ABSTRACT
Calcium carbonate minerals produced by marine organisms play a central role in the global carbon cycle and carbonate sedimentation, which influence the climate by regulating atmospheric CO2 levels. Foraminifera are important marine single-celled organisms that have produced calcite shells for over 300 million years. Here, we present new observations promoting our understanding for foraminiferal biocalcification by studying Amphistegina lessonii. We integrated in vivo confocal autofluorescence and dye fluorescence imaging with elemental analysis of the cell supporting the concept that the calcite shells of foraminifera are produced via deposition of intracellularly formed Mg-rich amorphous calcium carbonate (Mg-ACC) particles that transform into a stable mineral phase. This process is likely accompanied by the activity of endosymbiotic microalgae and seawater-derived endocytic vesicles that provide calcification substrates such as DIC, Ca2+, and Mg2+. The final transformation of semi-liquid amorphous nanoparticles into a crystalline shell was associated with Mg2+ liberation.
ABSTRACT
The close association between animals and their associated microbiota is usually beneficial for both partners. Here, we used a simple marine model invertebrate, the flatworm Macrostomum lignano, to characterize the host-microbiota interaction in detail. This analysis revealed that the different developmental stages each harbor a specific microbiota. Studies with gnotobiotic animals clarified the physiological significance of the microbiota. While no fitness benefits were mediated by the microbiota when food was freely available, animals with microbiota showed significantly increased fitness with a reduced food supply. The microbiota of M. lignano shows circadian rhythmicity, affecting both the total bacterial load and the behavior of specific taxa. Moreover, the presence of the worm influences the composition of the bacterial consortia in the environment. In summary, the Macrostomum-microbiota system described here can serve as a general model for host-microbe interactions in marine invertebrates.
Subject(s)
Microbiota , Platyhelminths , Animals , Platyhelminths/physiology , Regeneration/physiology , PeriodicityABSTRACT
The global degradation of coral reefs is steadily increasing with ongoing climate change. Yet coral larvae settlement, a key mechanism of coral population rejuvenation and recovery, is largely understudied. Here, we show how the lipophilic, settlement-inducing bacterial pigment cycloprodigiosin (CYPRO) is actively harvested and subsequently enriched along the ectoderm of larvae of the scleractinian coral Leptastrea purpura. A light-dependent reaction transforms the CYPRO molecules through photolytic decomposition and provides a constant supply of hydrogen peroxide (H2O2), leading to attachment on the substrate and metamorphosis into a coral recruit. Micromolar concentrations of H2O2 in seawater also resulted in rapid metamorphosis, but without prior larval attachment. We propose that the morphogen CYPRO is responsible for initiating attachment while simultaneously acting as a molecular generator for the comprehensive metamorphosis of pelagic larvae. Ultimately, our approach opens a novel mechanistic dimension to the study of chemical signaling in coral settlement and provides unprecedented insights into the role of infochemicals in cross-kingdom interactions.
Subject(s)
Acceptance and Commitment Therapy , Anthozoa , Animals , Photolysis , Hydrogen Peroxide , Coral Reefs , LarvaABSTRACT
Many invertebrate species inhabit coastal areas where loads of plastic debris and microplastics are high. In the current case study, we exemplarily illustrate the principal processes taking place in the Atlantic ditch shrimp, Palaemon varians, upon ingestion of microplastics. In the laboratory, shrimp readily ingested fluorescent polystyrene microbeads of 0.1-9.9 µm, which could be tracked within the widely translucent body. Ingested food items as well as micro-particles cumulate in the stomach where they are macerated and mixed with digestive enzymes. Inside the stomach, ingested particles are segregated by size by a complex fine-meshed filter system. Liquids and some of the smallest particles (0.1 µm) pass the filter and enter the midgut gland where resorption of nutrients as well as synthesis and release of digestive enzymes take place. Large particles and most of the small particles are egested with the feces through the hindgut. Small particles, which enter the midgut gland, may interact with the epithelial cells and induce oxidative stress, as indicated by elevated activities of superoxide dismutase and cellular markers of reactive oxygen species. The shrimp indiscriminately ingest microparticles but possess efficient mechanisms to protect their organs from overloading with microplastics and other indigestible particles. These include an efficient sorting mechanism within the stomach and the protection of the midgut gland by the pyloric filter. Formation of detrimental radical oxygen species is counteracted by the induction of enzymatic antioxidants.
