Pulsative venous return from the branchial vessels to the heart of the bivalve Mytilus galloprovincialis supports the constant-volume mechanism.

In bivalves and gastropods, ventricle contraction causes a negative pressure in the auricles and increases venous return from the afferent oblique vein (AOV): the constant-volume (CV) mechanism. The flow in the AOV should be a pulsative flow synchronized with the ventricular contraction. The flow in the heart and adjacent vessels of Mytilus galloprovincialis were measured by magnetic resonance imaging to confirm this hypothesis. Under a regular heartbeat, pulsative flows in the AOV and branchial vessels (BVs) were almost completely synchronized with the flow in the aorta, while filling of the ventricle was in the opposite phase. Flows in the BVs were directed to the posterior direction, and a pair of BVs in the gill axes (the efferent BVs) were connected to the AOV. Based on the images of the whole pathway of the AOV in an oblique slice, we confirmed that haemolymph flow was evoked from the efferent BVs and flow into the ventricle via the auricle was completed in a single heartbeat. Therefore, the walls of the AOV and BVs could resist negative transmural pressure caused by the ventricular contraction. In conclusion, the auricle, the AOV and the BVs, including the gill filaments, act as a suction pump. The pulsative venous return is driven by the negative pressure of the AOV as in the CV mechanism, and the negative pressure in the efferent BVs could draw haemolymph from the sinus via the gill and the afferent BVs. Therefore, Mytilus can start and stop its heartbeat as necessary.

The Special Developmental Biology of Craniofacial Tissues Enables the Understanding of Oral and Maxillofacial Physiology and Diseases.

Maxillofacial hard tissues have several differences compared to bones of other localizations of the human body. These could be due to the different embryological development of the jaw bones compared to the extracranial skeleton. In particular, the immigration of neuroectodermally differentiated cells of the cranial neural crest (CNC) plays an important role. These cells differ from the mesenchymal structures of the extracranial skeleton. In the ontogenesis of the jaw bones, the development via the intermediate stage of the pharyngeal arches is another special developmental feature. The aim of this review was to illustrate how the development of maxillofacial hard tissues occurs via the cranial neural crest and pharyngeal arches, and what significance this could have for relevant pathologies in maxillofacial surgery, dentistry and orthodontic therapy. The pathogenesis of various growth anomalies and certain syndromes will also be discussed.

An unexpected cyst of the ear lobule.

Branchial cleft anomalies (BCA) are among the most common congenital anomalies found in the pediatric head and neck. The embryology of these congenital anomalies is well understood, which allows clinicians to anticipate their diagnosis when a pediatric patient presents with a head or neck mass. The predictable anatomy of the various types of BCA allows for improved surgical planning to prevent recurrence and ensure complete resection. This report details an unusual location of a first BCA located in the ear lobule of a 10-month old male. There has been no documented first BCA at the ear lobule in the literature.

Paraneuronal pseudobranchial neurosecretory system in tank goby Glossogobius giuris with special reference to novel neurohaemal contact complex.

Various putative oxygen chemosensory cells are reported to be present throughout the vertebrate body performing pivotal roles in respiration by initiating responses during acute hypoxia. Since air-breathing fishes often are exposed to the oxygen-deficient milieu, in such conditions various chemosensory cells operate in an orchestrated fashion. The Pseudobranchial neurosecretory system (PSNS) a newly discovered system, is one of these. It has been placed in the category of "Diffuse NE systems (DNES)". It is found in all the catfish species and in some other non-catfish group of teleosts. In catfishes, it is present in close association with the carotid labyrinth- a chemosensory structure, known in fish and amphibians. The presence of this system in Glossogobius giuris, in association with the pseudobranch, a structure considered to be precursor of carotid labyrinth, is a significant finding. In an attempt to study the structure and organization of the pseudobranchial neurosecretory system in a non-catfish species of teleost, the present investigation was undertaken on a goby G. giuris. The histological observations, using a neurosecretion-specific stain, revealed the presence of this system in G. giuris. The findings are discussed in the light of the association of PSNS with pseudobranch and the type of "neurohaemal contact complex" formed between this neurosecretory system and the elements of the circulatory system.

Retinoic Acid Organizes the Zebrafish Vagus Motor Topographic Map via Spatiotemporal Coordination of Hgf/Met Signaling.

