The gut microbiome and its potential role in paradoxical anaerobism in pupfishes of the Mojave Desert.

BACKGROUND: Pupfishes frequently enter paradoxical anaerobism in response to endogenously produced or exogenously supplied ethanol in a dose-dependent manner. To decipher the role of the gut microbiota in ethanol-associated paradoxical anaerobism, gut microbial communities were depleted using a cocktail of antibiotics and profiled using 16S rRNA gene sequencing. RESULTS: Compared to the control group (n = 12), microbiota-depleted fish (n = 12) spent more time in paradoxical anaerobism. Our analysis indicated that the bacterial phyla Proteobacteria, Fusobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Patescibacteria, and Dependentiae dominated the pupfish gut, which is consistent with other fish gut microbiota. Although the gut microbial communities with and without antibiotic treatment were similarly diverse, they were distinct and the greatest contribution to the dissimilarity (27.38%) was the common fish commensal Cetobacterium. CONCLUSIONS: This study reports the first characterization of gut microbial communities of pupfish and suggests the microbiome may play a critical role in regulating metabolic strategies that are critical for survival in extremes of temperature and oxygen concentration. We speculate that Cetobacterium, a primary fermenter, also consumes ethanol through secondary fermentation via an alcohol dehydrogenase and therefore regulates the transition from paradoxical anaerobism to aerobic respiration in fish. Given the wide distribution and abundance of Cetobacterium in warm-water fishes, this process may be of broad importance, and suggests that the microbiome be carefully considered for both conservation and aquaculture.

The effect of temperature on development and behaviour of relict leopard frog tadpoles.

Relict leopard frog (Rana [Lithobates] onca) tadpoles were obtained shortly after hatching at Gosner stage 25 and raised in aquaria maintained at 15, 20, 25, 30 and 35°C. Development was arrested in the 15°C group, and survivorship declined to 64% after 191 days. However, 80% of the surviving larvae remained alive after the temperature was increased to 25°C. Of these, 96% reached metamorphosis. Survivorship of the 20, 25 and 30°C acclimation groups was 82, 94 and 66%, respectively, whereas none survived at 35°C. Time to metamorphosis was significantly shorter for the 25°C group (67 ± 1 days), followed by the 30°C (98 ± 2 days) and 20°C (264 ± 7 days) groups. A linear 66 cm thermal gradient was used to identify temperature ranges selected by tadpoles in the different acclimation groups. Five 10°C gradients (10-20, 15-25, 20-30, 25-35 and 30-40°C) were used, and time spent in the cooler, middle and warmer thirds of the gradient was compared for 10 individuals from each acclimation group. In the coolest gradient, tadpoles from all acclimation groups selected the warmer third (>17°C) of the gradient. In the warmer gradients, tadpoles from the 20 and 25°C acclimation groups selected temperatures <29°C, while those from the 30°C acclimation group selected temperatures <33°C. Maximal burst speed for all groups was greater at experimental temperatures of 25 than 15°C. Efforts to reintroduce this species to its historical range should select habitats where water temperatures between 25 and 30°C are available during the post-hatching period.

Localization of water channels in the skin of two species of desert toads, Anaxyrus (Bufo) punctatus and Incilius (Bufo) alvarius.

Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.

The water-absorption region of ventral skin of several semiterrestrial and aquatic anuran amphibians identified by aquaporins.

Regions of specialization for water absorption across the skin of Bufonid and Ranid anurans were identified by immunohistochemistry and Western blot analysis, using antibodies raised against arginine vasotocin (AVT)-stimulated aquaporins (AQPs) that are specific to absorbing regions of Hyla japonica. In Bufo marinus, labeling for Hyla urinary bladder-type AQP (AQP-h2), which is also localized in the urinary bladder, occurred in the ventral surface of the hindlimb, pelvic, and pectoral regions. AQP-h2 was not detected in any skin regions of Rana catesbeiana, Rana japonica, or Rana nigromaculata. Hyla ventral skin-type AQP (AQP-h3), which is found in the ventral skin but not the bladder of H. japonica, was localized in the hindlimb, pelvic, and pectoral skins of Bufo marinus, in addition to AQP-h2. AQP-h3 was also localized in ventral skin of the hindlimb of all three Rana species and also in the pelvic region of R. catesbiana. Messenger RNA for AQP-x3, a homolog of AQP-h3, could be identified by RT-PCR from the hindlimb, pectoral, and pelvic regions of the ventral skin of Xenopus laevis, although AVT had no effect on water permeability. In contrast, 10(-8) M AVT-stimulated water permeability and translocation of AQP-h2 and AQP-h3 into the apical membrane of epithelial cells in regions of the skin of species where they had been localized by immunohistochemistry and Western blot analysis. Finally, water permeability of the hindlimb skin of B. marinus and all the Rana species was stimulated by hydrins 1 and 2 to a similar level as seen for AVT. The present data demonstrate species differences in the occurrence, distribution, and regulation of AQPs in regions of skin specialized for rapid water absorption that can be associated with habitat and also phylogeny.

