The physiological response of the marine platyhelminth Macrostomum lignano to different environmental oxygen concentrations.

The respiration rate of meiofauna is difficult to measure, and the response to variations in the environmental oxygen concentration has so far been mainly addressed through behavioral investigation. We investigated the effect of different oxygen concentrations on the physiology of the marine platyhelminth Macrostomum lignano. Respiration was measured using batches of 20 animals in a glass microtiter plate equipped with optical oxygen sensor spots. At higher oxygen saturations (>12 kPa), the animals showed a clear oxyconforming behavior. However, below this value, the flatworms kept respiration rates constant at 0.064±0.001 nmol O2 l(-1) h(-1) individual(-1) down to 3 kPa PO2, and this rate was increased by 30% in animals that were reoxygenated after enduring a period of 1.5 h in anoxia. Physiological changes related to tissue oxygenation were assessed using live imaging techniques with different fluorophores in animals maintained in normoxic (21 kPa), hyperoxic (40 kPa) or near-anoxic (~0 kPa) conditions and subjected to anoxia-reoxygenation. The pH-sensitive dyes Ageladine-A and BCECF both indicated that pHi under near-anoxia increases by about 0.07-0.10 units. Mitochondrial membrane potential, Δψm, was higher in anoxic and hyperoxic than in normoxic conditions (JC1 dye data). Staining with ROS-sensitive dyes - DHE for detection of superoxide anion (O2•(-)) formation and C-H DFFDA for other ROS species aside from O2•(-) (H2O2, HOO• and ONOO) - showed increased ROS formation following anoxia-reoxygenation treatment. Animals exposed to hyperoxic, normoxic and anoxic treatments displayed no significant differences in O2•(-) formation, whereas mitochondrial ROS formation as detected by C-H2DFFDA was higher after hyperoxic exposure and lowest under near-anoxia conditions compared with the normoxic control group. Macrostomum lignano seems to be a species that is tolerant of a wide range of oxygen concentrations (being able to maintain aerobic metabolism from extremely low PO2 up to hyperoxic conditions), which is an essential prerequisite for successfully dealing with the drastic environmental oxygen variations that occur within intertidal sediments.

The effect of predator exposure and reproduction on oxidative stress parameters in the Catarina scallop Argopecten ventricosus.

Predation is known to impact growth and reproduction, and the physiological state of the prey, including its susceptibility to oxidative stress. In this study, we investigated how prolonged exposure to predators modulates tissue specific antioxidant defense and oxidative damage in the short-lived epibenthic scallop Argopecten ventricosus (2years maximum lifespan). Scallops that were experimentally exposed to predators had not only lower antioxidant capacities (superoxide dismutase and catalase), but also lower oxidative damage (protein carbonyls and TBARS=thiobarbituric acid reactive substances including lipid peroxides) in gills and mantle compared to individuals not exposed to predators. In contrast, oxidative damage in the swimming muscle was higher in predator-exposed scallops. When predator-exposed scallops were on the verge of spawning, levels of oxidative damage increased in gills and mantle in spite of a parallel increase in antioxidant defense in both tissues. Levels of oxidative damage increased also in the swimming muscle whereas muscle antioxidant capacities decreased. Interestingly, post-spawned scallops restored antioxidant capacities and oxidative damage to immature levels, suggesting they can recover from spawning-related oxidative stress. Our results show that predator exposure and gametogenesis modulate oxidative damage in a tissue specific manner and that high antioxidant capacities do not necessarily coincide with low oxidative damage.

Molecular responses to ocean acidification in an Antarctic bivalve and an ascidian.

Southern Ocean organisms are considered particularly vulnerable to Ocean acidification (OA), as they inhabit cold waters where calcite-aragonite saturation states are naturally low. It is also generally assumed that OA would affect calcifying animals more than non-calcifying animals. In this context, we aimed to study the impact of reduced pH on both types of species: the ascidian Cnemidocarpa verrucosa sp. A, and the bivalve Aequiyoldia eightsii, from an Antarctic fjord. We used gene expression profiling and enzyme activity to study the responses of these two Antarctic benthic species to OA. We report the results of an experiment lasting 66 days, comparing the molecular mechanisms underlying responses under two pCO2 treatments (ambient and elevated pCO2). We observed 224 up-regulated and 111 down-regulated genes (FC ≥ 2; p-value ≤ 0.05) in the ascidian. In particular, the decrease in pH caused an upregulation of genes involved in the immune system and antioxidant response. While fewer differentially expressed (DE) genes were observed in the infaunal bivalve, 34 genes were up-regulated, and 69 genes were downregulated (FC ≥ 2; p-value ≤ 0.05) in response to OA. We found downregulated genes involved in the oxidoreductase pathway (such as glucose dehydrogenase and trimethyl lysine dioxygenase), while the heat shock protein 70 was up-regulated. This work addresses the effect of OA in two common, widely distributed Antarctic species, showing striking results. Our major finding highlights the impact of OA on the non-calcifying species, a result that differ from the general trend, which describes a higher impact on calcifying species. This calls for discussion of potential effects on non-calcifying species, such as ascidians, a diverse and abundant group that form extended three-dimensional clusters in shallow waters and shelf areas in the Southern Ocean.