Information flow through neural circuits often requires their organization into topographic maps in which the positions of cell bodies and synaptic targets correspond. To understand how topographic map development is controlled, we examine the mechanism underlying targeting of vagus motor axons to the pharyngeal arches in zebrafish. We reveal that retinoic acid organizes topography by specifying anterior-posterior identity in vagus motor neurons. We then show that chemoattractant signaling between Hgf and Met is required for vagus innervation of the pharyngeal arches. Finally, we find that retinoic acid controls the spatiotemporal dynamics of Hgf/Met signaling to coordinate axon targeting with the developmental progression of the pharyngeal arches and show that experimentally altering the timing of Hgf/Met signaling is sufficient to redirect axon targeting and disrupt the topographic map. These findings establish a mechanism of topographic map development in which the regulation of chemoattractant signaling in space and time guides axon targeting.

Skeletal kinematics of the hyoid arch in the suction-feeding shark Chiloscyllium plagiosum.

White-spotted bamboo sharks, Chiloscyllium plagiosum, generate strong suction-feeding pressures that rival the highest levels measured in ray-finned fishes. However, the hyostylic jaw suspension of these sharks is fundamentally different from the actinopterygian mechanism, including more mobile hyomandibulae, with the jaws and ceratohyal suspended from the hyomandibulae. Prior studies have proposed skeletal kinematics during feeding in orectolobid sharks from indirect measurements. Here, we tested these hypotheses using XROMM to measure cartilage motions directly. In agreement with prior hypotheses, we found extremely large retraction and depression of the ceratohyal, facilitated by large protraction and depression of the hyomandibula. Somewhat unexpectedly, XROMM also showed tremendous long-axis rotation (LAR) of both the ceratohyal and hyomandibula. This LAR likely increases the range of motion for the hyoid arch by keeping the elements properly articulated through their large arcs of motion. XROMM also confirmed that upper jaw protraction occurs before peak gape, similarly to actinopterygian suction feeders, but different from most other sharks in which jaw protrusion serves primarily to close the mouth. Early jaw protraction results from decoupling the rotations of the hyomandibula, with much of protraction occurring before peak gape with the other rotations lagging behind. In addition, the magnitudes of retraction and protraction of the hyoid elements are independent of the magnitude of depression, varying the shape of the mouth among feeding strikes. Hence, the large variation in suction-feeding behavior and performance may contribute to the wide dietary breadth of bamboo sharks.

Progenitor targeting by adult stem cells in Ciona homeostasis, injury, and regeneration.

In the ascidian Ciona intestinalis, oral siphon amputation activates adult stem cell niches in the branchial sac to divide and dispatch migratory progenitor cells to a regeneration blastema at the site of injury. This study shows that progenitor cells derived from branchial sac stem cell niches have roles in homeostasis, wound repair, and regeneration of the siphons and neural complex (NC). During homeostasis, progenitor cells targeted the pharyngeal stigmata to replace ciliated cells involved in filter feeding. After individual or double siphon amputations, progenitor cells specifically targeted the oral or atrial siphons or both siphons, and were involved in the replacement of siphon circular muscle fibers. After oral siphon wounding, progenitor cells targeted the wound sites, and in some cases a supernumerary siphon was formed, although progenitor cell targeting did not predict the induction of supernumerary siphons. Following NC ablation, progenitor cells specifically targeted the regenerating NC, and supplied the precursors of new brain and neural gland cells. The tissues and organs targeted by branchial sac stem cells exhibited apoptosis during homeostasis and injury. It is concluded that branchial sac progenitor cells are multipotent and show targeting specificity that is correlated with apoptosis during homeostatic growth, tissue repair, and regeneration.

Cohort-based multiscale analysis of hemodynamic-driven growth and remodeling of the embryonic pharyngeal arch arteries.