ABSTRACT
Harmful effects caused by the exposure to paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) on bivalves are frequently difficult to attribute to one or the other compound group. We evaluate and compare the distinct effects of PSTs extracted from Alexandrium catenella (Alex5) cells and extracellular lytic compounds (LCs) produced by A. tamarense (NX-57-08) on Mytilus edulis hemocytes. We used a 4 h dose-response in vitro approach and analyzed how these effects correlate with those observed in a previous in vivo feeding assay. Both bioactive compounds caused moderated cell death (10-15%), being dose-dependent for PST-exposed hemocytes. PSTs stimulated phagocytic activity at low doses, with a moderate incidence in lysosomal damage (30-50%) at all tested doses. LCs caused a dose-dependent impairment of phagocytic activity (up to 80%) and damage to lysosomal membranes (up to 90%). PSTs and LCs suppressed cellular ROS production and scavenged H2O2 in in vitro assays. Neither PSTs nor LCs affected the mitochondrial membrane potential in hemocytes. In vitro effects of PST extracts on M. edulis hemocytes were consistent with our previous study on in vivo exposure to PST-producing algae, while for LCs, in vivo and in vitro results were not as consistent.
Subject(s)
Hemocytes/drug effects , Marine Toxins/toxicity , Mytilus edulis , Animals , Cell Survival/drug effects , Dinoflagellida , Hemocytes/metabolism , Hemocytes/physiology , Lysosomes/drug effects , Membrane Potential, Mitochondrial/drug effects , Phagocytosis/drug effects , Reactive Oxygen Species/metabolism , Shellfish PoisoningABSTRACT
Baltic mussels (Mytilus spp.) were exposed to the explosive trinitrotoluene (TNT) for 96 h (0.31-10.0 mg/L) and 21 d (0.31-2.5 mg/L). Bioaccumulation of TNT and its degradation products (2- and 4-ADNT) as well as biological effects ranging from the gene and cellular levels to behaviour were investigated. Although no mortality occurred in the concentration range tested, uptake and metabolism of TNT and responses in antioxidant enzymes and histochemical biomarkers were observed already at the lowest concentrations. The characteristic shell closure behaviour of bivalves at trigger concentrations led to complex exposure patterns and non-linear responses to the exposure concentrations. Conclusively, exposure to TNT exerts biomarker reponses in mussels already at 0.31 mg/L while effects are recorded also after a prolonged exposure although no mortality occurs. Finally, more attention should be paid on shell closure of bivalves in exposure studies since it plays a marked role in definining toxicity threshold levels.
Subject(s)
Mytilus , Trinitrotoluene , Water Pollutants, Chemical , Animals , Baltic States , Biomarkers , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/toxicityABSTRACT
Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from Alexandrium ostenfeldii on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca]i). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca]i mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca]i, but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca]i when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.
Subject(s)
Calcium/metabolism , Heterocyclic Compounds, 4 or More Rings/pharmacology , Imines/pharmacology , Receptors, Muscarinic/metabolism , Receptors, Nicotinic/metabolism , Spiro Compounds/pharmacology , Animals , Calcium Channels/metabolism , Calcium Signaling/drug effects , Cell Line , Dinoflagellida/metabolism , Heterocyclic Compounds, 4 or More Rings/isolation & purification , Imines/isolation & purification , Marine Toxins/isolation & purification , Marine Toxins/pharmacology , Muscarinic Antagonists , Nicotinic Agonists , PC12 Cells , Rats , Spiro Compounds/isolation & purificationABSTRACT
Fluorescent natural compounds have been identified in several marine hosts of microalgae. Their prevalence, and the energy the host is expending on their synthesis, suggests an important, yet poorly understood ecological role. It has been suggested that some of these natural products may enhance the photosynthesis of microbial symbionts. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on the photosynthesis of nine microalgal species and strains was examined. The data showed that the variety of effects of Ag A additions differed between species, and even strains within a species. While in one strain of Synechococcus sp., the presence of Ag A increased gross photosynthesis under UV light exposure, it decreased in another. And while in the chlorophyte T. chuii overall metabolic activity was greatly reduced under all forms of lighting, photosynthesis in T. lutea was positively affected by the addition of Ag A. The variety of effects of Ag A on photosynthesis observed in this study indicate a complex interaction of Ag A with microalgal cells and suggests that a host may be able to shape its own symbiotic microbiome with self-produced natural products.
