Cellular biomarkers to elucidate global warming effects on Antarctic sea urchin Sterechinus neumayeri

Global warming is a reality and its effects have been widely studied. However, the consequences for marine invertebrates remain poorly understood. Thus, the present study proposed to evaluate the effect of elevated temperature on the innate immune system of Antarctic sea urchin Sterechinus neumayeri. Sea urchins were collected nearby Brazilian Antarctic Station ??Comandante Ferraz?? and exposed to 0 (control), 2 and 4°C for periods of 48?h, 2, 7 and 14?days. After the experimental periods, coelomic fluid was collected in order to perform the following analyses: coelomocytes differential counting, phagocytic response, adhesion and spreading coelomocytes assay, intranuclear iron crystalloid and ultra structural analysis of coelomocytes. The red sphere cell was considered a biomarker for heat stress, as they increased in acute stress. Besides that, a significant increase in phagocytic indexes was observed at 2°C coinciding with a significant increase of intranuclear iron crystalloid at the same temperature and same time period. Furthermore, significant alterations in cell adhesion and spreading were observed in elevated temperatures. The ultra structural analysis of coelomocytes showed no significant difference across treatments. This was the first time that innate immune response alterations were observed in response to elevated temperature in a Polar echinoid.     

Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii

Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers.     

Salinity sensitivity of early embryos of the Antarctic sea urchin, <Emphasis Type="Italic">Sterechinus neumayeri</Emphasis>

Embryos and larvae of the Antarctic sea urchin, Sterechinus neumayeri, have received considerable experimental attention assessing impacts of low temperature on development; however, salinity effects are not well documented because heretofore, the Antarctic coastal marine environment has been remarkably stenohaline. In this study, subtle decreases of 2 and 4 parts per 1,000 in standard salinity were tested to see if the developmental rate of S. neumayeri embryos would be impacted by a potential hyposmotic stress. At 30 psu, significantly fewer embryos (2 individuals out of 198 tested) reached morula stage by 36 h post-fertilization in comparison embryos in control treatments at 34 psu. Antarctic sea urchins are an important component of marine environments due to their grazing activities. Reductions in larval recruitment success due to the influx of freshwater from melting ice shelves resulting from global climate change could have far-reaching impacts on benthic ecosystem structure in Antarctica.     

Variation among females in egg lipid content and developmental success of echinoderms from McMurdo Sound,Antarctica

Egg lipid and protein content of different females of Antarctic echinoderms in McMurdo Sound, Antarctica, were measured to assess variation among females and developmental success. Egg triacylglycerol content of the Antarctic sea urchin Sterechinus neumayeri, when less than 70 ng, correlated with embryos that failed to develop past the 4-day-old blastula stage. In contrast asteroids (Odontaster meridionalis, O. validus, Acodontaster hodgsoni) all produced eggs that developed normally, even with variable egg lipid content. This difference might be related to dietary sources for more herbivorous sea urchins compared to more omnivorous and predatory asteroids. Low egg lipid content, with resulting poor embryonic survivorship, suggests that herbivorous sea urchins may be under unusual levels of nutritional stress in McMurdo Sound during the time frame studied (2004–2005). This nutritional stress might be related to the presence of large icebergs, known to have reduced primary production in the Ross Sea area.     

Ion homeostasis and interrenal stress responses in juvenile Pacific herring, Clupea pallasi, exposed to the water-soluble fraction of crude oil

