A multi-tasking stomach: functional coexistence of acid-peptic digestion and defensive body inflation in three distantly related vertebrate lineages.

Puffer and porcupine fishes (families Diodontidae and Tetraodontidae, order Tetradontiformes) are known for their extraordinary ability to triple their body size by swallowing and retaining large amounts of seawater in their accommodating stomachs. This inflation mechanism provides a defence to predation; however, it is associated with the secondary loss of the stomach's digestive function. Ingestion of alkaline seawater during inflation would make acidification inefficient (a potential driver for the loss of gastric digestion), paralleled by the loss of acid-peptic genes. We tested the hypothesis of stomach inflation as a driver for the convergent evolution of stomach loss by investigating the gastric phenotype and genotype of four distantly related stomach inflating gnathostomes: sargassum fish, swellshark, bearded goby and the pygmy leatherjacket. Strikingly, unlike in the puffer/porcupine fishes, we found no evidence for the loss of stomach function in sargassum fish, swellshark and bearded goby. Only the pygmy leatherjacket (Monochanthidae, Tetraodontiformes) lacked the gastric phenotype and genotype. In conclusion, ingestion of seawater for inflation, associated with loss of gastric acid secretion, is restricted to the Tetraodontiformes and is not a selective pressure for gastric loss in other reported gastric inflating fishes.

Critical thermal maxima of early life stages of three tropical fishes: Effects of rearing temperature and experimental heating rate.

Marine ectotherms are often sensitive to thermal stress, and certain life stages can be particularly vulnerable (e.g., larvae or spawners). In this study, we investigated the critical thermal maxima (CTmax) of larval and early juvenile life stages of three tropical marine fishes (Acanthochromis polyacanthus, Amphiprion melanopus, and Lates calcarifer). We tested for potential effects of developmental acclimation, life stage, and experimental heating rates, and we measured metabolic enzyme activities from aerobic (citrate synthase, CS) and anaerobic pathways (lactate dehydrogenase, LDH). A slightly elevated rearing temperature neither influenced CTmax nor CS activity, which otherwise could have indicated thermal acclimation. However, we found CTmax to either remain stable (Acanthrochromis polyacanthus) or increase with body mass during early ontogeny (Amphiprion melanopus and Lates calcarifer). In all three species, faster heating rates lead to higher CTmax. Acute temperature stress did not change CS or LDH activities, suggesting that overall aerobic and anaerobic metabolism remained stable. Lates calcarifer, a catadromous species that migrates from oceanic to riverine habitats upon metamorphosis, had higher CTmax than the two coral reef fish species. We highlight that, for obtaining conservative estimates of a fish species' upper thermal limits, several developmental stages and body mass ranges should be examined. Moreover, upper thermal limits should be assessed using standardized heating rates. This will not only benefit comparative approaches but also aid in assessing geographic (re-) distributions and climate change sensitivity of marine fishes.

Impacts of increased ocean temperatures on a low-latitude coral reef fish - Processes related to oxygen uptake and delivery.

Increasing temperatures are expected to significantly affect the physiological performance of ectotherms, particularly in tropical locations. The shape of an organism's thermal reaction norm can provide important information on its capacity to persist under climate change scenarios; however, difficulty lies in choosing a measurable trait that best depicts physiological performance. This study investigated the effects of elevated temperatures on processes related to oxygen uptake and delivery, including oxygen consumption, haematology, and tissue health for a low-latitude population of coral reef damselfish. Acanthochromis polyacanthus were collected from the Torres Strait (10°31-46'S, 142°20-35'E) and maintained at current average ocean temperatures (+0 °C; seasonally cycling), + 1.5 °C and + 3 °C higher than present day temperatures for 10 months. Aerobic performance indicated a limit to metabolic function at + 3 °C (33 °C), following an increase in aerobic capacity at + 1.5 °C (31.5 °C). Neither haematological parameters nor gill morphology showed the same improvement in performance at + 1.5 °C. Gill histopathology provided the first indicator of a decline in organism health, which corresponded with mortality observations from previous research. Findings from this study suggest thermal specialisation in this low-latitude population as well as variation in thermal sensitivity, depending on the physiological trait.

Altered brain ion gradients following compensation for elevated CO2 are linked to behavioural alterations in a coral reef fish.