Subject(s)
Agelas/microbiology , Microalgae/physiology , Photosynthesis/drug effects , Pyrroles/pharmacology , Symbiosis/physiology , Animals , Microalgae/classification , Pyrroles/metabolism , Symbiosis/drug effectsABSTRACT
In several marine hosts of microalgae, fluorescent natural products may play an important role. While the ecological function of these compounds is not well understood, an interaction of these molecules with the photosynthesis of the symbionts has been suggested. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on microalgal fluorescence was examined. The spectra showed an accumulation of Ag A within the cells, but with variable impacts on fluorescence. While in two Synechococcus strains, fluorescence of phycoerythrin increased significantly, the fluorescence of other Synechococcus strains was not affected. In four out of the five eukaryote species examined, chlorophyll a (Chl a) fluorescence intensity was modulated. In Tisochrysis lutea, for example, the position of the fluorescence emission maximum of Chl a was shifted. The variety of these effects of Ag A on microalgal fluorescence suggests that fluorophores derived from animals could play a crucial role in shaping the composition of marine host/symbiont systems.
Subject(s)
Agelas/chemistry , Microalgae/drug effects , Pyrroles/pharmacology , Symbiosis , Animals , Chlorophyll A/chemistry , Fluorescence , Micrasterias/drug effects , Micrasterias/metabolism , Microalgae/metabolism , Photosynthesis/drug effects , Photosynthesis/radiation effects , Phycobilisomes/chemistry , Phycobilisomes/drug effects , Phycoerythrin/chemistry , Pigments, Biological/chemistry , Pyrroles/isolation & purification , Species Specificity , Spectrometry, Fluorescence , Synechococcus/drug effects , Synechococcus/metabolism , Ultraviolet RaysABSTRACT
Fluorescence measurements of the marine flatworm Macrostomum lignano were performed during exposure to the explosive TNT and its main derivatives 2-ADNT and 4-ADNT, using calcein AM, the acetoxymethylester of calcein, and the autofluorescence of its food (diatoms). Lethality was found to depend on temperature and exposure time. After 12 days of exposure to a concentration of 33,3 mg/L 2-ADNT and 4-ADNT, the lethality at 30 °C (100%) was strongly increased compared to 21 °C (~60%). First deaths were observed after four days of exposure. Using lower concentrations (≤3,33 mg/L) of all three compounds, the activity of ABC transporters (ATP binding cassette transporter) was determined using calcein as reporter dye. Worms exposed to toxicants for 72 h showed a significant upregulation of ABC transporter activity during exposure to 3,33 mg/L 2-ADNT and 4-ADNT, and 3 mg/L TNT demonstrating the efficacy of this cellular first line defense. A distinct behavioral defense of the worms decreased the uptake of 2-ADNT and 4-ADNT (0,033 mg/L) as they reduced feeding shown by diminished autofluorescence of algae in the gut.
Subject(s)
Platyhelminths , Trinitrotoluene , Aniline Compounds , Animals , FluorescenceABSTRACT
The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-synthase, Na+/K+-ATPase, H+-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly hatched larvae. Treatment-related embryo mortality until hatching (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacity. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although this is probably associated with energetic trade-offs. Interestingly, whole-larvae enzyme activity (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.
Subject(s)
Gadus morhua , Seawater , Animals , Gills , Homeostasis , Hydrogen-Ion Concentration , TemperatureABSTRACT
The contamination of coastal regions with different toxicants, including heavy metal ions such as copper and cadmium jeopardize health and survival of organisms exposed to this habitat. To study the effects of high copper and cadmium concentrations in these marine environments, we used the flatworm Macrostomum lignano as a model. This platyhelminth lives in shallow coastal water and is exposed to high concentrations of all toxicants that accumulate in these sea floors. We could show that both, cadmium and copper show toxicity at higher concentrations, with copper being more toxic than cadmium. At concentrations below acute toxicity, a reduced long-term survival was observed for both metal ions. The effects of sublethal doses comprise reduced physical activities, an increase in ROS levels within the worms, and alterations of the mitochondrial biology. Moreover, cell death events were substantially increased in response to sublethal concentrations of both metal ions and stem cell activity was reduced following exposure to higher cadmium concentrations. Finally, the expression of several genes involved in xenobiotic metabolism was substantially altered by this intervention. Taken together, M. lignano has been identified as a suitable model for marine toxicological studies as it allows to quantify several relevant life-history traits as well as of physiological and behavioral read-outs.
