Immunohistochemical and ultrastructural characterization of the inner ear epithelial cells of splitnose rockfish (Sebastes diploproa).

The inner ear of teleost fish regulates the ionic and acid-base chemistry and secretes protein matrix into the endolymph to facilitate otolith biomineralization, which is used to maintain vestibular and auditory functions. The otolith is biomineralized in a concentric ring pattern corresponding to seasonal growth, and this calcium carbonate (CaCO3) polycrystal has become a vital aging and life-history tool for fishery managers, ecologists, and conservation biologists. Moreover, biomineralization patterns are sensitive to environmental variability including climate change, thereby threatening the accuracy and relevance of otolith-reliant toolkits. However, the cellular biology of the inner ear is poorly characterized, which is a hurdle for a mechanistic understanding of the underlying processes. This study provides a systematic characterization of the cell types in the inner ear of splitnose rockfish (Sebastes diploproa). Scanning electron microscopy revealed the apical morphologies of six inner ear cell types. In addition, immunostaining and confocal microscopy characterized the expression and subcellular localization of the proteins Na+-K+-ATPase, carbonic anhydrase, V-type H+-ATPase, Na+-K+-2Cl--cotransporter, otolith matrix protein 1, and otolin-1 in six inner ear cell types bordering the endolymph. This fundamental cytological characterization of the rockfish inner ear epithelium illustrates the intricate physiological processes involved in otolith biomineralization and highlights how greater mechanistic understanding is necessary to predict their multistressor responses to future climate change.

A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill.

The gills of most teleost fishes lack plasma-accessible carbonic anhydrase (paCA) that could participate in CO2 excretion. We tested the prevailing hypothesis that paCA would interfere with red blood cell (RBC) intracellular pH regulation by ß-adrenergic sodium-proton exchangers (ß-NHE) that protect pH-sensitive haemoglobin-oxygen (Hb-O2) binding during an acidosis. In an open system that mimics the gills, ß-NHE activity increased Hb-O2 saturation during a respiratory acidosis in the presence or absence of paCA, whereas the effect was abolished by NHE inhibition. However, in a closed system that mimics the tissue capillaries, paCA disrupted the protective effects of ß-NHE activity on Hb-O2 binding. The gills are an open system, where CO2 generated by paCA can diffuse out and is not available to acidifying the RBCs. Therefore, branchial paCA in teleosts may not interfere with RBC pH regulation by ß-NHEs, and other explanations for the evolutionary loss of the enzyme must be considered.

Evolving views of ionic, osmotic and acid-base regulation in aquatic animals.

The regulation of ionic, osmotic and acid-base (IOAB) conditions in biological fluids is among the most fundamental functions in all organisms; being surrounded by water uniquely shapes the IOAB regulatory strategies of water-breathing animals. Throughout its centennial history, Journal of Experimental Biology has established itself as a premier venue for publication of comparative, environmental and evolutionary studies on IOAB regulation. This Review provides a synopsis of IOAB regulation in aquatic animals, some of the most significant research milestones in the field, and evolving views about the underlying cellular mechanisms and their evolutionary implications. It also identifies promising areas for future research and proposes ideas for enhancing the impact of aquatic IOAB research.

The V-type ATPase enhances photosynthesis in marine phytoplankton and further links phagocytosis to symbiogenesis.

Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean.1 These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis.2 However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood. Since intracellular digestive vacuoles are ubiquitously acidified by V-type H+-ATPase (VHA),3 proton pumps were proposed to acidify the microenvironment around secondary chloroplasts to promote the dehydration of dissolved inorganic carbon (DIC) into CO2, thus enhancing photosynthesis.4,5 We report that VHA is localized around the chloroplasts of centric diatoms and that VHA significantly contributes to their photosynthesis across a wide range of oceanic irradiances. Similar results in a pennate diatom, dinoflagellate, and coccolithophore, but not green or red microalgae, imply the co-option of phagocytic VHA activity into a carbon-concentrating mechanism (CCM) is common to secondary endosymbiotic phytoplankton. Furthermore, analogous mechanisms in extant photosymbiotic marine invertebrates6,7,8 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Based on the contribution of diatoms to ocean biogeochemical cycles, VHA-mediated enhancement of photosynthesis contributes at least 3.5 Gtons of fixed carbon per year (or 7% of primary production in the ocean), providing an example of a symbiosis-derived evolutionary innovation with global environmental implications.

Distinct genomic routes underlie transitions to specialised symbiotic lifestyles in deep-sea annelid worms.