Growth and remodeling of the primitive pharyngeal arch artery (PAA) network into the extracardiac great vessels is poorly understood but a major source of clinically serious malformations. Undisrupted blood flow is required for normal PAA development, yet specific relationships between hemodynamics and remodeling remain largely unknown. Meeting this challenge is hindered by the common reductionist analysis of morphology to single idealized models, where in fact structural morphology varies substantially. Quantitative technical tools that allow tracking of morphological and hemodynamic changes in a population-based setting are essential to advancing our understanding of morphogenesis. Here, we have developed a methodological pipeline from high-resolution nano-computed tomography imaging and live-imaging flow measurements to multiscale pulsatile computational models. We combine experimental-based computational models of multiple PAAs to quantify hemodynamic forces in the rapidly morphing Hamburger Hamilton (HH) stage HH18, HH24 and HH26 embryos. We identify local morphological variation along the PAAs and their association with specific hemodynamic changes. Population-level mechano-morphogenic variability analysis is a powerful strategy for identifying stage-specific regions of well and poorly tolerated morphological and/or hemodynamic variation that may protect or initiate cardiovascular malformations.

Physical modeling of vortical cross-step flow in the American paddlefish, Polyodon spathula.

Vortical cross-step filtration in suspension-feeding fish has been reported recently as a novel mechanism, distinct from other biological and industrial filtration processes. Although crossflow passing over backward-facing steps generates vortices that can suspend, concentrate, and transport particles, the morphological factors affecting this vortical flow have not been identified previously. In our 3D-printed models of the oral cavity for ram suspension-feeding fish, the angle of the backward-facing step with respect to the model's dorsal midline affected vortex parameters significantly, including rotational, tangential, and axial speed. These vortices were comparable to those quantified downstream of the backward-facing steps that were formed by the branchial arches of preserved American paddlefish in a recirculating flow tank. Our data indicate that vortices in cross-step filtration have the characteristics of forced vortices, as the flow of water inside the oral cavity provides the external torque required to sustain forced vortices. Additionally, we quantified a new variable for ram suspension feeding termed the fluid exit ratio. This is defined as the ratio of the total open pore area for water leaving the oral cavity via spaces between branchial arches that are not blocked by gill rakers, divided by the total area for water entering through the gape during ram suspension feeding. Our experiments demonstrated that the fluid exit ratio in preserved paddlefish was a significant predictor of the flow speeds that were quantified anterior of the rostrum, at the gape, directly dorsal of the first ceratobranchial, and in the forced vortex generated by the first ceratobranchial. Physical modeling of vortical cross-step filtration offers future opportunities to explore the complex interactions between structural features of the oral cavity, vortex parameters, motile particle behavior, and particle morphology that determine the suspension, concentration, and transport of particles within the oral cavity of ram suspension-feeding fish.

The interbranchial lymphoid tissue likely contributes to immune tolerance and defense in the gills of Atlantic salmon.

Central and peripheral immune tolerance is together with defense mechanisms a hallmark of all lymphoid tissues. In fish, such tolerance is especially important in the gills, where the intimate contact between gill tissue and the aqueous environment would otherwise lead to continual immune stimulation by innocuous antigens. In this paper, we focus on the expression of genes associated with immune regulation by the interbranchial lymphoid tissue (ILT) in an attempt to understand its role in maintaining immune homeostasis. Both healthy and virus-challenged fish were investigated, and transcript levels were examined from laser-dissected ILT, gills, head kidney and intestine. Lack of Aire expression in the ILT excluded its involvement in central tolerance and any possibility of its being an analogue to the thymus. On the other hand, the ILT appears to participate in peripheral immune tolerance due to its relatively high expression of forkhead box protein 3 (Foxp3) and other genes associated with regulatory T cells (Tregs) and immune suppression.

Asymmetry in dentition and shape of pharyngeal arches in the clonal fish Chrosomus eos-neogaeus: Phenotypic plasticity and developmental instability.

