Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology最新文献

Temperature and mineralization are among the most important environmental factors affecting all processes of aquatic ecosystems, including geographical distribution of water animals. Previously we showed that a brackish water population of Gammarus lacustris, a widespread amphipod, demonstrates substantially higher thermotolerance than a freshwater population. A possible reason for this difference is the fact that brackish water conditions are closer to internal media mineralization. Here we aimed to test this hypothesis and relate the observed effects in animal survival under the heat shock to the status of cellular defence systems. We acclimated four groups of amphipods from the same freshwater population to 0.5 ‰ and 15 ‰ at the temperatures of 6°С or 15°С. Acclimation at 6°С, but not at 15°С, to 15 ‰ significantly increased resistance of the amphipods to heat shock at 30°C. At 6°С activities of antioxidant enzymes and levels of the lipid peroxidation products in G. lacustris did not react to the increase in mineralization and the heat shock, while the level of HSP70 elevated two-fold in amphipods acclimated to mineralization of 15 ‰ compared to animals acclimated to 0.5 ‰. Thus, the observed increase in thermotolerance could be explained by the higher initial level of HSP70 and potentially other heat shock proteins caused by a less energy-demanding, more isotonic level.

In terrestrial vertebrates, the outermost layer of the skin, the stratum corneum (SC), provides a durable and flexible interface with the environment and is comprised of corneocytes embedded in lipids. However, the morphology and lipid composition of the SC varies throughout evolutionary history. Because crocodilians and birds phylogenetically bracket the Archosaurian clade, lipid composition in crocodilian SC may be compared with that of birds and other vertebrates to make inferences about broader phylogenetic patterns within Archosaurs while highlighting adaptations in vertebrate skin. We identified and quantified lipid classes in the SC of the American Alligator (Alligator mississippiensis) from three skin regions varying in mobility. Our results find similarities in lipid composition between alligator and avian SC, including a high percentage of cerebrosides, a polar lipid previously found only in the SC of birds and bats. Furthermore, polar lipids were more abundant in the most mobile region of the SC. Because polar lipids bind with water to increase skin hydration and therefore its pliability under physical stress, we hypothesize that selection for lipids in Archosaurian SC was driven by the unique distribution of proteins in the SC of this clade, and cerebrosides may have served as pre-adaptations for flight.

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister – an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH₄⁺ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na⁺/K⁺-ATPase detection was observed. The V-type H⁺-ATPase – a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

Male Japanese quails (Coturnix japonica) have been found to exhibit a three-phase metabolic change when subjected to prolonged fasting, during which basal thermogenesis is significantly reduced. A study had shown that there is a significant difference in the body temperature between male and female Japanese quails. However, whether female Japanese quails also show the same characteristic three-phase metabolic change during prolonged fasting and the underlying thermogenesis mechanisms associated with such changes are still unclear. In this study, female Japanese quails were subjected to prolonged starvation, and the body mass, basal metabolic rate (BMR), body temperature, mass of tissues and organs, body fat content, the state-4 respiration (S4R) and cytochrome c oxidase (CCO) activity in the muscle and liver of these birds were measured to determine the status of metabolic changes triggered by the starvation. In addition, the levels of glucose, triglyceride (TG) and uric acid, and thyroid hormones (T₃ and T₄) in the serum and the mRNA levels of myostatin (MSTN) and avian uncoupling protein (av-UCP) in the muscle were also measured. The results revealed the existence of a three-phase stage similar to that found in male Japanese quails undergoing prolonged starvation. Fasting resulted in significantly lower body mass, BMR, body temperature, tissues masses and most organs masses, as well as S4R and CCO activity in the muscle and liver. The mRNA level of av-UCP decreased during fasting, while that of MSTN increased but only during Phase I and II and decreased significantly during Phase III. Fasting also significantly lowered the T₃ level and the ratio of T₃/T₄ in the serum. These results indicated that female Japanese quails showed an adaptive response in basal thermogenesis at multiple hierarchical levels, from organismal to biochemical, enzyme and cellular level, gene and endocrine levels and this integrated adjustment could be a part of the adaptation used by female quails to survive long-term fasting.