Bacterial symbioses allow annelids to colonise extreme ecological niches, such as hydrothermal vents and whale falls. Yet, the genetic principles sustaining these symbioses remain unclear. Here, we show that different genomic adaptations underpin the symbioses of phylogenetically related annelids with distinct nutritional strategies. Genome compaction and extensive gene losses distinguish the heterotrophic symbiosis of the bone-eating worm Osedax frankpressi from the chemoautotrophic symbiosis of deep-sea Vestimentifera. Osedax's endosymbionts complement many of the host's metabolic deficiencies, including the loss of pathways to recycle nitrogen and synthesise some amino acids. Osedax's endosymbionts possess the glyoxylate cycle, which could allow more efficient catabolism of bone-derived nutrients and the production of carbohydrates from fatty acids. Unlike in most Vestimentifera, innate immunity genes are reduced in O. frankpressi, which, however, has an expansion of matrix metalloproteases to digest collagen. Our study supports that distinct nutritional interactions influence host genome evolution differently in highly specialised symbioses.

Effects of ocean acidification on dopamine-mediated behavioral responses of a coral reef damselfish.

We investigated whether CO2-induced ocean acidification (OA) affects dopamine receptor-dependent behavior in bicolor damselfish (Stegastes partitus). Damselfish were kept in aquaria receiving flow through control (pH ~ 8.03; pCO2 ~ 384 µatm) or OA (pH ~ 7.64; CO2 ~ 1100 µatm) seawater at a rate of 1 L min-1. Despite this relatively fast flow rate, fish respiration further acidified the seawater in both control (pH ~7.88; pCO2 ~ 595 µatm) and OA (pH ~7.55; pCO2 ~ 1450 µatm) fish-holding aquaria. After five days of exposure, damselfish locomotion, boldness, anxiety, and aggression were assessed using a battery of behavioral tests using automated video analysis. Two days later, these tests were repeated following application of the dopamine D1 receptor agonist SKF 38393. OA-exposure induced ceiling anxiety levels that were significantly higher than in control damselfish, and SKF 38393 increased anxiety in control damselfish to a level not significantly different than that of OA-exposed damselfish. Additionally, SKF 38393 decreased locomotion and increased boldness in control damselfish but had no effect in OA-exposed damselfish, suggesting an alteration in activity of dopaminergic pathways that regulate behavior under OA conditions. These results indicate that changes in dopamine D1 receptor function affects fish behavior during exposure to OA. However, subsequent measurements of seawater sampled using syringes during the daytime (~3-4 pm local time) from crevasses in coral reef colonies, which are used as shelter by damselfish, revealed an average pH of 7.73 ± 0.03 and pCO2 of 925.8 ± 62.2 µatm; levels which are comparable to Representative Concentration Pathway (RCP) 8.5 predicted end-of-century mean OA levels in the open ocean. Further studies considering the immediate environmental conditions experienced by fish as well as individual variability and effect size are required to understand potential implications of the observed OA-induced behavioral effects on damselfish fitness in the wild.

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal.

Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here, we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pHi) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single-cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatiotemporal expression of sAC during skeletogenesis. Live pHi imaging of PMCs revealed that the downregulation of sAC activity with two structurally unrelated small molecules inhibited pHi regulation of PMCs, an effect that was rescued by the addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth and spicule regeneration, establishing a link between PMC pHi regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton remineralization and exposure to CO2-induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote remineralization and to compensate for acidic stress. This work highlights the central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal.

Optimizing immunostaining of archival fish samples to enhance museum collection potential.

Immunohistochemistry (IHC) is a powerful biochemical technique that uses antibodies to specifically label and visualize proteins of interests within biological samples. However, fluid-preserved specimens within natural history collection often use fixatives and protocols that induce high background signal (autofluorescence), which hampers IHC as it produces low signal-to-noise ratio. Here, we explored techniques to reduce autofluorescence using sodium borohydride (SBH), citrate buffer, and their combination on fish tissue preserved with paraformaldehyde, formaldehyde, ethanol, and glutaraldehyde. We found SBH was the most effective quenching technique, and applied this pretreatment to the gill or skin of 10 different archival fishes - including specimens that had been preserved in formaldehyde or ethanol for up to 65 and 37 years, respectively. The enzyme Na+/K+-ATPase (NKA) was successfully immunostained and imaged using confocal fluorescence microscopy, allowing for the identification and characterization of NKA-rich ionocytes essential for fish ionic and acid-base homeostasis. Altogether, our SBH-based method facilitates the use of IHC on archival samples, and unlocks the historical record on fish biological responses to environmental factors (such as climate change) using specimens from natural history collections that were preserved decades to centuries ago.

