Terrestrial locomotion characteristics of climbing perch (Anabas testudineus).

The evolution and utilization of limbs facilitated terrestrial vertebrate movement on land, but little is known about how other lateral structures enhance terrestrial locomotion in amphibian fishes without terrestrialized limb structures. Climbing perch (Anabas testudineus) exhibit sustained terrestrial locomotion using uniaxial rotating gill covers instead of appendages. To investigate the role of such simple lateral structures in terrestrial locomotion and the motion-generating mechanism of the corresponding locomotor structure configuration (gill covers and body undulation), we measured the terrestrial kinematics of climbing perch and quantitatively analysed its motion characteristics. The digitized locomotor kinematics showed a unique body postural adjustment ability that enables the regulation of the posture of the caudal peduncle for converting lateral bending force into propulsion. An analysis of the coordination characteristics demonstrated that the motion of the gill cover is kinematically independent of axial undulation, suggesting that the gill cover functions as an anchored simple support pole while axial undulation actively mediates body posture and produces propulsive force. The two identified feature shapes explained more than 87% of the complex lateral undulation in multistage locomotion. The kinematic characteristics enhance our understanding of the underlying coordinating mechanism corresponding to locomotor configurations. Our work provides quantitative insight into the terrestrial locomotor adaptation of climbing perch and sheds light on terrestrial motion potential of locomotor configurations containing a typical aquatic body and restricted lateral structure.

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.

Impact of turbidity on the gill morphology and hypoxia tolerance of eastern sand darter (Ammocrypta pellucida).

Anthropogenic stressors such as agriculture and urbanization can increase river turbidity, which can negatively impact fish gill morphology and growth due to reduced oxygen in the benthic environment. We assessed the gill morphology, field metabolic rate (FMR), and two hypoxia tolerance metrics (oxygen partial pressure at loss of equilibrium, PO2 at LOE, and critical oxygen tension, Pcrit) of eastern sand darter (Ammocrypta pellucida), a small benthic fish listed as threatened under the Species at Risk Act in Canada, from rivers in southern Ontario. Field trials were conducted streamside in the Grand River (August 2019; mean NTU 8) and in the comparatively more turbid Thames River (August 2020; mean NTU 94) to test the effect of turbidity on each physiological endpoint. Gills were collected from incidental mortalities and museum specimens, and were assessed using hematoxylin and eosin and immunofluorescent staining. The between-river comparison indicated that turbidity significantly increased interlamellar space and filament width but had no significant influence on other gill morphometrics or FMR. Turbidity significantly increased PO2 at LOE (i.e., fish had a lower hypoxia tolerance) but did not significantly impact Pcrit. Therefore, although turbidity influences hypoxia tolerance through LOE, turbidity levels were not sufficiently high in the study rivers to contribute to measurable changes in gill morphology or metabolism in the wild. Determining whether changes in gill morphology or metabolism occur under higherturbidity levels would help resolve the ecological importance of turbidity on species physiology in urban and agricultural ecosystems.

The role of salinity on genome-wide DNA methylation dynamics in European sea bass gills.

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change.

A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex.

To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neuron to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have investigated the anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.

Persistent augmentation of fictive air breathing by hypoxia: An in vitro study of the role of GABAB signaling in pre-metamorphic tadpoles.

Tadpole development is influenced by environmental cues and hypoxia can favor the emergence of the neural networks driving air breathing. Exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (∼0% O2; 15 min) leads to a progressive increase in fictive air breaths (∼3 fold) in the hours that follow stimulation. Here, we first determined whether this effect persists over longer periods (<18 h); we then evaluated maturity of the motor output by comparing the breathing pattern of hypoxia-exposed brainstems to that of preparations from adult bullfrogs under basal conditions. Because progressive withdrawal of GABAB-mediated inhibition contributes to the developmental increase in fictive lung ventilation, we then hypothesised that hypoxia reduces respiratory sensitivity to baclofen (selective GABAB-agonist). Experiments were performed on isolated brainstem preparations from pre-metamorphic tadpoles (TK stages IV to XIV); respiratory-related neural activity was recorded from cranial nerves V/VII and X before and 18 h after exposure to hypoxia (0% O2 + 2% CO2; 25 min). Time-control experiments (no hypoxia) were performed. Exposing pre-metamorphic tadpoles to hypoxia did not affect gill burst frequency, but augmented the frequency of fictive lung bursts and the incidence of episodic breathing levels intermediate between pre-metamorphic and adult preparations. Addition of baclofen to the aCSF (0,2 µM - 20 min) reduced lung burst frequency, but the response of hypoxia-exposed brainstems was greater than controls. We conclude that acute hypoxia facilitates development and maturation of the motor command driving air breathing. We propose that a greater number of active rhythmogenic neurons expressing GABAb receptors contributes to this effect.