Neurosensory and behavioural disruptions are some of the most consistently reported responses upon exposure to ocean acidification-relevant CO2 levels, especially in coral reef fishes. The underlying cause of these disruptions is thought to be altered current across the GABAA receptor in neuronal cells due to changes in ion gradients (HCO3(-) and/or Cl(-)) that occur in the body following compensation for elevated ambient CO2. Despite these widely-documented behavioural disruptions, the present study is the first to pair a behavioural assay with measurements of relevant intracellular and extracellular acid-base parameters in a coral reef fish exposed to elevated CO2. Spiny damselfish (Acanthochromis polyacanthus) exposed to 1900 µatm CO2 for 4 days exhibited significantly increased intracellular and extracellular HCO3(-) concentrations and elevated brain pHi compared to control fish, providing evidence of CO2 compensation. As expected, high CO2 exposed damselfish spent significantly more time in a chemical alarm cue (CAC) than control fish, supporting a potential link between behavioural disruption and CO2 compensation. Using HCO3(-) measurements from the damselfish, the reversal potential for GABAA (EGABA) was calculated, illustrating that biophysical properties of the brain during CO2 compensation could change GABAA receptor function and account for the behavioural disturbances noted during exposure to elevated CO2.

Validation of the i-STAT system for the analysis of blood gases and acid-base status in juvenile sandbar shark (Carcharhinus plumbeus).

Accurate measurements of blood gases and acid-base status require an array of sophisticated laboratory equipment that is typically not available during field research; such is the case for many studies on the stress physiology, ecology and conservation of elasmobranch fish species. Consequently, researchers have adopted portable clinical analysers that were developed for the analysis of human blood characteristics, but often without thoroughly validating these systems for their use on fish. The aim of our study was to test the suitability of the i-STAT system, the most commonly used portable clinical analyser in studies on fish, for analysing blood gases and acid-base status in elasmobranchs, over a broad range of conditions and using the sandbar shark (Carcharhinus plumbeus) as a model organism. Our results indicate that the i-STAT system can generate useful measurements of whole blood pH, and the use of appropriate correction factors may increase the accuracy of results. The i-STAT system was, however, unable to generate reliable results for measurements of partial pressure of oxygen (PO2) and the derived parameter of haemoglobin O2 saturation. This is probably due to the effect of a closed-system temperature change on PO2 within the i-STAT cartridge and the fact that the temperature correction algorithms used by i-STAT assume a human temperature dependency of haemoglobin-O2 binding; in many ectotherms, this assumption will lead to equivocal i-STAT PO2 results. The in vivo partial pressure of CO2 (PCO2) in resting sandbar sharks is probably below the detection limit for PCO2 in the i-STAT system, and the measurement of higher PCO2 tensions was associated with a large measurement error. In agreement with previous work, our results indicate that the i-STAT system can generate useful data on whole blood pH in fishes, but not blood gases.

Alterations in gill structure in tropical reef fishes as a result of elevated temperatures.

Tropical regions are expected to be some of the most affected by rising sea surface temperatures (SSTs) because seasonal temperature variations are minimal. As temperatures rise, less oxygen dissolves in water, but metabolic requirements of fish and thus, the demand for effective oxygen uptake, increase. Gill remodelling is an acclimation strategy well documented in freshwater cyprinids experiencing large seasonal variations in temperature and oxygen as well as an amphibious killifish upon air exposure. However, no study has investigated whether tropical reef fishes remodel their gills to allow for increased oxygen demands at elevated temperatures. We tested for gill remodelling in five coral reef species (Acanthochromis polyacanthus, Chromis atripectoralis, Pomacentrus moluccensis, Dascyllus melanurus and Cheilodipterus quinquelineatus) from populations in northern Papua New Guinea (2° 35.765' S; 150° 46.193' E). Fishes were acclimated for 12-14 days to 29 and 31°C (representing their seasonal range) and 33 and 34°C to account for end-of-century predicted temperatures. We measured lamellar perimeter, cross-sectional area, base thickness, and length for five filaments on the 2nd gill arches and qualitatively assessed 3rd gill arches via scanning electron microscopy (SEM). All species exhibited significant differences in the quantitative measurements made on the lamellae, but no consistent trends with temperature were observed. SEM only revealed alterations in gill morphology in P. moluccensis. The overall lack of changes in gill morphology with increasing temperature suggests that these near-equatorial reef fishes may fail to maintain adequate O2 uptake under future climate scenarios unless other adaptive mechanisms are employed.

A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes.