Juvenile Pacific herring, Clupea pallasi, were exposed both acutely (96 h) and chronically (9 weeks) to three concentrations of the water-soluble fraction (WSF) of North Slope crude oil. Mean (± S.E.) total PAH (TPAH) concentrations at the beginning of the acute exposure experiment were: 9.7 ± 6.5, 37.9 ± 8.6 and 99.3 ± 5.6 μg/L. TPAH concentrations declined with time and the composition of the WSF shifted toward larger and more substituted PAHs. Significant induction of hepatic cytochrome P450 content, ethoxyresorufin O-deethylase and glutathione-S-transferase activities in WSF-exposed fish indicated that hydrocarbons were biologically available to herring. Significant but temporary, elevations in plasma cortisol (4.9-fold and 8.5-fold increase over controls in the 40 and 100 μg/L groups, respectively), lactate (2.2-fold and 3.1-fold over controls in the 40 and 100 μg/L groups) and glucose (1.3-fold, 1.4-fold and 1.6-fold over controls in the 10, 40 and 100 μg/L groups) occurred in fish exposed acutely to WSF. All values returned to baseline levels by 96 h. Similar responses were seen with the first of several sequential WSF pulses in the chronic exposure study. Subsequent WSF pulses resulted in muted cortisol responses and fewer significant elevations in both plasma lactate and glucose concentrations. Hematocrit, leucocrit, hemoglobin concentration and liver glycogen content were not affected by acute or chronic WSF exposure. Plasma [Cl⁻], [Na⁺] and [K⁺] were significantly higher in the 100 μg/L WSF-exposed group by 96 h compared to control fish, and continued to be elevated through the entire chronic exposure period. Unlike the measured stress parameters, ionoregulatory dysfunction was not modulated by WSF pulses. The results of this study suggest that chronic exposure to WSF affects at least two important physiological systems in herring: the ability of fish to maintain ion homeostasis and the interrenally-mediated organismal stress response.     

Cultivation of the heart urchin Echinocardium cordatum and validation of its use in marine toxicity testing for environmental risk assessment

To study environmental risk assessment, echinoderms provide a useful model for ecotoxicological testing. However, limited knowledge of the life history of field collected heart urchins is a problem and the use of cultured urchins has been investigated here. The present study describes a culture method for the heart urchin Echinocardium cordatum under controlled laboratory conditions, providing organisms with a low biological variation. Based on our optimized growth protocol both larvae and juveniles have a growth rate comparable to E. cordatum in the wild. The toxicological response of cultured and field-collected E. cordatum was compared in standard saltwater toxicity bioassays. Using ammonium chloride as a water-soluble reference toxicant, mean 96 h LC₅₀ values for cultured heart urchins versus field collected animals were 37.4 ± 7.6 mg NH₄+/l (n = 5) versus 22.5 ± 4.9 mg NH₄+/l (n = 19), respectively. Additional toxicity experiments with tributyl tin (TBT) spiked sediments revealed 14d LC₅₀ values of 1,242 (95% confidence interval 986–1,564) and 964 (95% confidence interval 843–1,102) µg Sn/kg dw respectively in cultured and field collected E. cordatum. From this it was concluded that cultured heart urchins are less sensitive to TBT than field collected E. cordatum. Furthermore in whole sediment toxicity tests, survival of cultured sea urchins was higher or at least similar to that of field collected E. cordatum. The increased sensitivity of field urchins compared to cultured urchins in various toxicity tests may be due to multiple environmental stressors reducing their overall performance. Overall it was demonstrated that the use of cultured E. cordatum provides a significant advance for urchin-based bioassays for marine environmental toxicity testing, resulting in a more homogeneous, vital population with experimental data displaying reduced variability.     

Biological weighting functions for DNA damage in sea urchin embryos exposed to ultraviolet radiation

Laboratory experiments examining the effects of ultraviolet radiation (UVR, 290-400 nm) on DNA damage were carried out using the embryos of three species of sea urchins from different habitats; Strongylocentrotus droebachiensis from the Gulf of Maine, Sterechinus neumayeri from the Antarctic, and Evechinus chloroticus from New Zealand. All three species exhibited significant amounts of accumulated DNA damage, measured as cyclobutane pyrimidine dimers (CPD) photoproducts, when exposed to UVR in the laboratory. Biological weighting functions (BWFs) revealed that S. neumayeri has significantly higher sensitivity to UVR-induced DNA damage across most of the UVR spectrum compared to the other two species, and all species were observed to have weightings in the ultraviolet-A (UVA, 320-400 nm) portion of the spectrum. The increased sensitivity to ultraviolet-B (290-320 nm) and UVA in S. neumayeri is correlated with the lowest concentration of UVR absorbing compounds observed in the embryos of the three species of urchin used in this study. Sea urchin embryos and larvae in the respective habitats of the species tested are known to occur within 5 m of the surface of the ocean where both UVB and UVA wavelengths occur. Solar irradiances of UVR at a depth of 5 m, weighted using the urchin DNA damage BWFs, show that E. chloroticus receives the greatest amount of biologically effective UVR despite having the lowest wavelength dependent weightings for DNA damage when compared to the other two species.     