Plasma and brain angiotensin concentrations associated with water response behavior in the desert anuran, Scaphiopus couchii under natural conditions in the field.

Terrestrial amphibians obtain water by absorption across a specialized region of the ventral skin and exhibit a behavior, the water absorption response (WR) to place that region in contact with moist surfaces. Spadefoot toads (Scaphiopus couchii) spend dry months of the year in burrows, then emerge during brief periods of summer rainfall and seek water sources for rehydration and reproduction. We tested the hypothesis that these toads have changes in plasma and/or central angiotensin concentrations that are associated with seasonal emergence and WR behavior. Immunoreactive concentrations of combined angiotensin II and III (ir-ANG) were measured in plasma samples and microdissected regions of brain tissue taken from toads moving across the road or toads showing WR behavior in shallow puddles on the road. Plasma ir-ANG concentrations were not significantly different between these groups, but were significantly higher in the periventricular region of the hypothalamus in toads showing WR behavior. Concentrations in other brain regions, while highly variable among individuals, were not different between groups. Within the context of the natural history of a specialized desert toad, these results support the hypothesis that ir-ANG is associated with WR behavior in spadefoot toads in a manner analogous to oral drinking exhibited by other vertebrate clades.

Stretch-activated cation channel from larval bullfrog skin.

Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current-voltage plots demonstrated linear or inward rectification and single channel conductances of 44-56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (-V(p)) of 20-40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin.

Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.

BACKGROUND: Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis. MATERIALS AND METHODS: Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined. RESULTS: The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis. CONCLUSIONS: DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.

Behavioral and neural responses of toads to salt solutions correlate with basolateral membrane potential of epidermal cells of the skin.

Dehydrated toads initiated water absorption response (WR) behavior and absorbed water from dilute NaCl solutions. With 200-250 mM NaCl, WR behavior and water absorption were both suppressed. With 200-250 mM Na-gluconate, WR initiation was significantly greater than with NaCl but water loss was greater. Neural recordings from spinal nerve #6 showed a greater integrated response to 250 mM NaCl than to 250 mM Na-gluconate, whereas a larger rinse response was seen with Na-gluconate. Studies with isolated epithelium showed a large increase in conductance (G(t)) when 250 mM NaCl replaced NaCl Ringer's as the apical bathing solution that was accompanied by depolarization of the transepithelial potential (V(t)) and basolateral membrane potential (V(b)). Depolarization of V(b) corresponded with the neural response to 250 mM NaCl. When 250 mM Na-gluconate replaced Ringer's as the apical solution G(t) remained low, V(b) transiently hyperpolarized to values near the equilibrium potential for K(+) and corresponded with the reduced neural response. These results support the hypothesis that chemosensory function of the skin is analogous to that of mammalian taste cells but utilizes paracellular ion transport to a greater degree.

Vascular aspects of water uptake mechanisms in the toad skin: perfusion, diffusion, confusion.

Blood cell flow (BCF) in the water absorbing "seat patch" region of toad skin was measured with laser Doppler flow cytometry. BCF of dehydrated toads increased by a factor of 6-8 when water contact was made and declined gradually as toads rehydrated. Water absorption was initially stimulated and declined in parallel with BCF. Water absorption measured during the initial rehydration period did not correlate with BCF and hydrated toads injected with AVT increased water absorption without an increase in BCF indicating the lack of an obligate relation between blood flow and water absorption. Aquaporins 1-3 were characterized by RT-PCR analysis of seat patch skin. AQP 1 was localized in the endothelium of subepidermal capillaries and serves as a pathway for water absorption in series with the apical and basolateral membranes of the epithelium. Dehydrated toads rehydrated more rapidly from dilute NaCl solutions than from deionized water despite the reduced osmotic gradient. BCF of toads rehydrating on 50 mM NaCl was not different than on deionized water and blocking Na+ transport with 100 microM amiloride did not reduce water absorption from 50 mM NaCl. Thus, neither circulation nor solute coupling explains the greater absorption from dilute salt solutions. Rehydration from 10 mM CaCl2 was stimulated above that of DI water by a similar degree as with 50 mM NaCl suggesting the anion might control water permeability of the skin.

Chemosensory function of salt and water transport by the amphibian skin.

