other to form a single merged pneumatic space within the adult braincase. However, the development of these complex branching and reticulating epithelia has not been examined in detail. In this study, we describe the comprehensive developmental pattern of the paratympanic sinus and its associated soft tissues in a model bird, Japanese quail (Coturnix japonica). The data are derived from three-dimensional reconstructions based on histological sections and soft tissue enhanced micro-CT data. Those data provide the foundation of the complex hierarchical developmental pattern of the paratympanic sinus system. Moreover, associations with other tissues help establish key morphologies that identify each pneumatic entity. This study clarifies the developmental relationships of the ventral portions of the paratympanic sinus system, the siphoneal diverticulum and marginal sinus, based on the ligaments associated with the Eustachian tube. In addition, detailed histological pneumatic morphologies reveal hitherto unknown epithelial diversity, which may be indicative of equally complex developmental processes. We use the pneumatization of the quadrate as an example to support a close relationship with vascular growth and pneumatic epithelia invasion into ossified bone. We confirm pneumatic diverticula never enter into cartilages, possibly due to the absence of vasculature in these tissues. Lastly, we use the concept of a morphogenetic tree as a tool to help present the complex developmental pattern of the paratympanic sinus system and apply it toward inferring pneumatic morphologies in a nonavian theropod braincase.

Multimodal virtual histology of rabbit vocal folds by nonlinear microscopy and nano computed tomography.

Human vocal folds (VFs) possess a unique anatomical structure and mechanical properties for human communication. However, VFs are prone to scarring as a consequence of overuse, injury, disease or surgery. Accumulation of scar tissue on VFs inhibits proper phonation and leads to partial or complete loss of voice, with significant consequences for the patient's quality of life. VF regeneration after scarring provides a significant challenge for tissue engineering therapies given the complexity of tissue microarchitecture. To establish an effective animal model for VF injury and scarring, new histological methods are required to visualize the wound repair process of the tissue in its three-dimensional native environment. In this work, we propose the use of a combination of nonlinear microscopy and nanotomography as contrast methods for virtual histology of rabbit VFs. We apply these methods to rabbit VF tissue to demonstrate their use as alternatives to conventional VF histology that may enable future clinical studies of this injury model.

Quantitative analysis of microscopic X-ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining.

Rapid three-dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I2 KI and alcoholic iodine stains) combined with microscopic X-ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I2 KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X-ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I2 KI protocol. We present comparative microscopic X-ray computed tomography analyses of ethanol I2 KI and I2 KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X-ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28 days staining for I2 KI and ethanol I2 KI, respectively. Ethanol I2 KI provided superior soft tissue contrasts by reducing overstaining of fluid-filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three-dimensional visualization.