The effect of the environment may result in different developmental outcomes. Extrinsic signals can modify developmental pathways and result in alternative phenotypes (phenotypic plasticity). The environment can also be interpreted as a stressor and increase developmental instability (developmental noise). Directional and fluctuating asymmetry provide a conceptual background to discriminate between these results. This study aims at assessing whether variation in dentition and shape of pharyngeal arches of the clonal fish Chrosomus eos-neogaeus results from developmental instability or environmentally induced changes. A total of 262 specimens of the Chrosomus eos-neogaeus complex from 12 natural sites were analysed. X-ray microcomputed tomography (X-ray micro-CT) was used to visualize the pharyngeal arches in situ with high resolution. Variation in the number of pharyngeal teeth is high in hybrids in contrast to the relative stability observed in both parental species. The basal dental formula is symmetric while the most frequent alternative dental formula is asymmetric. Within one lineage, large variation in the proportion of individuals bearing basal or alternative dental formulae was observed among sites in the absence of genetic difference. Both dentition and arch shape of this hybrid lineage were explained significantly by environmental differences. Only individuals bearing asymmetric dental formula displayed fluctuating asymmetry as well as directional left-right asymmetry for the arches. The hybrids appeared sensitive to environmental signals and intraspecific variation on pharyngeal teeth was not random but reflects phenotypic plasticity. Altogether, these results support the influence of the environment as a trigger for an alternative developmental pathway resulting in left-right asymmetry in dentition and shape of pharyngeal arches.

Fish mouths as engineering structures for vortical cross-step filtration.

Suspension-feeding fishes such as goldfish and whale sharks retain prey without clogging their oral filters, whereas clogging is a major expense in industrial crossflow filtration of beer, dairy foods and biotechnology products. Fishes' abilities to retain particles that are smaller than the pore size of the gill-raker filter, including extraction of particles despite large holes in the filter, also remain unexplained. Here we show that unexplored combinations of engineering structures (backward-facing steps forming d-type ribs on the porous surface of a cone) cause fluid dynamic phenomena distinct from current biological and industrial filter operations. This vortical cross-step filtration model prevents clogging and explains the transport of tiny concentrated particles to the oesophagus using a hydrodynamic tongue. Mass transfer caused by vortices along d-type ribs in crossflow is applicable to filter-feeding duck beak lamellae and whale baleen plates, as well as the fluid mechanics of ventilation at fish gill filaments.

The NDST gene family in zebrafish: role of NDST1B in pharyngeal arch formation.

Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout.

Rh protein expression in branchial neuroepithelial cells, and the role of ammonia in ventilatory control in fish.

Bill Milsom has made seminal contributions to our understanding of ventilatory control in a wide range of vertebrates. Teleosts are particularly interesting, because they produce a 3rd, potentially toxic respiratory gas (ammonia) in large amounts. Fish are well known to hyperventilate under high environmental ammonia (HEA), but only recently has the potential role of ammonia in normal ventilatory control been investigated. It is now clear that ammonia can act directly as a ventilatory stimulant in trout, independent of its effects on acid-base balance. Even in ureotelic dogfish sharks, acute elevations in ammonia cause increases in ventilation. Peripherally, the detection of elevated ammonia resides in gill arches I and II in trout, and in vitro, neuroepithelial cells (NECs) from these arches are sensitive to ammonia, responding with elevations in intracellular Ca(2+) ([Ca(2+)]i). Centrally, hyperventilatory responses to ammonia correlate more closely with concentrations of ammonia in the brain than in plasma or CSF. After chronic HEA exposure, ventilatory responsiveness to ammonia is lost, associated with both an attenuation of the [Ca(2+)]i response in NECs, and the absence of elevation in brain ammonia concentration. Chronic exposure to HEA also causes increases in the mRNA expression of several Rh proteins (ammonia-conductive channels) in both brain and gills. "Single cell" PCR techniques have been used to isolate the individual responses of NECs versus other gill cell types. We suggest several circumstances (post-feeding, post-exercise) where the role of ammonia as a ventilatory stimulant may have adaptive benefits for O2 uptake in fish.

Growth and hemodynamics after early embryonic aortic arch occlusion.