Role of cytosolic carbonic anhydrase Ca17a in cardiorespiratory responses to CO2 in developing zebrafish (Danio rerio).

The sensing of environmental fluctuations and initiation of appropriate physiological responses is crucial to homeostasis. Neuroepithelial cells (NECs) in fishes are putative chemoreceptors, resembling mammalian Type I (glomus) cells, that respond in vitro to changes in O2, CO2, NH3, and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO2 sensing owing to its presence in NECs. Zebrafish (Danio rerio) lacking functional Ca17a were generated via CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO2 (hypercapnia). Unfortunately, the homozygous knockout mutants (ca17a-/-) did not survive more than ∼12-14 days postfertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation (fV) and cardiac (fH) frequency in response to hypercapnia (1% CO2) in wild-type (ca17a+/+), heterozygous (ca17a+/-) and ca17a-/- fish were used to investigate Ca17a-dependent CO2 sensing and downstream signaling. Wild-type fish exhibited hyperventilation during hypercapnia as indicated by an increase in fV. In the ca17a-/- fish, the hyperventilatory response was attenuated markedly but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a-/- fish exhibited a significantly lower resting fH that became more pronounced as the fish aged. The decrease in resting fH was prevented ("rescued") when ca17a-/- embryos were injected with ca17a mRNA. Collectively, the results of this study support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Breathing versus feeding in the Pacific hagfish.

Hagfish represent the oldest extant connection to the ancestral vertebrates, but their physiology is not well understood. Using behavioural (video), physiological (respirometry, flow measurements), classical morphological (dissection, silicone injection) and modern imaging approaches (micro-MRI, DICE micro-CT), we examined the interface between feeding and the unique breathing mechanism (nostril opening, high-frequency velum contraction, low-frequency gill pouch contraction and pharyngo-cutaneous duct contraction) in the Pacific hagfish, Eptatretus stoutii. A video tour via micro-MRI is presented through the breathing and feeding passages. We have reconciled an earlier disagreement as to the position of the velum chamber, which powers inhalation through the nostril, placing it downstream of the merging point of the food and water passage, such that the oronasal septum terminates at the anterior end of the velum chamber. When feeding occurs by engulfment of large chunks by the dental plates, food movement through the chamber may transiently interfere with breathing. Swallowing is accelerated by peristaltic body undulation involving the ventral musculature, and is complete within 5 s. After a large meal (anchovy, 20% body mass), hagfish remain motionless, defaecating bones and scales at 1.7 days and an intestinal peritrophic membrane at 5 days. O2 consumption rate approximately doubles within 1 h of feeding, remaining elevated for 12-24 h. This is achieved by combinations of elevated O2 utilization and ventilatory flow, the latter caused by varying increases in velar contraction frequency and stroke volume. Additional imaging casts light on the reasons for the trend for greater O2 utilization by more posterior pouches and the pharyngo-cutaneous duct in fasted hagfish.

The osmorespiratory compromise in the fish gill.