Changes in oxidative stress parameters in relation to age, growth and reproduction in the short-lived catarina scallop Argopecten ventricosus reared in its natural environment.

Increase in oxidative damage and decrease in cellular maintenance is often associated with aging, but, in marine ectotherms, both processes are also strongly influenced by somatic growth, maturation and reproduction. In this study, we used a single cohort of the short-lived catarina scallop Argopecten ventricosus, to investigate the effects of somatic growth, reproduction and aging on oxidative damage parameters (protein carbonyls, TBARS and lipofuscin) and cellular maintenance mechanisms (antioxidant activity and apoptosis) in scallops, caged in their natural environment. The concentrations of protein carbonyls and TBARS increased steeply during the early period of fast growth and during reproduction in one-year-old scallops. However, oxidative damage was transient, and apoptotic cell death played a pivotal role in eliminating damage in gill, mantle and muscle tissues of young scallops. Animals were able to reproduce again in the second year, but the reduced intensity of apoptosis impaired subsequent removal of damaged cells. In late survivors low antioxidant capacity and apoptotic activity together with a fast accumulation of the age pigment lipofuscin was observed. Rates of oxygen consumption and oxidative stress markers were strongly dependent on somatic growth and reproductive state but not on temperature. Compared to longer-lived bivalves, A. ventricosus seems more susceptible to oxidative stress with higher tissue-specific protein carbonyl levels and fast accumulation of lipofuscin in animals surviving the second spawning. Superoxide dismutase activity and apoptotic cell death intensity were however higher in this short-lived scallop than in longer-lived bivalves. The life strategy of this short-lived and intensely predated scallop supports rapid somatic growth and fitness as well as early maturation at young age at the cost of fast cellular degradation in second year scallops.

Antarctic ascidians under increasing sedimentation: Physiological thresholds and ecosystem hysteresis.

Glacier melting sediment inputs affect coastal ecosystems on the Antarctic Peninsula. In Potter Cove (South Shetland Islands, Antarctica), the shift from an "ascidian dominated" to a "mixed" assemblage has been linked to sedimentation. However, in recently described newly ice-free areas ascidians became dominant in spite of total suspended particulate matter (TSPM) concentrations, which are the highest measured in Potter Cove. Here, we compared the gut content and energy reserve of three ascidian species at three stations under different TSPM regimes. All analysed species had a higher gut content with lower %OM at these newly areas. A theoretical relationship between the scope for growth for the targeted ascidians and TSPM explained assemblages' recorded change but failed to explain current ascidians distribution. The results may indicate the existence of a TSPM threshold that allows the spatial coexistence of alternative stable states at benthic Potter Cove system.

Molecular mechanisms underlying responses of the Antarctic coral Malacobelemnon daytoni to ocean acidification.

Benthic organisms of the Southern Ocean are particularly vulnerable to ocean acidification (OA), as they inhabit cold waters where calcite-aragonite saturation states are naturally low. OA most strongly affects animals with calcium carbonate skeletons or shells, such as corals and mollusks. We exposed the abundant cold-water coral Malacobelemnon daytoni from an Antarctic fjord to low pH seawater (LpH) (7.68 ± 0.17) to test its physiological responses to OA, at the level of gene expression (RT-PCR) and enzyme activity. Corals were exposed in short- (3 days) and long-term (54 days) experiments to two pCO2 conditions (ambient and elevated pCO2 equaling RCP 8.5, IPCC 2019, approximately 372.53 and 956.78 µatm, respectively). Of the eleven genes studied through RT-PCR, six were significantly upregulated compared with control in the short-term in the LpH condition, including the antioxidant enzyme superoxide dismutase (SOD), Heat Shock Protein 70 (HSP70), Toll-like receptor (TLR), galaxin and ferritin. After long-term exposure to low pH conditions, RT-PCR analysis showed seven genes were upregulated. These include the mannose-binding C-Lectin and HSP90. Also, the expression of TLR and galaxin, among others, continued to be upregulated after long-term exposure to LpH. Expression of carbonic anhydrase (CA), a key enzyme involved in calcification, was also significantly upregulated after long-term exposure. Our results indicated that, after two months, M. daytoni is not acclimatized to this experimental LpH condition. Gene expression profiles revealed molecular impacts that were not evident at the enzyme activity level. Consequently, understanding the molecular mechanisms behind the physiological processes in the response of a coral to LpH is critical to understanding the ability of polar species to cope with future environmental changes. Approaches integrating molecular tools into Antarctic ecological and/or conservation research make an essential contribution given the current ongoing OA processes.