Subject(s)
Cadmium/toxicity , Copper/toxicity , Oxidative Stress/drug effects , Platyhelminths/drug effects , Water Pollutants, Chemical/toxicity , Animals , Dose-Response Relationship, Drug , Platyhelminths/genetics , Platyhelminths/metabolism , Toxicity TestsABSTRACT
Dinoflagellates are microbial eukaryotes that have exceptionally large nuclear genomes; however, their organelle genomes are small and fragmented and contain fewer genes than those of other eukaryotes. The genus Amoebophrya (Syndiniales) comprises endoparasites with high genetic diversity that can infect other dinoflagellates, such as those forming harmful algal blooms (e.g., Alexandrium). We sequenced the genome (~100 Mb) of Amoebophrya ceratii to investigate the early evolution of genomic characters in dinoflagellates. The A. ceratii genome encodes almost all essential biosynthetic pathways for self-sustaining cellular metabolism, suggesting a limited dependency on its host. Although dinoflagellates are thought to have descended from a photosynthetic ancestor, A. ceratii appears to have completely lost its plastid and nearly all genes of plastid origin. Functional mitochondria persist in all life stages of A. ceratii, but we found no evidence for the presence of a mitochondrial genome. Instead, all mitochondrial proteins appear to be lost or encoded in the A. ceratii nucleus.
Subject(s)
Dinoflagellida/genetics , Dinoflagellida/metabolism , Genome, Mitochondrial , Mitochondria/physiology , Phylogeny , Aerobiosis , Cell Nucleus/genetics , Cluster Analysis , DNA, Complementary/metabolism , Evolution, Molecular , Gene Library , Genome , Likelihood Functions , Microscopy, Confocal , Sequence Analysis, DNAABSTRACT
This study is a proof of concept that the sponge derived pyrrole-imidazole alkaloid Ageladine A acts as an additional light harvesting molecule for photosynthesis of symbionts of marine sponges. The absorbance of Ageladine A is in the UV range and fluoresces blue, matching the blue absorbance of chlorophyll a. A joint modeling and experimental approach demonstrates that Ageladine A increases photosynthetic O2 production of Synechococcus bacillaris WH5701 (CCMP1333), when the cells are exposed to UV light, which is marginally used for photosynthesis. Due to the presence of Ageladine A, production of O2 increased 2.54 and 3.1-fold, in the experiments and the model, respectively.
Subject(s)
Photosynthesis , Porifera/metabolism , Pyrroles/chemistry , Synechococcus/metabolism , Animals , Oxygen/chemistry , Oxygen/metabolism , Photosynthesis/radiation effects , Photosystem II Protein Complex/metabolism , Symbiosis , Ultraviolet RaysABSTRACT
Although the emergence of complex biomineralized forms has been investigated for over a century, still little is known on how single cells control morphology of skeletal structures, such as frustules, shells, spicules, or scales. We have run experiments on the shell formation in foraminifera, unicellular, mainly marine organisms that can build shells by successive additions of chambers. We used live imaging to discover that all stages of chamber/shell formation are controlled by dedicated actin-driven pseudopodial structures. Successive reorganization of an F-actin meshwork, associated with microtubular structures, is actively involved in formation of protective envelope, followed by dynamic scaffolding of chamber morphology. Then lamellar dynamic templates create a confined space and control mineralization separated from seawater. These observations exclude extracellular calcification assumed in selected foraminiferal clades, and instead suggest a semiintracellular biomineralization pattern known from other unicellular calcifying and silicifying organisms. These results give a challenging prospect to decipher the vital effect on geochemical proxies applied to paleoceanographic reconstructions. They have further implications for understanding multiscale complexity of biomineralization and show a prospect for material science applications.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Biomineralization/physiology , Foraminifera/metabolism , Protozoan Proteins/metabolismABSTRACT
Multiple toxic and bioactive compounds produced by Alexandrium spp. cause adverse effects on bivalves, but these effects are frequently difficult to attribute to a single compound class. To disentangle the effect of neurotoxic vs lytic secondary metabolites, we exposed blue mussels to either a paralytic shellfish toxin (PST) producing Alexandrium spp. strain, or to an exclusively lytic compound (LC) producing strain, or a strain containing both compound classes, to evaluate the time dependent effects after 3 and 7 days of feeding. Tested parameters comprised signs of paralysis, feeding activity, and immune cell integrity (hemocyte numbers and viability; lysosomal membrane destabilization) and function (ROS production). Both compound classes caused paralysis and immune impairment. The only effect attributable exclusively to PST was increased phagocytic activity after 3 days and impaired feeding activity after 7 days, which curtailed toxin accumulation in digestive glands. Lysosomal membrane destabilization were more closely, but not exclusively, matched with LC exposure. Effects on circulating hemocyte integrity and immune related functions were mostly transient or remained stable within 7 days; except for increased lysosomal labialization and decreased extracellular ROS production when mussels were exposed to the toxin combination. M. edulis displays adaptive fitness traits to survive and maintain immune capacity upon prolonged exposure to environmentally relevant concentrations of PST and/or LC producing Alexandrium strains.