The silent loss of cell physiology hampers marine biosciences.

An ongoing loss of experts in marine cellular biochemistry and physiology (CBP) is stagnating the generation of knowledge upon which rapidly growing "omics" approaches rely, ultimately hampering our ability to predict organismal responses to climate change.

Dining on the dead in the deep: Active NH4 + excretion via Na+ /H+ (NH4 + ) exchange in the highly ammonia tolerant Pacific hagfish, Eptatretus stoutii.

AIM: Pacific hagfish are exceptionally tolerant to high environmental ammonia (HEA). Here, we elucidated a cellular mechanism that enables hagfish to actively excrete ammonia against steep ammonia gradients expected to be found inside a decomposing whale carcass. METHODS: Hagfish were exposed to varying concentrations of HEA in the presence or absence of environmental Na+ , while plasma ammonia levels were tracked. 14 C-methylammonium was used as a proxy for NH4 + to measure efflux in whole animals and in isolated gill pouches; the latter allowed us to assess the effects of amiloride specifically on Na+ /H+ exchangers (NHEs) in gill cells. Western blotting and immunohistochemistry were utilized to evaluate the abundance and sub-cellular localization of Rhesus glycoprotein (Rh) channels in the response to HEA. RESULTS: Hagfish actively excreted NH4 + against steep inwardly directed ENH4 + (ΔENH4 + ~ 35 mV) and pNH3 (ΔpNH3 ~ 2000 µtorr) gradients. Active NH4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na+ , indicating a Na+ /NH4 + exchange mechanism. Active NH4 + excretion across isolated gill pouches was amiloride-sensitive. Exposure to HEA resulted in decreased abundance of Rh channels in the apical membrane of gill ionocytes. CONCLUSIONS: During HEA exposure, hagfish can actively excrete ammonia against a steep concentration gradient using apical NHEs energized by Na+ -K+ -ATPase in gill ionocytes. Additionally, apical Rh channels are removed from the apical membrane, presumably to reduce ammonia loading from the environment. We suggest that this mechanism allows hagfish to maintain tolerable ammonia levels while feeding inside decomposing carrion, allowing them to exploit nutrient-rich food-falls.

A Rhesus channel in the coral symbiosome membrane suggests a novel mechanism to regulate NH3 and CO2 delivery to algal symbionts.

Reef-building corals maintain an intracellular photosymbiotic association with dinoflagellate algae. As the algae are hosted inside the symbiosome, all metabolic exchanges must take place across the symbiosome membrane. Using functional studies in Xenopus oocytes, immunolocalization, and confocal Airyscan microscopy, we established that Acropora yongei Rh (ayRhp1) facilitates transmembrane NH3 and CO2 diffusion and that it is present in the symbiosome membrane. Furthermore, ayRhp1 abundance in the symbiosome membrane was highest around midday and lowest around midnight. We conclude that ayRhp1 mediates a symbiosomal NH4+-trapping mechanism that promotes nitrogen delivery to algae during the day-necessary to sustain photosynthesis-and restricts nitrogen delivery at night-to keep algae under nitrogen limitation. The role of ayRhp1-facilitated CO2 diffusion is less clear, but it may have implications for metabolic dysregulation between symbiotic partners and bleaching. This previously unknown mechanism expands our understanding of symbioses at the immediate animal-microbe interface, the symbiosome.

V-type H+-ATPase in the symbiosome membrane is a conserved mechanism for host control of photosynthesis in anthozoan photosymbioses.

In reef-building corals (order Scleractinia) and giant clams (phylum Molluca), V-type H+-ATPase (VHA) in host cells is part of a carbon concentrating mechanism (CCM) that regulates photosynthetic rates of their symbiotic algae. Here, we show that VHA plays a similar role in the sea anemone Anemonia majano, a member of the order Actinaria and sister group to the Scleractinia, which in contrast to their colonial calcifying coral relatives is a solitary, soft-bodied taxa. Western blotting and immunofluorescence revealed that VHA was abundantly present in the host-derived symbiosome membrane surrounding the photosymbionts. Pharmacological inhibition of VHA activity in individual anemones resulted in an approximately 80% decrease of photosynthetic O2 production. These results extend the presence of a host-controlled VHA-dependent CCM to non-calcifying cnidarians of the order Actiniaria, suggesting it is widespread among photosymbiosis between aquatic invertebrates and Symbiodiniaceae algae.