August Krogh made fundamental discoveries about both respiratory gas exchange and osmo/iono-regulation in fish gills. Dave Randall and co-workers identified a tradeoff between these two functions such that high functional surface area and low diffusion distance would favour O2 uptake (e.g. exercise, hypoxia), whereas low functional surface area and high diffusion distance would favour osmo/iono-regulation (rest, normoxia). Today we call this concept the "osmorespiratory compromise" and realize that it is much more complex than originally envisaged. There are at least 6 mechanisms by which fish can change functional branchial area and diffusion distance. Three involve reorganizing blood flow pathways: (i) flow redistribution within the secondary (respiratory) lamellae; (ii) flow shunting between "respiratory" and "ionoregulatory" pathways in the filament; (iii) opening up more distal lamellae on the filament and closing non-respiratory pathways. Three more involve "reversible gill remodeling": (iv) proliferation of the interlamellar gill cell mass (ILCM); (v) proliferation of ionocytes up the sides of the lamellae; (vi) covering over the apical exposure of ionocytes by extension of pavement cells. In ways that remain incompletely understood, these mechanisms allow dynamic regulation of the osmorespiratory compromise, such that ion and water fluxes can be decoupled from O2 uptake during continuous exercise. Furthermore, hypoxia-tolerant species can reduce branchial ion and water fluxes below normoxic levels despite hyperventilating during hypoxia. In marine fish, the osmorespiratory conflict is intensified by the greater ionic and osmotic gradients from seawater to blood, but underlying mechanisms remain poorly understood.

How insects transition from water to air: Respiratory insights from dragonflies.

The transition of animal life from water onto land is associated with well-documented changes in respiratory physiology and blood chemistry, including a dramatic increase in blood pCO2 and bicarbonate, and changes in ventilatory control. However, these changes have primarily been documented among ancestrally aquatic animal lineages that have evolved to breathe air. In contrast, the physiological consequences of air-breathing animals secondarily adopting aquatic gas exchange are not well explored. Insects are arguably the most successful air-breathing animals, but they have also re-evolved the ability to breathe water multiple times. The juvenile life stages of many insect lineages possess tracheal gills for aquatic gas exchange, but all shift back to breathing air in their adult form. This makes these amphibiotic insects an instructive contrast to most other animal groups, being not only an ancestrally air-breathing group of animals that have re-adapted to life in water, but also a group that undergoes an ontogenetic shift from water back to air across their life cycle. This graphical review summarizes the current knowledge on how blood acid-base balance and ventilatory control change in the dragonfly during its water-to-air transition, and highlights some of the remaining gaps to be filled.

Cutaneous respiration and osmoregulation in amphibious fishes.

In his early career, August Krogh made fundamental discoveries of the properties of cutaneous respiration in fish, frogs and other vertebrates. Following Krogh's example, the study of amphibious fishes provides an excellent model to understand how the skin morphology and physiological mechanisms evolved to meet the dual challenges of aquatic and terrestrial environments. The skin of air-exposed fishes takes on many of the functions that are typically associated with the gills of fish in water: gas exchange, gas sensing, iono- and osmoregulation, and nitrogen excretion. The skin of amphibious fishes has capillaries close to the surface in the epidermis. Skin ionocytes or mitochondrial-rich cells (MRCs) in the epidermis are thought to be responsible for ion exchange, as well as ammonia excretion in the amphibious mangrove rivulus Kryptolebias marmoratus. Ammonia gas (NH3) moves down the partial pressure gradient from skin capillaries to the surface through ammonia transporters (e.g., Rhcg) and NH3 is volatilized from the mucus film on the skin. Future studies are needed on the skin of amphibious fishes from diverse habitats to understand more broadly the role of the skin as a multifunctional organ.

Acute and chronic temperature dependence of Na+/H+ exchange activity of Pimephales promelas gills.

