Regional differences in mRNA responses in blue mussels within the Baltic proper.

Mussels (Mytilus sp.) from two regions along the permanent salinity gradient within the Baltic proper were exposed to copper (35 ppb) or petrol (0.3 mL/L) for 10 days and analyzed for mRNA expressions in gill tissue. Expression of mRNAs for the heat shock proteins HSP70 and HSP90 was significantly induced by copper, but not by petrol. For the metallothioneins MT10 and MT20, regional differences in mRNA expressions could be seen. In mussels from the northern Baltic proper, MT20 expression increased 2.8 and 3.4 times, after exposure to copper and petrol, respectively. In contrast, no change could be seen in MT20 expression for mussels from the southern Baltic proper. MT10 showed a peculiar expression not previously described. For some mussels, no expression at all was detected, some showed a weak expression and for some individuals a strong expression could be seen. For the mussels from the southern Baltic proper, the number of individuals with a strong expression of MT10 increased from 1 out of 18 (control), to 7 and 8, after exposure to copper and petrol, respectively. The results clearly show that responses vary between different regions within the Baltic proper, which emphasises the importance to study interactions between contaminants, populations and regions.

Evidence of population genetic effects of long-term exposure to contaminated sediments-a multi-endpoint study with copepods.

In the environment, pollution generally acts over long time scales and exerts exposure of multiple toxicants on the organisms living there. Recent findings show that pollution can alter the genetics of populations. However, few of these studies have focused on long-term exposure of mixtures of substances. The relatively short generation time (ca. 4-5 weeks in sediments) of the harpacticoid copepod Attheyella crassa makes it suitable for multigenerational exposure studies. Here, A. crassa copepods were exposed for 60 and 120 days to naturally contaminated sediments (i.e., Svindersviken and Trosa; each in a concentration series including 50% contaminated sediment mixed with 50% control sediment and 100% contaminated sediment), and for 120 days to control sediment spiked with copper. We assayed changes in F(ST) (fixation index), which indicates if there is any population subdivision (i.e., structure) between the samples, expected heterozygosity, percent polymorphic loci, as well as abundance. There was a significant decrease in total abundance after 60 days in both of the 100% naturally contaminated sediments. This abundance bottleneck recovered in the Trosa treatment after 120 days but not in the Svindersviken treatment. After 120 days, there were fewer males in the 100% naturally contaminated sediments compared to the control, possibly caused by smaller size of males resulting in higher surface: body volume ratio in contact with toxic chemicals. In the copper treatment there was a significant decrease in genetic diversity after 120 days, although abundance remained unchanged. Neither of the naturally contaminated sediments (50 and 100%) affected genetic diversity after 120 days but they all had high within treatment F(ST) values, with highest F(ST) in both 100% treatments. This indicates differentiation between the replicates and seems to be a consequence of multi-toxicant exposure, which likely caused selective mortality against highly sensitive genotypes. We further assayed two growth-related measures, i.e., RNA content and cephalothorax length, but none of these endpoints differed between any of the treatments and the control. In conclusion, the results of the present study support the hypothesis that toxicant exposure can reduce genetic diversity and cause population differentiation. Loss of genetic diversity is of great concern since it implies reduced adaptive potential of populations in the face of future environmental change.

Oxidative stress in response to xenobiotics in the blue mussel Mytilus edulis L.: evidence for variation along a natural salinity gradient of the Baltic Sea.