Elucidating the acid-base mechanisms underlying otolith overgrowth in fish exposed to ocean acidification.

Over a decade ago, ocean acidification (OA) exposure was reported to induce otolith overgrowth in teleost fish. This phenomenon was subsequently confirmed in multiple species; however, the underlying physiological causes remain unknown. Here, we report that splitnose rockfish (Sebastes diploproa) exposed to ~1600 µatm pCO2(pH ~7.5) were able to fully regulated the pH of both blood and endolymph (the fluid that surrounds the otolith within the inner ear). However, while blood was regulated around pH 7.80, the endolymph was regulated around pH ~8.30. These different pH setpoints result in increased pCO2diffusion into the endolymph, which in turn leads to proportional increases in endolymph [HCO3-] and [CO32-]. Endolymph pH regulation despite the increased pCO2suggests enhanced H+removal. However, a lack of differences in inner ear bulk and cell-specific Na+/K+-ATPase and vacuolar type H+-ATPase protein abundance localization pointed out to activation of preexisting ATPases, non-bicarbonate pH buffering, or both, as the mechanism for endolymph pH-regulation. These results provide the first direct evidence showcasing the acid-base chemistry of the endolymph of OA-exposed fish favors otolith overgrowth, and suggests that this phenomenon will be more pronounced in species that count with more robust blood and endolymph pH regulatory mechanisms.

A novel K+ -dependent Na+ uptake mechanism during low pH exposure in adult zebrafish (Danio rerio): New tricks for old dogma.

AIM: To determine whether Na+ uptake in adult zebrafish (Danio rerio) exposed to acidic water adheres to traditional models reliant on Na+ /H+ Exchangers (NHEs), Na+ channels and Na+ /Cl- Cotransporters (NCCs) or if it occurs through a novel mechanism. METHODS: Zebrafish were exposed to control (pH 8.0) or acidic (pH 4.0) water for 0-12 hours during which 22 Na+ uptake ( JNain ), ammonia excretion, net acidic equivalent flux and net K+ flux ( JHnet ) were measured. The involvement of NHEs, Na+ channels, NCCs, K+ -channels and K+ -dependent Na+ /Ca2+ exchangers (NCKXs) was evaluated by exposure to Cl- -free or elevated [K+ ] water, or to pharmacological inhibitors. The presence of NCKXs in gill was examined using RT-PCR. RESULTS: JNain was strongly attenuated by acid exposure, but gradually recovered to control rates. The systematic elimination of each of the traditional models led us to consider K+ as a counter substrate for Na+ uptake during acid exposure. Indeed, elevated environmental [K+ ] inhibited JNain during acid exposure in a concentration-dependent manner, with near-complete inhibition at 10 mM. Moreover, JHnet loss increased approximately fourfold at 8-10 hours of acid exposure which correlated with recovered JNain in 1:1 fashion, and both JNain and JHnet were sensitive to tetraethylammonium (TEA) during acid exposure. Zebrafish gills expressed mRNA coding for six NCKX isoforms. CONCLUSIONS: During acid exposure, zebrafish engage a novel Na+ uptake mechanism that utilizes the outwardly directed K+ gradient as a counter-substrate for Na+ and is sensitive to TEA. NKCXs are promising candidates to mediate this K+ -dependent Na+ uptake, opening new research avenues about Na+ uptake in zebrafish and other acid-tolerant aquatic species.

A pH-powered mechanochemical engine regulates the buoyancy of Chaoborus midge larvae.

The freshwater aquatic larvae of the Chaoborus midge are the world's only truly planktonic insects, regulating their buoyancy using two pairs of internal air-filled sacs, one in the thorax and the other in the seventh abdominal segment. In 1911, August Krogh demonstrated the larvae's ability to control their buoyancy by exposing them to an increase in hydrostatic pressure.1 However, how these insects control the volume of their air-sacs has remained a mystery. Gas is not secreted into the air-sacs, as the luminal gas composition is always the same as that dissolved in the surrounding water.1,2 Instead, the air-sac wall was thought to play some role.3-6 Here we reveal that bands of resilin in the air-sac's wall are responsible for the changes in volume. These bands expand and contract in response to changes in pH generated by an endothelium that envelops the air-sac. Vacuolar type H+ V-ATPase (VHA) in the endothelium acidifies and shrinks the air-sac, while alkalinization and expansion are regulated by the cyclic adenosine monophosphate signal transduction pathway. Thus, Chaoborus air-sacs function as mechanochemical engines, transforming pH changes into mechanical work against hydrostatic pressure. As the resilin bands interlaminate with bands of cuticle, changes in resilin volume are constrained to a single direction along the air-sac's longitudinal axis. This makes the air-sac functionally equivalent to a cross-striated pH muscle and demonstrates a unique biological role for resilin as an active structural element.