Na+/H+ exchangers (NHE) mediate at least part of Na+ entry into gill epithelia via Na+/NH4+ exchange. For homeostasis, Na+ entry into and exit via Na+/K+ ATPase from gill epithelia must balance. Na+/K+ ATPase activity is reduced in cold- compared to warm-acclimated freshwater temperate fish. We hypothesized gill NHE activity is greater in warm- than cold-acclimated fish when measured at acclimation temperatures, and NHE activity displays a temperature dependence similar to Na+/K+ ATPase. Since NHE mRNA expression does not differ, we measured the Na+-dependence of pH-induced Na+ fluxes in gill vesicles from warm- and cold-acclimated fathead minnows at 20o and 7 °C, and calculated maximum transport rates (Vmax) and Na+ K1/2s. We also measured NH4+-induced Na+ fluxes and Na+-induced H+ fluxes. In vesicles from warm-acclimated fish, NHE Vmaxs were 278 ± 33 and 149 ± 23 arbitrary unit/s (au/s) and Na+ K1/2s were 12 ± 4 and 6 ± 4 mmol/l when assayed at 20o and 7 °C (p < 0.004), respectively. In vesicles from cold-acclimated fish, Vmaxs were 288 ± 35 and 141 ± 13 au/s and Na+ K1/2s 17 ± 5 and 7 ± 2 mmol/l when assayed at 20o and 7 °C (p < 0.002), respectively. Na+-induced H+ fluxes were 98 ± 8 and 104 ± 26 au/s in warm- and cold-acclimated fish assayed at 20 °C, respectively. Na+/NH4+ exchange was 120 ± 11 and 158 ± 13 au/s in warm- and cold-acclimated fish, respectively. Conclusions: Gill NHE activity was greater in warm- than cold-acclimated fish assayed at acclimation temperatures. The temperature dependence of NHE activity was similar in both groups, but differed from that reported for Na+/K+ ATPase suggesting complex mechanisms to maintain Na+ homeostasis.

Combined hypoxia and high temperature affect differentially the response of antioxidant enzymes, glutathione and hydrogen peroxide in the white shrimp Litopenaeus vannamei.

Low oxygen concentration in water (hypoxia) and high temperature are becoming more frequent due to climate change, forcing animals to endure stress or decease. Hypoxia and high temperature stress can lead to reactive oxygen species (ROS) accumulation and oxidative damage to the organisms. The shrimp Litopenaeus vannamei is the most cultivated crustacean worldwide. The aim of this study was to evaluate the expression and enzymatic activity of glutathione peroxidase (GPx), catalase (CAT) and cytosolic manganese superoxide dismutase (cMnSOD) in gills and hepatopancreas from L. vannamei in response to two combined stressors: hypoxia and reoxygenation at control and high temperature (28 vs 35 °C, respectively). In addition, glutathione and hydrogen peroxide content were analyzed. The changes were mainly tissue-specific. In gills, cMnSOD expression and enzymatic activity increased in response to the interactions between oxygen variation and thermal stress, while GPx and CAT were maintained. More changes occurred in GPx, CAT and MnSOD in hepatopancreas than in gills, mainly due to the effect of the individual stress factors of thermal stress or oxygen variations. On the other hand, the redox state of glutathione indicated that during high temperature, changes in the GSH/GSSG ratio occurred due to the fluctuations of GSSG. Hydrogen peroxide concentration was not affected by thermal stress or oxygen variations in hepatopancreas, whereas in gills, it was not detected. Altogether, these results indicate a complex pattern of antioxidant response to hypoxia, reoxygenation, high temperature and their combinations.

Salt transport by the gill Na+-K+-2Cl- symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes.

Palaemonid shrimps inhabit osmotic niches from marine to continental waters. They hyper-regulate hemolymph osmolality and ionic concentrations in dilute media, hypo-regulating in concentrated media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. To examine Cl- hypo-regulatory capability and phylogenetic correlations between gill NKCC mRNA levels and protein expression, we used palaemonids ranging from marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium). We established the species' upper critical salinity limits (UL50) and short- (24 h) and long-term (120h) hypo-regulatory abilities at salinities of 80% of their UL50's (80%UL50). The Palaemon species exhibited the highest UL50's and greatest hypo-regulatory capabilities; among the Macrobrachium species, UL50's were higher in the diadromous than in the hololimnetic species. While basal transcript levels of gill NKCC mRNA were highest in P. pandaliformis, levels were unaffected by salinity or exposure time in all species. However, gill NKCC protein abundance increased after 120-h exposure at the 80%UL50 in all Macrobrachium species, except M. potiuna. Unexpectedly, hemolymph hyper-osmoregulatory capability in acclimatization media correlated with gill NKCC protein synthesis, while gill NKCC mRNA expression correlated with hemolymph hyper-Cl- regulation in Macrobrachium. These findings, together with the evolutionary history of osmoregulation in this shrimp clade, suggest a role for the gill NKCC symporter in both salt uptake and secretion. The evolution of NKCC protein expression responsiveness, unlike hemolymph hypo-regulation and NKCC mRNA expression, may have been driven by environmental salinity during niche radiation. SUMMARY STATEMENT: While mRNA expression of the gill Na+-K+-2Cl- symporter is unchanged during acclimation of palaemonid shrimps to saline media, protein expression is up regulated, revealing a role in chloride secretion.