Euryhaline organisms can accumulate organic osmolytes to maintain osmotic balance between their internal and external environments. Proline is a pivotal organic small molecule and plays an important role in osmoregulation that enables marine shellfish to tolerate high-salinity conditions. During high-salinity challenge, NAD kinase (NADK) is involved in de novo synthesis of NADP(H) in living organisms, which serves as a reducing agent for the biosynthetic reactions. However, the role of shellfish NADK in proline biosynthesis remains elusive. In this study, we show the modulation of NADK on proline synthesis in the razor clam (Sinonovacula constricta) in response to osmotic stress. Under acute hypersaline conditions, gill tissues exhibited a significant increase in the expression of ScNADK. To elucidate the role of ScNADK in proline biosynthesis, we performed dsRNA interference in the expression of ScNADK in gill tissues to assess proline content and the expression levels of key enzyme genes involved in proline biosynthesis. The results indicate that the knock-down of ScNADK led to a significant decrease in proline content (P<0.01), as well as the expression levels of two proline synthetase genes P5CS and P5CR involved in the glutamate pathway. Razor clams preferred to use ornithine as substrate for proline synthesis when the glutamate pathway is blocked. Exogenous administration of proline greatly improved cell viability and mitigated cell apoptosis in gills. In conclusion, our results demonstrate the important role of ScNADK in augmenting proline production under high-salinity stress, by which the razor clam is able to accommodate salinity variations in the ecological niche.

Kisspeptin is a multifunctional neurohormone, primarily involved in the regulation of reproduction. We tested whether peripheral administration of kisspeptin10 (KP-10) via intraperitoneal injection or slow release affects reproductive hormones and metabolites in Sterlet sturgeon (Acipenser ruthenus). Plasma and mucus 17β-estradiol (E₂), and testosterone (T), plasma and follicular vitellogenin (VTG) and calcium (Ca) as well as glucose and lipids were determined. Mature Sterlet sturgeon were grouped into six groups: saline i.p injection (control), human kisspeptin (hKP-10) i.p injection; acipenser kisspeptin (aKP-10) i.p injection; hKP-10 (slow release); aKP-10 (slow-release) and no treatment control. No effect for KP-10 on sturgeon body weight was found after 4 weeks of treatment. Multivariate analysis revealed a significant disparity in plasma E₂ levels. It was significantly different between groups (time, P = 0.0022). E₂ in epithelia mucosa showed significant difference between and within groups in the acute group (time, P = 0.0252; treatment, P = 0.0423; time × treatment, P = 0.0429). T levels were unaffected by treatments (P > 0.05). The presence of synthetic aKP-10 led to an elevation in oocyte and plasma VTG levels (P < 0.05). Prolonged exposure to this peptide resulted in an increase in plasma calcium levels. Simultaneously, there was an augmentation in the number of mature follicles. Regardless of the duration of exposure, aKP-10 significantly elevated plasma glucose levels in Sterlet (P < 0.0). Additionally, KP-10 led to an increase in plasma lipids and cholesterol in Sterlet. Overall, our data support an involvement for KP-10 in the regulation of gonadal steroid hormones, oocyte maturation and metabolite levels in sturgeon, suggesting a positive role for this peptide in the reproductive physiology of this species.

Vertebrates elevate heart rate when metabolism increases during digestion. Part of this tachycardia is due to a non-adrenergic-non-cholinergic (NANC) stimulation of the cardiac pacemaker, and it has been suggested these NANC factors are circulating hormones that are released from either gastrointestinal or endocrine glands. The NANC stimulation is particularly pronounced in species with large metabolic responses to digestion, such as reptiles. To investigate the possibility that the pancreas may release hormones that exert positive chronotropic effects on the digesting Burmese python heart, a species with very large postprandial changes in heart rate and oxygen uptake, we evaluate how pancreatectomy affects postprandial heart rate before and after autonomic blockade of the muscarinic and the beta-adrenergic receptors. We also measured the rates of oxygen consumption and evaluated the short-term control of the heart using the spectral analysis of heart rate variability and the baroreflex sequence method. Digestion caused the ubiquitous tachycardia, but the intrinsic heart rate (revealed after the combination of atropine and propranolol) was not affected by pancreatectomy and therefore hormones, such as glucagon and insulin, do not appear to contribute to the regulation of heart rate during digestion in Burmese pythons.