Teleost fishes constitute 95% of extant aquatic vertebrates, and we suggest that this is related in part to their unique mode of tissue oxygenation. We propose the following sequence of events in the evolution of their oxygen delivery system. First, loss of plasma-accessible carbonic anhydrase (CA) in the gill and venous circulations slowed the Jacobs-Stewart cycle and the transfer of acid between the plasma and the red blood cells (RBCs). This ameliorated the effects of a generalised acidosis (associated with an increased capacity for burst swimming) on haemoglobin (Hb)-O2 binding. Because RBC pH was uncoupled from plasma pH, the importance of Hb as a buffer was reduced. The decrease in buffering was mediated by a reduction in the number of histidine residues on the Hb molecule and resulted in enhanced coupling of O2 and CO2 transfer through the RBCs. In the absence of plasma CA, nearly all plasma bicarbonate ultimately dehydrated to CO2 occurred via the RBCs, and chloride/bicarbonate exchange was the rate-limiting step in CO2 excretion. This pattern of CO2 excretion across the gills resulted in disequilibrium states for CO2 hydration/dehydration reactions and thus elevated arterial and venous plasma bicarbonate levels. Plasma-accessible CA embedded in arterial endothelia was retained, which eliminated the localized bicarbonate disequilibrium forming CO2 that then moved into the RBCs. Consequently, RBC pH decreased which, in conjunction with pH-sensitive Bohr/Root Hbs, elevated arterial oxygen tensions and thus enhanced tissue oxygenation. Counter-current arrangement of capillaries (retia) at the eye and later the swim bladder evolved along with the gas gland at the swim bladder. Both arrangements enhanced and magnified CO2 and acid production and, therefore, oxygen secretion to those specialised tissues. The evolution of ß-adrenergically stimulated RBC Na(+)/H(+) exchange protected gill O2 uptake during stress and further augmented plasma disequilibrium states for CO2 hydration/dehydration. Finally, RBC organophosphates (e.g. NTP) could be reduced during hypoxia to further increase Hb-O2 affinity without compromising tissue O2 delivery because high-affinity Hbs could still adequately deliver O2 to the tissues via Bohr/Root shifts. We suggest that the evolution of this unique mode of tissue O2 transfer evolved in the Triassic/Jurassic Period, when O2 levels were low, ultimately giving rise to the most extensive adaptive radiation of extant vertebrates, the teleost fishes.

Function and control of the fish secondary vascular system, a contrast to mammalian lymphatic systems.

Teleost fishes and mammalian lineages diverged 400 million years ago, and environmental requirements (water versus air) have resulted in marked differences in cardiovascular function between fish and mammals. Suggestions that the fish secondary vascular system (SVS) could be used as a model for the mammalian lymphatic system should be taken with caution. Despite molecular markers indicating similar genetic origin, functions of the SVS in teleost fish are probably different from those of the mammalian lymphatic system. We determined that, in resting glass catfish (Kryptopterus bicirrhis), plasma moves from the primary vascular system (PVS) to the SVS through small connecting vessels less than 10 µm in diameter, smaller than the red blood cells (RBCs). During and following hypoxia or exercise, flow increases and RBCs enter the SVS, possibly via ß-adrenoreceptor-mediated dilation of the connecting vessels. The volume of the SVS can be large and, as RBCs flow into the SVS, the haematocrit of the PVS falls by as much as 50% of the resting value. Possible functions of the SVS, including skin respiration, ionic and osmotic buffering, and reductions in heart work and RBC turnover, are discussed.

Reduced and reversed temperature dependence of blood oxygenation in an ectothermic scombrid fish: implications for the evolution of regional heterothermy?

Tunas (family Scombridae) are exceptional among most teleost fishes in that they possess vascular heat exchangers which allow heat retention in specific regions of the body (termed 'regional heterothermy'). Seemingly exclusive to heterothermic fishes is a markedly reduced temperature dependence of blood-oxygen (blood-O(2)) binding, or even a reversed temperature dependence where increasing temperature increases blood-O(2) affinity. These unusual binding properties have been documented in whole blood and in haemoglobin (Hb) solutions, and they are hypothesised to prevent oxygen loss from arteries to veins within the vascular heat exchangers and/or to prevent excessive oxygen unloading to the warm tissues and ensure an adequate supply of oxygen to tissues positioned efferent to the heat exchangers. The temperature sensitivity of blood-O(2) binding has not been characterised in an ectothermic scombrid (mackerels and bonitos), but the existence of the unusual binding properties in these fishes would have clear implications for their proposed association with regional heterothermy. Accordingly, the present study examined oxygenation of whole blood of the chub mackerel (Scomber japonicus) at 10, 20 and 30 degrees C and at 0.5, 1 and 2% CO(2). Oxygen affinity was generally highest at 20 degrees C for all levels of CO(2). Temperature-independent binding was observed at low (0.5%) CO(2), where the PO(2) at 50% blood-O(2) saturation (P (50)) was not statistically different at 10 and 30 degrees C (2.58 vs. 2.78 kPa, respectively) with an apparent heat of oxygenation (H degrees ) close to zero (-6 kJ mol(-1)). The most significant temperature-mediated difference occurred at high (2%) CO(2), where the P (50) at 10 degrees C was twofold higher than that at 20 degrees C with a corresponding H degrees of +43 kJ mol(-1). These results provide clear evidence of independent and reversed open-system temperature effects on blood oxygenation in S. japonicus, and it is therefore speculated that these unusual blood-O(2) binding characteristics may have preceded the evolution of vascular heat exchangers and regional heterothermy in fishes.

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss).

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.