Subject(s)
Dinoflagellida/physiology , Hemocytes/drug effects , Marine Toxins/pharmacology , Mytilus edulis/drug effects , Animals , Avoidance Learning/drug effects , Feeding Behavior/drug effects , Hemocytes/metabolism , Mytilus edulis/physiologyABSTRACT
Thermal tolerance windows serve as a powerful tool for estimating the vulnerability of marine species and their life stages to increasing temperature means and extremes. However, it remains uncertain to which extent additional drivers, such as ocean acidification, modify organismal responses to temperature. This study investigated the effects of CO2 -driven ocean acidification on embryonic thermal sensitivity and performance in Atlantic cod, Gadus morhua, from the Kattegat. Fertilized eggs were exposed to factorial combinations of two PCO2 conditions (400 µatm vs. 1100 µatm) and five temperature treatments (0, 3, 6, 9 and 12 °C), which allow identifying both lower and upper thermal tolerance thresholds. We quantified hatching success, oxygen consumption (MO2 ) and mitochondrial functioning of embryos as well as larval morphometrics at hatch and the abundance of acid-base-relevant ionocytes on the yolk sac epithelium of newly hatched larvae. Hatching success was high under ambient spawning conditions (3-6 °C), but decreased towards both cold and warm temperature extremes. Elevated PCO2 caused a significant decrease in hatching success, particularly at cold (3 and 0 °C) and warm (12 °C) temperatures. Warming imposed limitations to MO2 and mitochondrial capacities. Elevated PCO2 stimulated MO2 at cold and intermediate temperatures, but exacerbated warming-induced constraints on MO2 , indicating a synergistic interaction with temperature. Mitochondrial functioning was not affected by PCO2 . Increased MO2 in response to elevated PCO2 was paralleled by reduced larval size at hatch. Finally, ionocyte abundance decreased with increasing temperature, but did not differ between PCO2 treatments. Our results demonstrate increased thermal sensitivity of cod embryos under future PCO2 conditions and suggest that acclimation to elevated PCO2 requires reallocation of limited resources at the expense of embryonic growth. We conclude that ocean acidification constrains the thermal performance window of embryos, which has important implication for the susceptibility of cod to projected climate change.
Subject(s)
Climate Change , Gadus morhua , Acclimatization , Animals , Hydrogen-Ion Concentration , Oceans and Seas , TemperatureABSTRACT
OBJECTIVES: In search for methods of anesthesia of crustaceans, an implanted electrode into lobster and crayfish CNS enabled us to monitor signal propagation in the nerve system of animals undergoing different protocols. RESULTS: Cooling (tap water 0°C, sea water -1,8°C) and anesthesia with MgCl2 (10%) were both discarded as anesthetic procedures because responses to external stimuli were still detectable under treatment. Contrarily, bubbling the aquarium water with CO2 can be considered a "partially successful" anesthesia, because signal propagation is inhibited but before that the animals show discomfort. The procedure of "electro-stunning" induces epileptic-form seizures in the crustacean CNS (lobster, crayfish), which overlay but do not mitigate the response to external stimuli. After several minutes the activity declines before the nervous system starts to recover. A feasible way to sacrifice lobsters is to slowly raise the water temperature (1°C min-1), as all electrical activities in the CNS cease at temperatures above ~30°C, whereas below this temperature the animals do not show signs of stress or escape behavior (e.g. tail flips) in the warming water. CONCLUSION: CO2 is efficient to anaesthetize lobster and crayfish but due to low pH in water is stressful to the animals previous to anesthesia. Electrical stunning induces epileptiform seizures but paralyses the animals and leads to a reversible decline of nerve system activity after seizure. Electric stunning or slowly warming just before preparation may meet ethical expectations regarding anaesthesia and to sacrifice crustaceans.