Seasonal modulation of oxidative stress biomarkers in mangrove oyster (Crassostrea gasar) from an Amazon estuary.

Estuaries are the final destination of many pollutants derived from anthropogenic activity. Therefore, it is difficult to find this kind of ecosystem in a pristine condition. In this context, biomonitoring studies that characterize the organism's conditions against the environment' s natural variation are essential for future impact analysis due to anthropic activity. The present study aims to characterize the natural modulation of biochemical biomarkers in oysters Crassostrea gasar. The research was conducted in Japerica Bay, an estuary region located in the Eastern Amazon (Pará, Brazil), which has remained in pristine condition for many years. The samplings were carried out throughout one year during the rainy-dry transition period (June/2013), dry period (September/2013), dry-rainy transition period (November / 2013), and rainy period (February / 2014) in the lower and upper estuary. The activity of glutathione-S-transferase (GST) and total antioxidant capacity (ACAP) were evaluated as biomarkers of exposure and lipid peroxidation (LPO) as an effect biomarker. In gills, GST decreased during the rainy season in both sites and increased during the salinity peak (dry-rainy transition period) for the upper estuary's organisms. In this organ, the lowest levels of LPO occurred during the dry season for both points. There was an induction of ACAP in muscle during the rainy-dry transition period compared to the dry and dry-rainy transition periods for the lower estuary's organisms, and there were no differences for GST suggesting low tissue sensitivity. There was an increase in LPO during the rainy season compared to the rainy-dry transition period for the lower estuaries animals. Biomarkers in gills suggest a metabolic challenge to the rainy season and stability during the dry season. The species shows high viability of use in biomonitoring programs. However, these seasonality-induced alterations in biomarkers responses must be taken into account to interpret the results.

The osmotic response capacity of the Antarctic fish Harpagifer antarcticus is insufficient to cope with projected temperature and salinity under climate change.

Over the last decades, climate change has intensified. Temperatures have increased and seawater has become "fresher" in Antarctica, affecting fish such as Harpagifer antarcticus. Thus, this study aimed to evaluate changes in the osmoregulatory response of the Antarctic notothenioid fish Harpagifer antarcticus and evaluate how it will cope with the future climate change and environmental conditions in the Antarctic, and in the hypothetical case that its geographical distribution will be extended to the Magellanes region. The present study was undertaken to determine the interaction between temperature and salinity tolerance (2 °C and 33 psu as the control group, the experimental groups were 5, 8, and 11 °C and 28 and 23 psu) and their effect on the osmoregulatory status of H. antarcticus. We evaluated changes in gill-kidney-intestine NKA activity, gene expression of NKAα, NKCC, CFTR, Aquaporins 1 and 8 in the same tissues, muscle water percentage, and plasma osmolality to evaluate osmoregulatory responses. Plasma osmolality decreased with high temperature, also the gill-kidney-intestine NKA activity, gene expression of NKA α, NKCC, CFTR, Aquaporins 1, and 8 were modified by temperature and salinity. We demonstrated that H. antarcticus can not live in the Magallanes region, due to its incapacity to put up with temperatures over 5 °C and with over 8 °C being catastrophic.

The Gill-Oxygen Limitation Theory and size at maturity/maximum size relationships for salmonid populations occupying flowing waters.

