Gill ionocyte remodeling mediates blood pH regulation in rockfish (Sebastes diploproa) exposed to environmentally relevant hypercapnia.

Marine fishes excrete excess H+ using basolateral Na+-K+-ATPase (NKA) and apical Na+/H+ exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H+ excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish (Sebastes diploproa) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 µatm Pco2). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1) mRNAs coding for proteins involved in blood acid-base regulation, 2) total NKA and NHE3 protein abundance, and 3) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H+ excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems.NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

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.

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.

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.

Acid-base regulation in the air-breathing swamp eel (Monopterus albus) at different temperatures.

Vertebrates reduce arterial blood pH (pHa) when body temperature increases. In water breathers, this response occurs primarily by reducing plasma HCO3- levels with small changes in the partial pressure of CO2 (PCO2 ). In contrast, air breathers mediate the decrease in pHa by increasing arterial PCO2  (PaCO2 ) at constant plasma HCO3- by reducing lung ventilation relative to metabolic CO2 production. Much less is known about bimodal breathers, which utilize both water and air. Here, we characterized the influence of temperature on arterial acid-base balance and intracellular pH (pHi) in the bimodal-breathing swamp eel, Monopterus albus This teleost uses the buccopharyngeal cavity for gas exchange and has very reduced gills. When exposed to ecologically relevant temperatures (20, 25, 30 and 35°C) for 24 and 48 h, pHa decreased by -0.025 pH units (U) °C-1 in association with an increase in PaCO2 , but without changes in plasma [HCO3-]. pHi was also reduced with increased temperature. The slope of pHi of liver and muscle was -0.014 and -0.019 U °C-1, while the heart muscle showed a smaller reduction (-0.008 U °C-1). When exposed to hypercapnia (7 or 14 mmHg) at either 25 or 35°C, M. albus elevated plasma [HCO3-] and therefore seemed to defend the new pHa set-point, demonstrating an adjusted control of acid-base balance with temperature. Overall, the effects of temperature on acid-base balance in M. albus resemble those in air-breathing amniotes, and we discuss the possibility that this pattern of acid-base balance results from a progressive transition in CO2 excretion from water to air as temperature rises.

Acid secretion by the boring organ of the burrowing giant clam, Tridacna crocea.

The giant clam Tridacna crocea, native to Indo-Pacific coral reefs, is noted for its unique ability to bore fully into coral rock and is a major agent of reef bioerosion. However, T. crocea's mechanism of boring has remained a mystery despite decades of research. By exploiting a new, two-dimensional pH-sensing technology and manipulating clams to press their presumptive boring tissue (the pedal mantle) against pH-sensing foils, we show that this tissue lowers the pH of surfaces it contacts by greater than or equal to 2 pH units below seawater pH day and night. Acid secretion is likely mediated by vacuolar-type H+-ATPase, which we demonstrate (by immunofluorescence) is abundant in the pedal mantle outer epithelium. Our discovery of acid secretion solves this decades-old mystery and reveals that, during bioerosion, T. crocea can liberate reef constituents directly to the soluble phase, rather than producing sediment alone as earlier assumed.

Impacts of ash-induced environmental alkalinization on fish physiology, and their implications to wildfire-scarred watersheds.

Changes in land use, a warming climate and increased drought have amplified wildfire frequency and magnitude globally. Subsequent rainfall in wildfire-scarred watersheds washes ash into aquatic systems, increasing water pH and exposing organisms to environmental alkalinization. In this study, 15 or 20 °C-acclimated Chinook salmon (Oncorhynchus tshawytscha) yearlings were exposed to an environmentally-relevant ash concentration (0.25 % w/v), increasing water pH from ∼8.1 to ∼9.2. Salmon experienced significant disturbance to blood plasma pH (pHe) and red blood cell intracellular pH (RBC pHi) within 1 h, but recovered within 24 h. Impacts on plasma ion concentrations were relatively mild, and plasma glucose increased by 2- to 4-fold at both temperatures. Temperature-specific differences were observed: 20 °C salmon recovered their pHe more rapidly, perhaps facilitated by higher basal metabolism and anaerobic metabolic H+ production. Additionally, 20 °C salmon experienced dramatically greater spikes in plasma total ammonia, [NH3] and [NH4+] after 1 h of exposure that decreased over time, whereas 15 °C salmon experienced a gradual nitrogenous waste accumulation. Despite pHe and RBC pHi recovery and non-lethal nitrogenous waste levels, we observed 20 % and 33 % mortality in 15 and 20 °C treatments within 12 h of exposure, respectively. The mortalities cannot be explained by high water pH alone, nor was it likely to be singularly attributable to a heavy metal or organic compound released from ash input. This demonstrates post-wildfire ash input can induce lethal yet previously unexplored physiological disturbances in fish, and further highlights the complex interaction with warmer temperatures typical of wildfire-scarred landscapes.

Industrial-strength ecology: trade-offs and opportunities in algal biofuel production.

Microalgae represent one of the most promising groups of candidate organisms for replacing fossil fuels with contemporary primary production as a renewable source of energy. Algae can produce many times more biomass per unit area than terrestrial crop plants, easing the competing demands for land with food crops and native ecosystems. However, several aspects of algal biology present unique challenges to the industrial-scale aquaculture of photosynthetic microorganisms. These include high susceptibility to invading aquatic consumers and weeds, as well as prodigious requirements for nutrients that may compete with the fertiliser demands of other crops. Most research on algal biofuel technologies approaches these problems from a cellular or genetic perspective, attempting either to engineer or select algal strains with particular traits. However, inherent functional trade-offs may limit the capacity of genetic selection or synthetic biology to simultaneously optimise multiple functional traits for biofuel productivity and resilience. We argue that a community engineering approach that manages microalgal diversity, species composition and environmental conditions may lead to more robust and productive biofuel ecosystems. We review evidence for trade-offs, challenges and opportunities in algal biofuel cultivation with a goal of guiding research towards intensifying bioenergy production using established principles of community and ecosystem ecology.