Unusual trophic strategies of Hydractinia angusta (Cnidaria, Hydrozoa) from Terra Nova Bay, Antarctica

Hydractinia angusta Hartlaub, 1904 has been recorded at Terra Nova Bay (Ross Sea, Antarctica) as epizoic on shells of the Antarctic scallop Adamussium colbecki. The species can exploit different trophic resources: first, polyps are able to detach and ingest tube feet and pedicellariae from the sea urchins Sterechinus neumayeri, grazing on the scallop shell, and second, they also eat masses of benthic diatoms settled among the hydrorhiza of the colony. The particular relationship observed between the hydroid and one of the most common Antarctic sea urchins may prevent or reduce the damage to A. colbecki shells, otherwise caused by the grazing of sea urchins on the algal film of the upper valve of the scallops. H. angusta is the first known species of hydroid that exploits prey several times its own size and the second that does not ingest entire prey but portions of them. The use of benthic diatoms as a food resource has previously been documented for the sub-Antarctic marine hydroid Silicularia rosea. Accepted: 20 December 1999     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

Papyrus swamps and the respiratory ecology of Barbus neumayeri

Synopsis In seasonally flooding fresh waters, dissolved oxygen and fluctating water levels combine to create strong seasonal changes in habitat availability and dispersal pathways for fishes. In this study we demonstrate how respiratory mode can affect the use of and dispersal through hypoxic papyrus swamps for a small cyprinid fish, Barbus neumayeri, a species which uses aquatic surface respiration (ASR) in response to severe hypoxia. Monthly records of the distribution and relative abundance of B. neumayeri across 28 stations in a papyrus swamp in Uganda were used to examine seasonal patterns of habitat use and movement. The distribution of B. neumayeri was very restricted during the drier months. Most fish were captured in open pools and channels with large areas of open water surface and higher oxygen. Movement of B. neumayeri was limited to short periods during the wet season when peak water levels produced high levels of oxygen and pathways for movement. Three lines of evidence suggest that these patterns relate to its respiratory mode. These include: the positive :relationship between dissolved oxygen and the number of stations used per month, the positive relationship between fish density at dry season stations and dissolved oxygen, and the absence of fish from stations with. little open water surface area during the dry season. Significant differences in the gill morphology between B. neumayeri from the papyrus swamp and those from the main river suggest that papyrus swamps may contribute to the diversification of B. neumayeri by limiting movement and demanding specialization for extremely hypoxic waters.     

Effect of macrophyte diet and initial size on the survival and somatic growth of sub-adult <Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis>: a laboratory experimental approach

Recently recruited urchins from the same brood, but with different initial sizes, may respond differently to similar environmental factors. The aim of this study was to assess and compare the effects of starvation and diet on the survival, growth rates in size and weight, and gonad index among small and large sub-adult purple sea urchins, Strongylocentrotus purpuratus. Small urchins ranged from 7.3 to 7.8 mm and large urchins from 11.8 to 14.1 mm (test diameters). Two independent experiments were performed. In the first experiment, sea urchins were fed during 22 weeks on Egregia menziesii (ad libitum) and for only 1 day month⁻¹ (starved condition). Feeding regime significantly affected survival, somatic growth rate in size and weight, and gonad index, with higher means in the ad libitum treatments than in starving conditions. A recurrent cannibalism event by conspecifics occurred in small sea urchins under starving conditions. In the second experiment, sea urchins were fed during 13 weeks ad libitum with four diets: kelp (E. menziesii), coralline algae (Bossiella orbigniana), eelgrass (Phyllospadix scouleri) and a mixed diet of the three species. Survival was not affected by diet or urchin size, but diet significantly affected somatic growth rate in size and weight and gonad index. Kelp promoted the highest growth rate (2.23 ± 0.21 mm month⁻¹), the mixed diet produced an intermediate growth (1.26 ± 0.21 mm month⁻¹), while the lowest values corresponded to coralline algae and the eelgrass (0.30 ± 0.12 and 0.10 ± 0.03 mm month⁻¹, respectively, means ± SE). The mean growth rate of small urchins (averaging all diets) was higher than in large specimens (1.17 ± 0.37 and 0.77 ± 0.28 mm month⁻¹, respectively).     