The majority of severe clinically significant forms of congenital heart disease (CHD) are associated with great artery lesions, including hypoplastic, double, right or interrupted aortic arch morphologies. While fetal and neonatal interventions are advancing, their potential ability to restore cardiac function, optimal timing, location, and intensity required for intervention remain largely unknown. Here, we combine computational fluid dynamics (CFD) simulations with in vivo experiments to test how individual pharyngeal arch artery hemodynamics alter as a result of local interventions obstructing individual arch artery flow. Simulated isolated occlusions within each pharyngeal arch artery were created with image-derived three-dimensional (3D) reconstructions of normal chick pharyngeal arch anatomy at Hamburger-Hamilton (HH) developmental stages HH18 and HH24. Acute flow redistributions were then computed using in vivo measured subject-specific aortic sinus inflow velocity profiles. A kinematic vascular growth-rendering algorithm was then developed and implemented to test the role of changing local wall shear stress patterns in downstream 3D morphogenesis of arch arteries. CFD simulations predicted that altered pressure gradients and flow redistributions were most sensitive to occlusion of the IVth arches. To evaluate these simulations experimentally, a novel in vivo experimental model of pharyngeal arch occlusion was developed and implemented using two-photon microscopy-guided femtosecond laser-based photodisruption surgery. The right IVth arch was occluded at HH18, and resulting diameter changes were followed for up to 24 h. Pharyngeal arch diameter responses to acute hemodynamic changes were predicted qualitatively but poorly quantitatively. Chronic growth and adaptation to hemodynamic changes, however, were predicted in a subset of arches. Our findings suggest that this complex biodynamic process is governed through more complex forms of mechanobiological vascular growth rules. Other factors in addition to wall shear stress or more complex WSS rules are likely important in the long-term arterial growth and patterning. Combination in silico/experimental platforms are essential for accelerating our understanding and prediction of consequences from embryonic/fetal cardiovascular occlusions and lay the foundation for noninvasive methods to guide CHD diagnosis and fetal intervention.

New insights into the role of the pharyngeal jaw apparatus in the sound-producing mechanism of Haemulon flavolineatum (Haemulidae).

Grunts are fish that are well known to vocalize, but how they produce their grunting sounds has not been clearly identified. In addition to characterizing acoustic signals and hearing in the French grunt Haemulon flavolineatum, the present study investigates the sound-production mechanism of this species by means of high-speed X-ray videos and scanning electron microscopy of the pharyngeal jaw apparatus. Vocalizations consist of a series of stridulatory sounds: grunts lasting ~47 ms with a mean period of 155 ms and a dominant frequency of ~700 Hz. Auditory capacity was determined to range from 100 to 600 Hz, with greatest sensitivity at 300 Hz (105.0±11.8 dB re. 1 µPa). This suggests that hearing is not tuned exclusively to detect the sounds of conspecifics. High-speed X-ray videos revealed how pharyngeal jaws move during sound production. Traces of erosion on teeth in the fourth ceratobranchial arch suggest that they are also involved in sound production. The similarity of motor patterns of the upper and lower pharyngeal jaws between food processing and sound production indicates that calling is an exaptation of the food-processing mechanism.

Hcfc1b, a zebrafish ortholog of HCFC1, regulates craniofacial development by modulating mmachc expression.

Mutations in HCFC1 (MIM300019), have been recently associated with cblX (MIM309541), an X-linked, recessive disorder characterized by multiple congenital anomalies including craniofacial abnormalities. HCFC1 is a transcriptional co-regulator that modulates the expression of numerous downstream target genes including MMACHC, but it is not clear how these HCFC1 targets play a role in the clinical manifestations of cblX. To begin to elucidate the mechanism by which HCFC1 modulates disease phenotypes, we have carried out loss of function analyses in the developing zebrafish. Of the two HCFC1 orthologs in zebrafish, hcfc1a and hcfc1b, the loss of hcfc1b specifically results in defects in craniofacial development. Subsequent analysis revealed that hcfc1b regulates cranial neural crest cell differentiation and proliferation within the posterior pharyngeal arches. Further, the hcfc1b-mediated craniofacial abnormalities were rescued by expression of human MMACHC, a downstream target of HCFC1 that is aberrantly expressed in cblX. Furthermore, we tested distinct human HCFC1 mutations for their role in craniofacial development and demonstrated variable effects on MMACHC expression in humans and craniofacial development in zebrafish. Notably, several individuals with mutations in either HCFC1 or MMACHC have been reported to have mild to moderate facial dysmorphia. Thus, our data demonstrates that HCFC1 plays a role in craniofacial development, which is in part mediated through the regulation of MMACHC expression.

Anatomical and physiological studies of bigheaded carps demonstrate that the epibranchial organ functions as a pharyngeal taste organ.