Solute and water transport mechanisms of anuran skin mediate chemosensory functions that permit evaluation of ionic and osmotic properties of hydration sources in a manner similar to taste receptors in the mammalian tongue. Histochemical observations demonstrated apparent connections between spinal nerve endings and epithelial cells of the skin and we used neural and behavioral responses as measures of coupling between transport and chemosensation. The inhibition of transcellular Na+ transport by amiloride partially reduced the neural response and the avoidance of hyperosmotic NaCl but not KCl solutions. Cetylpyridinium chloride (CPC) reduced the neural response to hyperosmotic salt solutions, suggesting a chemosensory role for vanilloid receptors in the skin. Avoidance of hyperosmotic salt solutions was reduced by impermeant anions suggesting paracellular conductance is important for chemosensation. The effects of blocking the transcellular and paracellular pathways was additive but did not eliminate the avoidance of osmotically unfavorable solutions by dehydrated toads. The timing of the neural response to deionized water was similar to the onset of water absorption behavior and increased blood flow to the pelvic skin. Water absorption from 50 mM NaCl was greater than from deionized water when toads were fully immersed, but not when contact was limited to the ventral surface.

Cardiovascular and behavioural changes during water absorption in toads, Bufo alvarius and Bufo marinus.

Blood cell flux (BCF) in the pelvic skin of Bufo marinus was lower than Bufo alvarius when toads rehydrated from deionised water (DI) or 50 mmol l-1 NaCl (NaCl). Despite the lower BCF in B. marinus, water absorption was not different between the species when toads rehydrated from DI or NaCl. When fluid contact was limited to the pelvic skin, water uptake from NaCl was lower than from DI, but became greater than uptake from DI as the immersion level increased. Hydrophobic beeswax coating the lateral sides reduced absorption from NaCl but not from DI. Toads settled into water absorption response posture well after maximal BCF was attained in both DI and NaCl, indicating that the behavioural response requires neural integration beyond the increase in BCF. Water exposure increased BCF in hydrated B. alvarius with empty bladders but not in those with stored bladder water. Hydrated B. marinus with an empty bladder did not increase BCF when given water. Handling stress depressed BCF but increased central arterial flow (CAF), measured using a flow probe around the dorsal aorta. In undisturbed toads, CAF increased with the same time course as BCF while heart rate remained relatively constant, suggesting redistribution of blood flow.

Transcellular and paracellular elements of salt chemosensation in toad skin.

Dehydrated toads absorb water by pressing a specialized (seat patch) area of the skin to moist surfaces. This behavior, the water absorption response (WR), is preceded by periods of more limited skin contact (seat patch down, SPD) in which the suitability of the rehydration source is evaluated. WR and SPD behaviors were suppressed on 250 mM NaCl and 200 mM KCl solutions. Ten micromolar amiloride partially restored SPD and WR on NaCl solutions. The addition of 5 mM La(3+) also partially restored the initiation of WR and this effect was additive to the effect of amiloride, suggesting transcellular and paracellular pathways exist in parallel. Similarly, 5 mM La(3+) partially restored the initiation of WR on KCl solutions, to levels comparable to those with K(+)gluconate, suggesting a paracellular pathway for detection of K(+). Hyperosmotic (250 mM) NaCl solutions bathing the mucosal surface rapidly and reversibly increased the paracellular conductance of isolated skin and this increase was partially inhibited by 5 mM La(3+). These results suggest that the regulation of tight junctions has a chemosensory role in toad skin.

Comparison of dehydration and angiotensin II-stimulated cutaneous drinking in toads, Bufo punctatus.

Toads (Bufo punctatus) use a sequence of two postures to place the ventral skin on a moist surface and absorb water osmotically. First, the skin contacts the surface (seat patch down, SPD), and then the hindlimbs are abducted to maximize skin contact area (water absorption response, WR). Toads modulated behavior in response to hydration status and osmotic content of the hydration source. Dehydrated toads placed on water displayed both SPD and WR. Hydrated toads injected with angiotensin II (AII) displayed SPD longer than Ringer-injected controls but did not initiate WR and absorbed less water than dehydrated toads. These results suggest that dehydration has a more robust dipsogenic effect than AII. Dehydrated toads placed on 250 mM NaCl briefly initiated SPD but not WR. The addition of amiloride to the hyperosmotic salt solution resulted in brief display of WR but no water loss. Hydrated toads placed on 250 mM NaCl showed shorter periods of SPD behavior. The combination of AII injection and amiloride addition to the salt solution increased SPD initiation but SPD duration was short and water loss was prevented. Neither AII nor dehydration overrides chemosensory mechanisms in the skin that suppress cutaneous drinking from hypertonic solutions.