Subject(s)
Anesthesia , Crustacea/physiology , Animals , Crustacea/classification , Electrophysiological Phenomena , Species Specificity , TemperatureABSTRACT
BACKGROUND: Reactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues. Most analytical work uses tissue homogenates, and lacks spatial information on the tissue specific sites of actual ROS formation. Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes at cellular level. RESULTS: Together with oxidative stress measurements, here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of ROS and RNS formation in the living tissue. Our results indicate that a) mitochondria located in the basal parts of the epithelial cells close to the blood vessels are hyperpolarized with high Δψm, whereas b) the peripheral mitochondria close to the cilia have low (depolarized) Δψm. These mitochondria are densely packed (mitotracker Deep Red 633 staining), have acidic pH (Ageladine-A) and collocate with high formation of nitric oxide (DAF-2DA staining). NO formation is also observed in the endothelial cells surrounding the filament blood sinus. ROS (namely H2O2, HOO(â¢) and ONOO(-) radicals, assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. On the ventral bend of the gills, subepithelial mucus glands contain large mucous vacuoles showing higher fluorescence intensities for O2 (â¢-) than the rest of the tissue. Whether this O2 (â¢-) production is instrumental to mucus formation or serves antimicrobial protection of the gill surface is unknown. Cells of the ventral bends contain the superoxide forming mucocytes and show significantly higher protein carbonyl formation than the rest of the gill tissue. CONCLUSIONS: In summary, ROS and RNS formation is highly compartmentalized in bivalve gills under unstressed conditions. The main mechanisms are the differentiation of mitochondria membrane potential and basal ROS formation in inner and outer filament layers, as well as potentially antimicrobial ROS formation in the central blood vessel. Our results provide new insight into this subject and highlight the fact that studying ROS formation in tissue homogenates may not be adequate to understand the underlying mechanism in complex tissues.
ABSTRACT
Marine teleost fish sustain compensation of extracellular pH after exposure to hypercapnia by means of efficient ion and acid-base regulation. Elevated rates of ion and acid-base regulation under hypercapnia may be stimulated further by elevated temperature. Here, we characterized the regulation of transepithelial ion transporters (NKCC1, NBC1, SLC26A6, NHE1 and 2) and ATPases (Na(+)/K(+) ATPase and V-type H(+) ATPase) in gills of Atlantic cod (Gadus morhua) after 4 weeks of exposure to ambient and future PCO2 levels (550 µatm, 1200 µatm, 2200 µatm) at optimum (10 °C) and summer maximum temperature (18 °C), respectively. Gene expression of most branchial ion transporters revealed temperature- and dose-dependent responses to elevated PCO2. Transcriptional regulation resulted in stable protein expression at 10 °C, whereas expression of most transport proteins increased at medium PCO2 and 18 °C. mRNA and protein expression of distinct ion transport proteins were closely co-regulated, substantiating cellular functional relationships. Na(+)/K(+) ATPase capacities were PCO2 independent, but increased with acclimation temperature, whereas H(+) ATPase capacities were thermally compensated but decreased at medium PCO2 and 10 °C. When functional capacities of branchial ATPases were compared with mitochondrial F1Fo ATP-synthase strong correlations of F1Fo ATP-synthase and ATPase capacities generally indicate close coordination of branchial aerobic ATP demand and supply. Our data indicate physiological plasticity in the gills of cod to adjust to a warming, acidifying ocean within limits. In light of the interacting and non-linear, dose-dependent effects of both climate factors the role of these mechanisms in shaping resilience under climate change remains to be explored.