Negative effects of oil spillage on beach microalgae in Nigeria

In a concerted effort to apply epipsammic microalgae indices as a biological indicator of crude oil pollution and natural remediation in a tropical estuarine environment, the direct effect of a recent oil spill on the abundance of microalgae in the coastal shore of the Qua Iboe Estuary was investigated. A significant negative effect of contamination on the salinity, acidity and nutritive salts (CO₃²⁻, Cl⁻, and SO₄²⁻) levels of the sandy beach soil was observed. The Biological Index of Pollution (BIP) of the beach soil was raised from the previous slightly polluted level (18%) to 75, 88, 45 and 41% after contamination, at sampling distances of 5.5, 9.5, 11.5 and 15 m from the barrier used for pollution control. These corresponded with increases in the density of microalgae with distance from the barrier. This implies that the effect of oil pollution was more severe on microalgal cells that are close to the barrier. The overall effect was a distance-influenced reduction in the regeneration capabilities of the epipsammic microalgae. Some microalgal species, particularly the cyanobacterial species of Aphanizomenon flos-aquae, Lyngbya majusculata,and a centric diatom Actinoptychus undulatus may have been exposed to contamination levels exceeding normal homeostasis and compensation. They lost their existence in the sandy beach, and their absence is recommended for use as an indicator of the short term effect of oil pollution in coastal sandy beaches in a tropical estuarine environment.     

Sub-lethal levels of waterborne petroleum may depress routine metabolism in polar cod Boreogadus saida (Lepechin, 1774)

Petroleum-related activities in Arctic waters are rapidly increasing parallel to the ongoing thinning of the Arctic sea ice. As part of a series of studies on petroleum-induced stress in polar cod Boreogadus saida, we tested the effects of acute (~60 min) and chronic (4 weeks) exposure to the water soluble fraction (WSF) of petroleum on whole body metabolism inferred from measurements of oxygen consumption rates. The exposure of polar cod to WSF leads to a statistically significant depression in routine metabolism in the order Control (0.260 mg O₂ g fish⁻¹ h⁻¹; N = 6) > Chronic (0.191 mg O₂ g fish⁻¹ h⁻¹; N = 6) > Acute (0.110 mg O₂ g fish⁻¹ h⁻¹; N = 2), decoupling of routine metabolism and body mass but possibly also to a partial metabolic compensation after 4 weeks of exposure. The results are reviewed in context with similar studies on Antarctic and non-polar fishes.     

High-Antarctic regular sea urchins – the role of depth and feeding in niche separation

Regular sea urchins of the families Cidaridae and Echinidae are widespread and sympatrically occurring epibenthic species in Antarctic waters. Food preference and water depth distribution of the five most abundant species (Ctenocidaris gigantea, C. spinosa, Notocidaris mortenseni, Sterechinus antarcticus, S. neumayeri) were analysed based on trawl and photograph samples. Both diet and water depth contribute to niche separation among these species. All sea urchins consume bryozoans and sediment, but echinids feed predominantly on diatoms in the fluff, when available. Cidarids do not consume diatoms, most likely owing to morphological constraints; their typical food consists of sponges and hydroids. C. spinosa and S. neumayeri prefer shallow water depths, whereas N. mortenseni and S. antarcticus prefer deeper regions. C. gigantea is the most variable species regarding food composition and living depth.     