Blue mussels (Mytilus edulis L.) collected at three sampling sites in each of three geographical regions (South, Middle, North) along the permanent longitudinal South-North salinity gradient of the Baltic Sea, were exposed for 10 days to copper (35ppb) or 95 octane petrol (0.3 per thousand). During the experiment, they were maintained at the respective sampling site salinity. Scope for growth (SFG) was determined, and biochemical stress markers (protein carbonyl groups, disulfide bond formation, and glutathione transferase (GST), and catalase (CAT) activities) were investigated in gill tissue upon termination of the experiment. Treatment and regional effects for SFG and protein carbonyl groups were all significant for petrol. The largest increase in protein carbonyl groups was observed in the North. Mussels from the southern, more saline ( approximately 7 per thousand) region had the highest SFG, and displayed the largest SFG decrease in response to treatment, indicating that they had the most energy available for allocation to stress response. They also displayed the least increase in the level of protein carbonyl groups. Mussels from the Northern, less saline ( approximately 5%) region had the highest degree of protein carbonyl groups in response to both treatments, and lowest average SFG. Silver stained diagonal gels for samples from one sampling site in South and North, respectively, demonstrated differences in disulfide bond profiles for both stress treatments. There was also a regional difference in the number of protein disulfides observed on diagonal gels. The most diverse protein disulfide response was found in South. No treatment related effects on GST and CAT activities were observed. We suggest that both SFG and protein carbonyl groups show that geographical difference in stress susceptibility, previously established between the North and the Baltic Seas, also apply on a regional scale within the Baltic Sea, along the salinity gradient.

A multilevel approach to predict toxicity in copepod populations: assessment of growth, genetics, and population structure.

One of the goals of environmental risk assessment (ERA) is to understand effects of toxicant exposure on individual organisms and populations. We hypothesized that toxicant exposure can reduce genetic diversity and alter genotype composition, which may ultimately lead to a reduction in the average fitness of the exposed population. To test this hypothesis, we exposed a copepod, Nitocra psammophila, to a toxic reference compound and assayed resulting alterations in genetic structure, i.e. expected heterozygosity and percent polymorphic loci, as well as other population- and fitness-related measures, i.e. population abundance, demographic structure and juvenile growth. The copepods were exposed to 0.11-1.1 microg of the pentabromo-substituted diphenyl ether (BDE-47) mg(-1) freeze-dried algae for 24 days (i.e. >1 generation). There was no significant decline in total population abundance. However, there were significant alterations in population structure, manifested as diminished proportion of nauplii and increased proportion of copepodites. In addition, individual RNA content in copepodites decreased significantly in exposed individuals, indicating declined growth. Finally, in the exposed populations, heterozygosity was lower and genotype composition was altered compared to the controls. These results therefore confirm the hypothesized reduction in overall genetic variability resulting from toxicant exposure. Multilevel approaches, such as the one used in the present study, may help unravel subtle effects on the population level, thus increasing the predictive capacity of future ERA.

Variability of heat shock proteins and glutathione S-transferase in gill and digestive gland of blue mussel, Mytilus edulis.

Glutathione S-transferase (GST) and heat shock proteins (hsps) 40, 60, 70 and 90 were determined by immunoblotting using actin as an internal control in Mytilus edulis from one station outside (site1) and three stations within (sites 2-4) Cork Harbour, Ireland. Comparisons were made between gill and digestive gland and between sites. Gill shows generally higher hsp 60, 70 and 90 while digestive gland has higher hsp 40. Site 1 showed higher gill hsps 40 and 70 than sites 2-4 while gill GST was higher in sites 3 and 4 than 1 and 2. Comparison with sites in the North Sea (site 5: outside Tjärnö in The Koster archipelago in the Skagerack) and Baltic Sea (site 6: Askö island) also revealed lower hsps 40 and 70 in site 6 (low salinity) than site 5 (high salinity) although hsps 60, 70 and 90 were detectable in digestive gland unlike sites 1-4. Previously, only hsp 70 had been studied at these sites [Mar. Environ. Res. 39. (1995), 181]. At the mRNA level, gill hsp 70 is 80-fold higher at Tjärnö than Askö. These data suggest that, while salinity may slightly decrease hsp 40 and 70, both hsp 70 and GST are selectively up-regulated by approx. 10- and 3-fold, respectively, at Tjärnö compared to the other sites which we attribute to exposure to more widely fluctuating pollution levels.