Rapid blood acid-base regulation by European sea bass (Dicentrarchus labrax) in response to sudden exposure to high environmental CO2.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1 kPa CO2 (2000-10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ∼1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40 min, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2 h, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3- in blood, which increased from ∼4 to ∼22 mmol l-1. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3- and pH, probably because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

Adrenergically induced translocation of red blood cell ß-adrenergic sodium-proton exchangers has ecological relevance for hypoxic and hypercapnic white seabass.

White seabass (Atractoscion nobilis) increasingly experience periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically stimulated sodium-proton-exchangers (ß-NHEs). We hypothesized that RBC ß-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the cellular and subcellular response of RBCs to adrenergic stimulation, and quantified the protective effect of ß-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (Po2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). Novel findings from super-resolution microscopy revealed ß-NHE protein in vesicle-like structures and its translocation into the membrane after adrenergic stimulation. Microscopy data were corroborated by molecular and phylogenetic results and a functional characterization of ß-NHE activity. The activation of RBC ß-NHEs increased Hb-O2 saturation by ∼8% in normoxic hypercapnia and by up to ∼20% in hypoxic normocapnia. Our results provide novel insight into the cellular mechanism of adrenergic RBC stimulation within an ecologically relevant context. ß-NHE activity in white seabass has great potential to protect arterial O2 transport during hypoxia and hypercapnia but is less effective during combinations of these stressors.

Ion-transporting capacity and aerobic respiration of larval white seabass (Atractoscion nobilis) may be resilient to ocean acidification conditions.

Ocean acidification (OA) has been proposed to increase the energetic demand for acid-base regulation at the expense of larval fish growth. Here, white seabass (Atractoscion nobilis) eggs and larvae were reared at control (542 ± 28 µatm) and elevated pCO2 (1831 ± 105 µatm) until five days post-fertilization (dpf). Skin ionocytes were identified by immunodetection of the Na+/K+-ATPase (NKA) enzyme. Larvae exposed to elevated pCO2 possessed significantly higher skin ionocyte number and density compared to control larvae. However, when ionocyte size was accounted for, the relative ionocyte area (a proxy for total ionoregulatory capacity) was unchanged. Similarly, there were no differences in relative NKA abundance, resting O2 consumption rate, and total length between control and treatment larvae at 5 dpf, nor in the rate at which relative ionocyte area and total length changed between 2 and 5 dpf. Altogether, our results suggest that OA conditions projected for the next century do not significantly affect the ionoregulatory capacity or energy consumption of larval white seabass. Finally, a retroactive analysis of the water in the recirculating aquarium system that housed the broodstock revealed the parents had been exposed to average pCO2 of ~1200 µatm for at least 3.5 years prior to this experiment. Future studies should investigate whether larval white seabass are naturally resilient to OA, or if this resilience is the result of parental chronic acclimation to OA, and/or from natural selection during spawning and fertilization in elevated pCO2.

Changes in gill and air-breathing organ characteristics during the transition from water- to air-breathing in juvenile Arapaima gigas.

The obligate air-breathing Amazonian fish, Arapaima gigas, hatch as water-breathing larvae but with development, they modify their swim bladder to an air-breathing organ (ABO) while reducing their gill filaments to avoid oxygen loss. Here, we show that significant changes already take place between 4 weeks (1.6 g) and 11 weeks (5 g) post hatch, with a reduction in gill lamellar surface area, increase in gill diffusion distance, and proliferation of the parenchyma in the ABO. By using a variety of methods, we quantified the surface area and diffusion distances of the gills and skin, and the swim bladder volume and anatomical complexity from hatch to 11-week-old juveniles. In addition, we identified the presence of two ionocyte types in the gills and show how these change with development. Until 1.6 g, A. gigas possess only the H+ -excreting/Na+ -absorbing type, while 5-g fish and adults have an additional ionocyte which likely absorbs H+ and Cl- and excretes HCO3- . The ionocyte density on the gill filaments increased with age and is likely a compensatory mechanism for maintaining ion transport while reducing gill surface area. In the transition from water- to air-breathing, A. gigas likely employs a trimodal respiration utilizing gills, skin, and ABO and thus avoid a respiratory-ion regulatory compromise at the gills.