The Value of the Serum Neurofilament Protein Heavy Chain as a Biomarker for Peri-operative Brain Injury After Carotid Endarterectomy

This prospective study examined the value of serum neurofilament protein levels for detecting peri-operative brain damage following carotid endarterectomy. An ELISA was used for quantification of neurofilament protein heavy chain (NfH^SMI35) levels from patients undergoing endarterectomy for symptomatic (n = 17) and asymptomatic high-grade internal carotid artery stenosis (n = 30). All patients underwent diffusion-weighted brain imaging before and after the procedure. NfH^SMI35 levels were significantly higher in patients with a symptomatic carotid artery stenosis (0.131 ng/ml) if compared to asymptomatic patients (0.055 ng/ml, P = 0.01). However, serum NfH^SMI35 levels were not related to signs of brain ischemia on routine brain imaging techniques. Our pilot data suggests that raised NfH^SMI35 serum levels in patients with symptomatic carotid artery disease may be a sensitive biomarker for diffuse ischemic damage to the CNS. We conclude that NfH^SMI35 failed to qualify as a biomarker for peri-operative brain injury in CEA and factors that may have compromised the validation of this biomarker are discussed and need to be taken into account for the design of further studies.     

Effects of serum on phagocytic activity and proteomic analysis of tilapia (Oreochromis mossambicus) serum after acute osmotic stress

The objective of the study was to analyze the effect of serum from freshwater (FW) exposed tilapia or from 25 ppt seawater (SW) exposed tilapia on the ability to mediate the phagocytic activity of tilapia phagocytes. To analyze the phagocytic activity, head kidney (HK) and spleen leukocytes were tested in 300 or 500 mOsm medium using three different treatment groups (a) control, (b) addition of 25% serum from freshwater (FW) exposed tilapia, and (c) addition of 25% of serum from 25 ppt seawater (SW) exposed tilapia. HK leukocytes cultured in 300 and 500 mOsm media for 4 h showed an increase of phagocytic ability in the control group as compared to the addition of serum from either FW or SW exposed tilapia. HK leukocytes exposed to 500 mOsm medium showed a higher phagocytic ability than those leukocytes exposed to 300 mOsm medium in each corresponding group. Concurrently, spleen leukocytes in the control group showed a higher phagocytic ability than those leukocytes with the addition of serum from FW or SW exposed tilapia. As compared to spleen leukocytes cultured in 300 mOsm medium, leukocytes cultured in 500 mOsm medium showed an increase of phagocytic ability within their respective group. To further investigate the observed phenomenon, 2D-gel electrophoresis was performed for analyzing the differentially expressed proteins in serum that was thought to influence the phagocytic ability. Up-regulated serum proteins in SW exposed tilapia contained complement C3 protein, NADH dehydrogenase (Ubiquinone) Fe–S protein 3, Mg²⁺-dependent neutral sphingomyelinase, Semaphorins, and Caspase 3. Taken together these results suggest that addition of serum decreased the phagocytic activity in HK and spleen leukocytes in vitro, furthermore, induced proteins semaphorin, complement C3, Mg²⁺-dependent neutral sphingomyelinase, and Caspase 3 are up-regulated in the serum, which might have decreased the phagocytic activity upon exposure to hyperosmotic solutions.     

Hemocyte parameters of the Pacific oyster Crassostrea gigas a year after the Hebei Spirit oil spill off the west coast of Korea

In marine bivalves, hemocytes support various physiological functions, including immune defense, nutrient transport, shell repair, and homeostatic maintenance. Although the effects of marine contaminants on the immunological functions of bivalves have been extensively investigated, the impacts of oil spills are not well understood. Therefore, we investigated hemocyte parameters in the Pacific oyster Crassostrea gigas 13 months after the Hebei Spirit oil spill (December 2007) off the west coast of Korea. The parameters studied included hemocyte concentration and mortality, relative proportion of hemocyte populations, and immunological functions such as phagocytosis and oxidative activity using flow cytometry. These immune-related parameters in oysters damaged by the oil spill were also compared to control oysters that were collected from an area unaffected by the spill. The flow cytometry study indicated that granulocyte population, phagocytic capacity, and reactive oxygen species production in oysters exposed to crude oil 13 months prior were depressed compared to the unexposed control oysters. Our data suggest that immunocompetence in oysters affected by the oil spill had not fully recovered 1 year after the accident, although more detailed studies on the physiology and disease resistance should be performed.     

1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure

The metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (¹H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm². Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm²) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm² DDT dose and both the 0.5 and 1.0 μg/cm² endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Environmental Metabolomics Special Issue of Metabolomics.