The slowing of growth as fish age has long been believed to be related to energy expenditure for maturation, and this rationalization has been used to explain why, across nearly all fish species, the relationship between size at first maturity (Lm ) and maximum (Lmax ) or asymptotic length (L∞ ) is relatively constant. In contrast, the Gill-Oxygen Limitation Theory (GOLT) postulates that (a) fish growth slows because as they grow, their two-dimensional ability to extract oxygen from the water diminishes relative to their three-dimensional weight gain, and (b) they can only invest energy for maturation if oxygen supply at their size at first maturity (Qm ) exceeds that needed for maintenance metabolism (Q∞ ). It has been reported previously across dozens of marine fish species that the relationship between Qm and Q∞ is linear and, further, it can be mathematically converted to Lm vs. L∞ by raising both terms to the power of D (the gill surface factor), resulting in a slope of 1.36. If the GOLT is universal, a similar slope should exist for Lm D vs. L∞ D relationships for freshwater species across multiple individual populations that reside in disparate habitats, although to our knowledge this has never been evaluated. For analysis, we used existing data from previous studies conducted on 51 stream-dwelling populations of redband trout Oncorhynchus mykiss gairdneri, Yellowstone cutthroat trout O. clarkii bouvieri and mountain whitefish Prosopium williamsoni. The resulting Lm D vs. L∞ D slopes combining all data points (1.35) or for all species considered separately (range = 1.29-1.40) were indeed equivalent to the slope originally produced for the marine species from which the GOLT-derived relationship was first reported. We briefly discuss select papers both supporting and resisting various aspects of the GOLT, note that it could potentially explain shrinking sizes of marine fish, and call for more concerted research efforts combining laboratory and field expertise in fish growth research.

Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms.

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na+/H+ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.

Fgf- and Bmp-signaling regulate gill regeneration in Ambystoma mexicanum.

Gill regeneration has not been well studied compared to regeneration of other appendages, such as limb and tail regeneration. Here, we focused on axolotl gill regeneration and found that Fgf- and Bmp-signaling are involved in their gill regeneration mechanism. Axolotls have three pairs of gill rami, and each gill ramus has multiple gill filaments. The gills consist of mesenchyme rich in extracellular matrix and epidermis. The gill nerves are supplied from the trigeminal ganglia located in the head. Denervation resulted in no gill regeneration responses. Nerves and gills express Bmp and Fgf genes, and treating animals with Fgf- and Bmp-signaling inhibitors results in phenotypes similar to those seen in denervated gills. Inducing an accessory appendage is a standard assay in amphibian regeneration research. In our study, an accessory gill could be induced by lateral wounding, suggesting that thin axon fibers and mesenchymal Fgfs and Bmps contributed to the induction of the accessory structure. Such accessory gill induction was inhibited by the denervation. Exogenous Fgf2+Fgf8+Bmp7, which have been determined to function as a regeneration inducer in urodele amphibians, could compensate for the effects denervation has on accessory blastema formation. Our findings suggest that regeneration of appendages in axolotls is regulated by common Fgf- and Bmp-signaling cascades.

Calcified gill filaments increase respiratory function in fishes.

The morphology of fish gills is closely linked to aerobic capacity and tolerance of environmental stressors such as hypoxia. The importance of gill surface area is well studied, but little is known about how the mechanical properties of gill tissues determine function. In some fishes, the bases of the gill filaments are surrounded by a calcified 'sheath' of unknown function. We tested two non-exclusive hypotheses: (i) calcified gill filaments enhance water flow through the gill basket, improving aquatic respiratory function, and (ii) in amphibious fishes, calcification provides support for gills out of water. In a survey of more than 100 species of killifishes and related orders, we found filament calcification was widespread and thus probably arose before the evolution of amphibious lifestyles in killifishes. Calcification also did not differ between amphibious and fully aquatic species, but terrestrial acclimation caused calcium deposition on the filaments of the killifish Kryptolebias marmoratus, suggesting a possible structural role when out of water. We found strong evidence supporting a role for filament calcification in enhancing aquatic respiratory function. First, acclimation to increased respiratory demands (hypoxia, elevated temperatures) induced calcium deposition on the filaments of K. marmoratus. Next, gentle removal of filament calcification decreased branchial resistance to water flow, indicating disruption of gill basket positioning. Thus, the mechanical properties of the gill filaments appear to play an important and previously unappreciated role in determining fish respiratory function.