Is the umbo matrix of bivalve shells (Laternula elliptica) a climate archive?

Heavy metal accumulation into bivalve soft tissues has received increasing interest in recent years with respect to biomonitoring of environmental change including pollution. To a lesser extent, accretion of elements from the environment into bivalve hard structures (shells) has been investigated, although the importance of the shells as environmental archives has been acknowledged. Here we report element distribution within consecutive growth bands in the shells of the Antarctic soft shell clam Laternula elliptica, which is currently exposed to vast environmental change in Antarctic Peninsula coastal environments that undergo rapid climate warming. We performed a high spatial resolution analysis for Al, Fe, Mn, Cu, Pb and U in the shell umbo, by means of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Element ratios within the umbo did not resemble either the ratios in the surrounding seawater, the sedimenting material in Potter Cove, or even the Earth's crust basal composition. Mn and Cu were preferentially incorporated into the umbo. A strong decrease of element accretion with time could be related to lifetime respiration mass (R) of the animals. This indicates element accretion into the umbo and shell matrix to be largely a function of animal ecophysiology and life history, and these effects need to be considered in the context of potential usefulness of L. elliptica shells as environmental archives.

Pelagic and benthic communities of the Antarctic ecosystem of Potter Cove: Genomics and ecological implications.

Molecular technologies are more frequently applied in Antarctic ecosystem research and the growing amount of sequence-based information available in databases adds a new dimension to understanding the response of Antarctic organisms and communities to environmental change. We apply molecular techniques, including fingerprinting, and amplicon and metagenome sequencing, to understand biodiversity and phylogeography to resolve adaptive processes in an Antarctic coastal ecosystem from microbial to macrobenthic organisms and communities. Interpretation of the molecular data is not only achieved by their combination with classical methods (pigment analyses or microscopy), but furthermore by combining molecular with environmental data (e.g., sediment characteristics, biogeochemistry or oceanography) in space and over time. The studies form part of a long-term ecosystem investigation in Potter Cove on King-George Island, Antarctica, in which we follow the effects of rapid retreat of the local glacier on the cove ecosystem. We formulate and encourage new approaches to integrate molecular tools into Antarctic ecosystem research, environmental conservation actions, and polar ocean observatories.

Seasonal proliferation rates and the capacity to express genes involved in cell cycling and maintenance in response to seasonal and experimental food shortage in Laternula elliptica from King George Island.

Melting of coastal glaciers at the West Antarctic Peninsula (WAP) causes shorter winter sea ice duration, intensified ice scouring, sediment erosion and surface freshening in summer, which alters coastal productivity and feeding conditions for the benthos. The soft shell clam Laternula elliptica is a fast growing and abundant filter feeder in coastal Antarctica and a key element for bentho-pelagic carbon recycling. Our aim was to assess the cellular growth and maintenance capacity of small and large clams during natural winter food shortage (seasonal sampling) and in response to experimental starvation exposure. We measured tissue specific proliferation rates, the expression of cell cycling genes, and the iron binding protein Le-ferritin in freshly collected specimens in spring (Nov 2008) and at the end of summer (March 2009). For the experimental approach, we focused on 14 cell cycling and metabolic genes using the same animal size groups. Mantle tissue of young bivalves was the only tissue showing accelerated proliferation in summer (1.7% of cells dividing per day in March) compared to 0.4% dividing cells in animals collected in November. In mantle, siphon and adductor muscle proliferation rates were higher in younger compared to older individuals. At transcript level, Le-cyclin D was upregulated in digestive gland of older animals collected in spring (Nov) compared to March indicating initiation of cell proliferation. Likewise, during experimental starvation Le-cyclin D expression increased in large clam digestive gland, whereas Le-cyclin D and the autophagic factor beclin1 decreased in digestive gland of smaller starved clams. The paper corroborates earlier findings of size and age dependent differences in the metabolic response and gene expression patterns in L. elliptica under energetic deprivation. Age structure of shallow water populations can potentially change due to differences in cellular response between young and old animals as environmental stress levels increase.

Mal perforans and spina bifida occulta.

A 26-year-old man had mal perforans pedis as the principal manifestation of spina bifida occulta with an associated intradural lipoma. Two lumbosacral dimples and a tuft of hair overlying the spina bifida led to the diagnosis. We include a review of the causes of mal perforans and a review of spina bifida occulta and associated abnormalities.

Mucha-Habermann's disease in children: treatment with erythromycin.