Chemical cues play important roles in mediating ecological interactions. Oxylipins, oxygenated metabolites of fatty acids, are one signalling molecule type that influences the physiology and function of species, suggesting their broader significance in chemical communication within aquatic systems. Yet, our current understanding of their function is restricted taxonomically and contextually making it difficult to infer their ecological significance. Snails and leeches are ubiquitous in freshwater ecosystems worldwide, yet little is known about their oxylipin profiles and the factors that cause their profiles to change. As snails and leeches differ taxonomically and represent different trophic groups, we postulated oxylipin profile differences. For snails, we hypothesized that ontogeny (non-reproductive vs reproductive) and predation (non-infested vs leech-infested) would affect oxylipin profiles. Oxylipins were characterized from water conditioned with the snail Planorbella duryi and leech Helobdella lineata, and included three treatment types (snails, leeches, and leech-infested snails) with the snails consisting of three size classes: small (5–6 mm, non-reproductive) and medium and large (13–14 and 19–20 mm, reproductive). The two species differed in the composition of their oxylipin profiles both in diversity and amounts. Further, ontogeny and predation affected the diversity of oxylipins emitted by snails. Our experimental profiles of oxylipins show that chemical cues within freshwater systems vary depending upon the species emitting the signals, the developmental stage of the species, as well as from ecological interactions such as predation. We also identified some candidates, like 9-HETE and PGE₂, that could be explored more directly for their physiological and ecological roles in freshwater systems.

The red-leg syndrome in amphibians is a condition commonly associated with the bacteria Aeromonas hydrophila and has led to population declines. However, there is little information concerning the inflammatory assemblage in infected anurans. We evaluated immune and endocrine alterations induced by stimulation with heat-killed A. hydrophila injected in Rhinella diptycha toads. Control animals were not manipulated, while the others were separated into groups that received intraperitoneal injection of 300 μl of saline or heat-killed bacteria: groups A1 (3 × 10⁷ cells), A2 (3 × 10⁸ cells), and A3 (3 × 10⁹ cells). Animals were bled and euthanized six hours post-injection. We evaluated neutrophil: lymphocyte ratio (NLR), plasma bacterial killing ability (BKA), testosterone (T), melatonin (MEL), and corticosterone (CORT) plasma levels. Heat-killed A. hydrophila increased CORT and NLR, and decreased MEL, especially at higher concentrations. There was no effect of treatment on T and BKA. We then selected the saline and A3 groups to conduct mRNA expression of several genes including glucocorticoid receptor (GR), toll-like receptor-4 (TLR-4), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-6, and IL-10. We found higher expression of IL-6, IL-1β, IL-10, and IFN-γ in group A3 compared to the saline group. These results indicate the beginning of an inflammatory assemblage, notably at the two highest concentrations of bacteria, and give a better understanding of how anurans respond to an infection within an integrated perspective, evaluating different physiological aspects. Future studies should investigate later phases of the immune response to elucidate more about the inflammation in amphibians challenged with A. hydrophila.

In a recent mechanistic study, octopamine was shown to promote proton transport over the branchial epithelium in green crabs, Carcinus maenas. Here, we follow up on this finding by investigating the involvement of octopamine in an environmental and physiological context that challenges acid-base homeostasis, the response to short-term high pCO₂ exposure (400 Pa) in a brackish water environment. We show that hyperregulating green crabs experienced a respiratory acidosis as early as 6 h of exposure to hypercapnia, with a rise in hemolymph pCO₂ accompanied by a simultaneous drop of hemolymph pH. The slightly delayed increase in hemolymph HCO₃⁻ observed after 24 h helped to restore hemolymph pH to initial values by 48 h. Circulating levels of the biogenic amine octopamine were significantly higher in short-term high pCO₂ exposed crabs compared to control crabs after 48 h. Whole animal metabolic rates, intracellular levels of octopamine and cAMP, as well as branchial mitochondrial enzyme activities for complex I + III and citrate synthase were unchanged in posterior gill #7 after 48 h of hypercapnia. However, application of octopamine in gill respirometry experiments suppressed branchial metabolic rate in posterior gills of short-term high pCO₂ exposed animals. Furthermore, branchial enzyme activity of cytochrome C oxidase decreased in high pCO₂ exposed crabs after 48 h. Our results indicate that hyperregulating green crabs are capable of quickly counteracting a hypercapnia-induced respiratory acidosis. The role of octopamine in the acclimation of green crabs to short-term hypercapnia seems to entail the alteration of branchial metabolic pathways, possibly targeting mitochondrial cytochrome C in the gill. Our findings help advancing our current limited understanding of endocrine components in hypercapnia acclimation.

Summary statement

Acid-base compensation upon short-term high pCO₂ exposure in hyperregulating green crabs started after 6 h and was accomplished by 48 h with the involvement of the biogenic amine octopamine, accumulation of hemolymph HCO₃⁻, and regulation of mitochondrial complex IV (cytochrome C oxidase).