A two-barrier compartment model for volume flow across amphibian skin.

The amphibian skin has long been used as a model tissue for the study of ion transport and osmotic water movement across tight epithelia. To understand the mechanism of water uptake across amphibian skin, we model the skin as a well-stirred compartment bounded by an apical barrier and a tissue barrier. The compartment represents the lateral intercellular space between cells in the stratum granulosum. The apical barrier represents the stratum corneum, the principal/mitochondria-rich cells, and the junctional area between cells. This barrier is hypothesized to have the ability to actively transport solutes through Na+-K+-ATPase. The actively transported solute flux is assumed to satisfy the Michaelis-Menten relationship. The tissue barrier represents a composite barrier comprising the stratum spinosum, the stratum germinativum, the basal lamina, and the dermis. Our model shows that 1) the predicted rehydration rates from apical bathing solutions are in good agreement with the experiment results in Hillyard and Larsen (J Comp Physiol 171: 283-292, 2001); 2) under their experimental conditions, there is a substantial volume flux coupled to the active solute flux and this coupled volume flux is nearly constant when the osmolality of the apical bathing solution is >100 mosmol/kgH2O; 3) the molar ratio of the actively transported solute flux to the coupled water flux is about 1:160, which is the same as that reported in Nielsen (J Membr Biol 159: 61-69, 1997).

Basolateral Cl- channels in the larval bullfrog skin epithelium.

The addition of 150 U/ml nystatin to the mucosal surface of isolated skin from larval bullfrogs increases apical membrane permeability and allows a voltage clamp to be applied to the basolateral membrane. With identical Ringer's solutions bathing either side of the tissue the short-circuit current (I(SC)) averaged 7.60+/-0.78 micro A/cm2, and this current could be increased or decreased by imposing a Cl- concentration gradient. Fluctuation analysis of the I(SC) gave power spectra that could be fit with low- and high-frequency Lorentzian functions having corner frequencies of 1.48+/-0.06 Hz and 48.5+/-11.4 Hz, respectively. The Lorentzian plateau was minimal at the lowest I(SC) and increased as the I(SC) became greater in the positive or negative direction. Current-voltage plots with identical Ringer's on either side of the tissue showed a pattern of outward rectification. Cell attached patches of cells isolated from the skin with collagenase-trypsin treatment showed spontaneous channel activity with a conductance of 20.9 pS at a pipette potential, -Vp=20 mV. Current-voltage plots of single channels showed a similar pattern of rectification to that of the intact skin, and partial replacement of Cl- by gluconate in the pipette solution shifted the reversal potential from zero to about 40 mV, which is close to the expected shift of the reversal potential of the chloride current through a Cl- selective ion channel. These results suggest that the basolateral Cl- conductance of the larval skin is mediated by a channel with properties that resemble a volume-sensing outward-rectifier anion channel that has been described in a variety of cell types

Electrophysiological properties of the tongue epithelium of the toad Bufo marinus.

The dorsal lingual epithelium from the tongue of the toad Bufo marinus was mounted in an Ussing-type chamber, and the short-circuit current (I(sc)) was measured using a low-noise voltage clamp. With NaCl Ringer bathing the mucosal and serosal surfaces of the isolated tissue, an outwardly directed (mucosa-positive) I(sc) was measured that averaged -10.71+/-0.82 microA cm(-2) (mean +/- S.E.M., N=24) with a resistance of 615+/-152 Omega cm(2) (mean +/- S.E.M., N=10). Substitution of chloride with sulfate as the anion produced no significant change in I(sc). Fluctuation analysis with either NaCl or Na(2)SO(4) Ringer bathing both sides of the tissue revealed a spontaneous Lorentzian component, suggesting that the I(sc) was the result of K(+) secretion through spontaneously fluctuating channels in the apical membrane of the epithelium. This hypothesis was supported by the reversible inhibition of I(sc) by Ba(2+) added to the mucosal Ringer. Analysis of the kinetics of Ba(2+) inhibition of I(sc) indicates that there might be more than one type of K(+) channel carrying the I(sc). This hypothesis was supported by power spectra obtained with a serosa-to-mucosa K(+) gradient, which could be fitted to two Lorentzian components. At present, the K(+) secretory current cannot be localized to taste cells or other cells that might be associated with the secretion of saliva or mucus. Nonetheless, the resulting increase in [K(+)] in fluid bathing the mucosal surface of the tongue could presumably affect the sensitivity of the taste cells. These results contrast with those from the mammalian tongue, in which a mucosa-negative I(sc) results from amiloride-sensitive Na(+) transport.