The epibranchial organ (EO) is an enigmatic tubular organ found in the pharyngeal cavity of many filter-feeding fishes. We investigated whether it might function as a taste organ that mediates aggregation and ingestion of planktonic food within the buccal cavity. The EO and associated structures of bighead and silver carps, two successful and invasive planktivorous fishes, were examined using histological and electrophysiological techniques. Both species possess finely structured gill rakers that extend directly via a series of protrusions into each of the four blind canals which are organized as the muscular EO, suggesting that the gill rakers and EO probably function in an integrated manner. Both the interior and exterior surfaces of the EOs of both species are covered with high densities of taste buds and solitary chemosensory cells (SCCs) as well as mucous cells. Conversely, taste buds are scarce in both the buccal cavities and external portions of the head and mouth of both species. Electrophysiological recordings from a caudal branch of the vagus nerve (cranial nerve X) found to innervate the EO showed it to be sensitive to chemicals found in a planktonic diet. l-Amino acids accounted for some, but not all of the neural activity. We conclude that taste buds and SCCs located on the EO and gill rakers probably serve to chemically detect food particles, which the EO then aggregates by mucus secretion before eventually expelling them onto the floor of the pharynx for ingestion. This specialized, pharyngeal chemosensory structure may explain the feeding success of these, and perhaps other planktivorous, filter-feeding fishes.

Effects of salinity on metabolic rate and branchial expression of genes involved in ion transport and metabolism in Mozambique tilapia (Oreochromis mossambicus).

This study investigated the effects of two rearing salinities, and acute salinity transfer, on the energetic costs of osmoregulation and the expression of metabolic and osmoregulatory genes in the gill of Mozambique tilapia. Using automated, intermittent-flow respirometry, measured standard metabolic rates (SMRs) of tilapia reared in seawater (SW, 130 mg O2 kg⁻¹ h⁻¹) were greater than those reared in fresh water (FW, 103 mg O2 kg⁻¹ h⁻¹), when normalized to a common mass of 0.05 kg and at 25±1°C. Transfer from FW to 75% SW increased SMR within 18h, to levels similar to SW-reared fish, while transfer from SW to FW decreased SMR to levels similar to FW-reared fish. Branchial gene expression of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), an indicator of SW-type mitochondria-rich (MR) cells, was positively correlated with SMR, while Na⁺-Cl⁻ cotransporter (NCC), an indicator of FW-type MR cells, was negatively correlated. Principal Components Analysis also revealed that branchial expression of cytochrome c oxidase subunit IV (COX-IV), glycogen phosphorylase (GP), and a putative mitochondrial biogenesis regulator in fish, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), were correlated with a higher SMR, plasma osmolality, and environmental salinity, while expression of glycogen synthase (GS), PGC-1ß, and nuclear respiratory factor 1 (NRF-1) had negative correlations. These results suggest that the energetic costs of osmoregulation are higher in SW than in FW, which may be related to the salinity-dependent differences in osmoregulatory mechanisms found in the gills of Mozambique tilapia.

Osmoregulation and branchial plasticity after acute freshwater transfer in red drum, Sciaenops ocellatus.

Red drum, Sciaenops ocellatus, is an estuarine-dependent fish species commonly found in the Gulf of Mexico and along the coast of the southeastern United States. This economically important species has demonstrated freshwater tolerance; however, the physiological mechanisms and costs related to freshwater exposure remain poorly understood. The current study therefore investigated the physiological response of red drum using an acute freshwater transfer protocol. Plasma osmolality, Cl⁻, Mg²âº and Ca²âº were all significantly reduced by 24h post-transfer; Cl⁻ and Mg²âº recovered to control levels by 7days post-transfer. No effect of transfer was observed on muscle water content; however, muscle Cl⁻ was significantly reduced. Interestingly, plasma and muscle Na⁺ content was unaffected by freshwater transfer. Intestinal fluid was absent by 24h post-transfer indicating cessation of drinking. Branchial gene expression analysis showed that both CFTR and NKCC1 exhibited significant down-regulation at 8 and 24h post-transfer, respectively, although transfer had no impact on NHE2, NHE3 or Na⁺, K⁺ ATPase (NKA) activity. These general findings are supported by immunohistochemical analysis, which revealed no apparent NKCC containing cells in the gills at 7days post transfer while NKA cells localization was unaffected. The results of the current study suggest that red drum can effectively regulate Na⁺ balance upon freshwater exposure using already present Na⁺ uptake pathways while also down-regulating ion excretion mechanisms.