Safe therapeutic measures for Mucha-Habermann's disease in children are lacking. Three patients with the disease were treated with erythromycin for systemic effect. Although the series is small and uncontrolled, this approach seemed effective. An anti-inflammatory mechanism related to inhibition of chemotaxis is speculated.

Ocular rosacea. Signs, symptoms, and tear studies before and after treatment with doxycycline.

OBJECTIVE: To examine ocular signs, symptoms, and results of tear analysis in patients with cutaneous rosacea before, during, and after doxycycline therapy. DESIGN: Before-after trial. SETTING: General community. PATIENTS OR OTHER PARTICIPANTS: Thirty-nine patients with cutaneous rosacea underwent dermatologic and ocular examinations, testing of tear break-up time, and Schirmer testing at baseline and 4, 8, and 12 weeks. Six patients did not complete the study. Baseline tear break-up time and results of Schirmer test were compared with those of 13 patients without rosacea who were matched for age and sex. INTERVENTION: Patients with rosacea were given doxycycline, 100 mg daily for 12 weeks. MAIN OUTCOME MEASURE: Statistically significant (P, .05) improvement in tear break-up time. RESULT: The most frequent ocular symptoms were dryness, itching, blurred vision, and photosensitivity, all of which improved significantly with treatment. All patients had signs of ocular disease, most commonly erythema and telangiectasia, meibomian gland dysfunction, and ciliary base injection. Significant improvement (P,.05) for scales, erythema and telangiectasia, ciliary base injection, bulbar injection, papillary hypertrophy, and punctate epithelial erosions was seen. Average tear break-up time for the patients with rosacea was 5.7 seconds, which improved to 10.8 seconds after 12 weeks of treatment (P = .007). Baseline tear break-up time was significantly lower than for the comparison group of normal subjects (P = .001). There was no correlation between severity of cutaneous disease and ocular disease. CONCLUSIONS: All patients with cutaneous rosacea had some degree of ocular involvement. Tear break-up time is abnormal in patients with rosacea. Ocular erythema and telangiectasia, meibomian gland dysfunction, and short tear break-up time in patients with cutaneous rosacea are indicators of ocular rosacea. Doxycycline, 100 mg daily, will improve ocular disease and increase the tear break-up time.

Transitory subependymal cysts in the developing rat rhombencephalon.

A bilaterally symmetrical cystic cavity is situated in the subependymal neuropil of the rostral rhombencephalon of the rat during the perinatal period of ontogeny. These cysts are formed by the confluence of enlarged extracellular spaces in this region between E18 and E20. The cysts are present for about 2 weeks but disappear on about P15 without trace. They have a maximal volume of about 0.004 to 0.006 mm3 on P2, with a rostrocaudal extension of about 200 microm. Their shape is characterized by a medial convexity and a lateral concavity, and they have their maximal circumference at about the middle of the rostrocaudal axis. The caudal portion is juxtaposed to the subependyma, while the rostral part lies in the neuropil of the presumptive griseum centrale pontis. In the lumen and the wall of the cysts are found numerous macrophages, glioblasts and some degenerating axons and dendrites. The significance of these cysts in the context of morphogenesis and the origin of the numerous macrophages within them are both unresolved.

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences.

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.

Age-dependent expression of stress and antimicrobial genes in the hemocytes and siphon tissue of the Antarctic bivalve, Laternula elliptica, exposed to injury and starvation.

Increasing temperatures and glacier melting at the Western Antarctic Peninsula (WAP) are causing rapid changes in shallow coastal and shelf systems. Climate change-related rising water temperatures, enhanced ice scouring, as well as coastal sediment runoff, in combination with changing feeding conditions and microbial community composition, will affect all elements of the nearshore benthic ecosystem, a major component of which is the Antarctic soft-shelled clam Laternula elliptica. A 454-based RNA sequencing was carried out on tissues and hemocytes of L. elliptica, resulting in 42,525 contigs, of which 48 % was assigned putative functions. Changes in the expression of putative stress response genes were then investigated in hemocytes and siphon tissue of young and old animals subjected to starvation and injury experiments in order to investigate their response to sedimentation (food dilution and starvation) and iceberg scouring (injury). Analysis of antioxidant defense (Le-SOD and Le-catalase), wound repair (Le-TIMP and Le-chitinase), and stress and immune response (Le-HSP70, Le-actin, and Le-theromacin) genes revealed that most transcripts were more clearly affected by injury rather than starvation. The upregulation of these genes was particularly high in the hemocytes of young, fed individuals after acute injury. Only minor changes in expression were detected in young animals under the selected starvation conditions and in older individuals. The stress response of L. elliptica thus depends on the nature of the environmental cue and on age. This has consequences for future population predictions as the environmental changes at the WAP will differentially impact L. elliptica age classes and is bound to alter population structure.