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.

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.

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.

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.

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.

Molecular and biochemical characterization of the bicarbonate-sensing soluble adenylyl cyclase from a bony fish, the rainbow trout Oncorhynchus mykiss.

Soluble adenylyl cyclase (sAC) is a HC O 3 - -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid-base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by HC O 3 - at physiologically relevant levels for fish internal fluids (EC50 ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid-base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.

Immunological characterization of two types of ionocytes in the inner ear epithelium of Pacific Chub Mackerel (Scomber japonicus).

The inner ear is essential for maintaining balance and hearing predator and prey in the environment. Each inner ear contains three CaCO3 otolith polycrystals, which are calcified within an alkaline, K+-rich endolymph secreted by the surrounding epithelium. However, the underlying cellular mechanisms are poorly understood, especially in marine fish. Here, we investigated the presence and cellular localization of several ion-transporting proteins within the saccular epithelium of the Pacific Chub Mackerel (Scomber japonicus). Western blotting revealed the presence of Na+/K+-ATPase (NKA), carbonic anhydrase (CA), Na+-K+-2Cl--co-transporter (NKCC), vacuolar-type H+-ATPase (VHA), plasma membrane Ca2+ ATPase (PMCA), and soluble adenylyl cyclase (sAC). Immunohistochemistry analysis identified two distinct ionocytes types in the saccular epithelium: Type-I ionocytes were mitochondrion-rich and abundantly expressed NKA and NKCC in their basolateral membrane, indicating a role in secreting K+ into the endolymph. On the other hand, Type-II ionocytes were enriched in cytoplasmic CA and VHA, suggesting they help transport HCO3- into the endolymph and remove H+. In addition, both types of ionocytes expressed cytoplasmic PMCA, which is likely involved in Ca2+ transport and homeostasis, as well as sAC, an evolutionary conserved acid-base sensing enzyme that regulates epithelial ion transport. Furthermore, CA, VHA, and sAC were also expressed within the capillaries that supply blood to the meshwork area, suggesting additional mechanisms that contribute to otolith calcification. This information improves our knowledge about the cellular mechanisms responsible for endolymph ion regulation and otolith formation, and can help understand responses to environmental stressors such as ocean acidification.

A novel acidification mechanism for greatly enhanced oxygen supply to the fish retina.

Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

Ontogenetic changes in cutaneous and branchial ionocytes and morphology in yellowfin tuna (Thunnus albacares) larvae.

The development of osmoregulatory and gas exchange organs was studied in larval yellowfin tuna (Thunnus albacares) from 2 to 25 days post-hatching (2.9-24.5 mm standard length, SL). Cutaneous and branchial ionocytes were identified using Na+/K+-ATPase immunostaining and scanning electron microscopy. Cutaneous ionocyte abundance significantly increased with SL, but a reduction in ionocyte size and density resulted in a significant decrease in relative ionocyte area. Cutaneous ionocytes in preflexion larvae had a wide apical opening with extended microvilli; however, microvilli retracted into an apical pit from flexion onward. Lamellae in the gill and pseudobranch were first detected ~ 3.3 mm SL. Ionocytes were always present on the gill arch, first appeared in the filaments and lamellae of the pseudobranch at 3.4 mm SL, and later in gill filaments at 4.2 mm SL, but were never observed in the gill lamellae. Unlike the cutaneous ionocytes, gill and pseudobranch ionocytes had a wide apical opening with extended microvilli throughout larval development. The interlamellar fusion, a specialized gill structure binding the lamellae of ram-ventilating fish, began forming by ~ 24.5 mm SL and contained ionocytes, a localization never before reported. Ionocytes were retained on the lamellar fusions and also found on the filament fusions of larger sub-adult yellowfin tuna; however, sub-adult gill ionocytes had apical pits. These results indicate a shift in gas exchange and NaCl secretion from the skin to branchial organs around the flexion stage, and reveal novel aspects of ionocyte localization and morphology in ram-ventilating fishes.

Quantification of Cutaneous Ionocytes in Small Aquatic Organisms.

Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na+/K+ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera's focal plane and the microscope's field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching-thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.

Acute fluoxetine exposure alters crab anxiety-like behaviour, but not aggressiveness.

Aggression and responsiveness to noxious stimuli are adaptable traits that are ubiquitous throughout the animal kingdom. Like vertebrate animals, some invertebrates have been shown to exhibit anxiety-like behaviour and altered levels of aggression that are modulated by the neurotransmitter serotonin. To investigate whether this influence of serotonin is conserved in crabs and whether these behaviours are sensitive to human antidepressant drugs; the striped shore crab, Pachygrapsus crassipes, was studied using anxiety (light/dark test) and aggression (mirror test) paradigms. Crabs were individually exposed to acute doses of the selective serotonin reuptake inhibitor, fluoxetine (5 or 25 mg/L), commonly known as Prozac®, followed by behavioural testing. The high dose of fluoxetine significantly decreased anxiety-like behaviour but had no impact on mobility or aggression. These results suggest that anxiety-like behaviour is more sensitive to modulation of serotonin than is aggressiveness in the shore crab.