The synthetic estrogen 17α-ethinylestradiol (EE2), ubiquitous in the aquatic environment and commonly detected in sewage effluents, interferes with the endocrine system in multiple ways. Exposure during sensitive windows of development causes persistent effects on fertility, reproductive and non-reproductive behavior in mammals and fish. In the present study, three-spined stickleback (Gasterosteus aculeatus) were exposed to nominal 0 and 20 ng/L EE2 from fertilization to 7 weeks post-hatch. After 8 months of remediation in clean water three non-reproductive behaviors, not previously analyzed in developmentally EE2-exposed progeny of wild-caught fish, were evaluated. Chemical analysis revealed that the nominal 0 and 20 ng/L exposure contained 5 and 30 ng/L EE2, respectively. Therefore, the use of control fish from previous experiments was necessary for comparisons. Fish exposed during development showed significant concentration-dependent reduction in anxiety-like behavior in the scototaxis (light/dark preference) test by means of shorter latency to first entrance to the white compartment, more visits in white, and longer total time in white compared to unexposed fish. In the novel tank test, developmental exposure significantly increased the number of transitions to the upper half of the aquaria. Exposure to EE2 during development did not alter shoal cohesion in the shoaling test compared with unexposed fish but fish exposed to 30 ng/L EE2 had significantly longer latency to leave the shoal and fewer transitions away from the shoal compared to fish exposed to 5 ng/L EE2. Skewed sex ratio with more females, sex reversal in genetic males as well as intersex in males was observed after exposure to 30, but not 5 ng/L EE2. In conclusion, EE2 exposure during development in three-spined stickleback resulted in persistent effects on anxiety-like behaviors. These long-term effects from developmental exposure are likely to be of higher relevance for natural populations than are short-term effects from adult exposure.

This project aims at assessment of learning outcome of teaching evolutionary theory to pre-service primary school teacher students. The quality of the students understanding and use of evolutionary theory after their participation in seminars and lectures was made by analysing written reflections of the students. The texts were primarily written by students to show that the knowledge requirements of the course were accomplished but they were here used to analyse their ability to communicate the content but also to evaluate the depth of their understanding of evolutionary theory. For this assessment two different methods were used aiming at the two different targets. One was focused on the students’ use of evolutionary terminology and to what extent they used the language of an evolutionist. Here, we also analysed whether the students gave accurate explanations or descriptions without using the traditional evolutionary terminology. Thus, mainly the students´ ability in their own writing to present evolutionary reflections in an everyday language was here investigated. This ability may be useful when teaching young students without using a theoretical framework. For the assessment of quality of the students’ own understanding of the theory Doll’s criteria, the four R´s, richness, recursion, relations, and rigor, were used. Richness refers to the depth, the layers of meaning in their texts. Recursion is here referring to the students’ use of making thoughts loop back on themselves and earlier experiences. The use of relations are important when developing thinking in different areas. Here, both the students’ references to relations to other persons as well as to objects, theories, places etc. were recorded. Rigor means in this context to purposely look for different alternatives, relations, connections, new combinations, interpretations and patterns.

The results may briefly be summarized as follows: 1) most of the students use evolutionary terminology and 2) are fairly good at presenting their thoughts within the theoretical framework in everyday language, although 3) they often show problems in distinguishing processes on molecular, individual, and population levels.

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.

The expression of protein biomarkers in Baltic Sea blue mussels was analyzed after three days exposure to low (2.8 mu g/animal/day), intermediate (28 mu g/animal/day), or high (280 mu g/animal/day) nominal doses of benzo[a]pyrene (BaP). Significant expression changes were found in the animals exposed to the low dose, the lowest reported dose for DNA adduct formation in the gills of Baltic Sea blue mussels. Up-regulated expression of the proliferating cell nuclear antigen (PCNA), quantified from Western blots, and no change in the 5-bromo-deoxyuridine (BrdU) staining pattern, determined by immunocytochemistry, indicated that the observed PCNA response was mainly non-proliferative, and thus possibly due to DNA damage. The expression of the multixenobiotic resistance polyglycoprotein (P-gp) was also up-regulated, proving its usefulness as an exposure marker to planar organic compounds. No effect of the BaP treatment with respect to the retinoblastoma 110 protein or heat shock proteins 60 and 70 was found. The variance in the medium and high dose data was too large to allow for the detection of significant expression changes. We suggest PCNA to be a marker for genotoxic stress derived from the polycyclic aromatic hydrocarbon BaP, irrespective of whether the stress leads to DNA repair or to cell proliferation.

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 (35 ppb) or 95 octane petrol (0.3%0). 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 (similar to 7%) 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 (similar to 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.

Proline-specific dipeptidyl peptidase (DPP TV) is an established enzyme known to degrade neuropeptides and peptide hormones in vertebrate tissues. DPP TV cleaves peptides at the Pro(2) residue. Because several neuropeptides of the cockroach Leucophaea maderae, such as LemTRP-1 (APSGFLGVRamide), are potential substrates for this peptidase, we investigated the occurrence of proline-specific DPP activity in cockroach tissues. Partly purified DPP activity was characterized from the brain and midgut of L. maderae by using Gly-Pro-4-nitroanilide as a substrate. The highest activity was obtained from the membrane fraction of intestine; about 10 times less activity (per milligram protein) was obtained from brain membranes. A smaller amount of soluble DPP activity could also be identified in both tissues. Gel chromatography of the solubilized intestinal DPP activity revealed a molecular mass of about 75 kDa. The enzyme had a pH optimum of 8.5. Diprotin A (Ile-Pro-Ile) was an efficient competitive inhibitor of the cockroach DPP, whereas other known DPP inhibitors were found to be less potent. When incubated with human and cockroach DPP IV, the cleavage products of LemTRP-1 were AP and SGFLGVRamide (des-AP-LemTRP-1) as determined by mass spectrometry of high-performance liquid chromatography (HPLC)-purified peptide fragments. The AP fragment was biologically inactive and the des-AP fragment had a drastically reduced myostimulatory activity on the hindgut of L. maderae. The blowfly TRP callitachykinin-I (CavTK-I; APTAFYGVRamide) was cleaved in two steps to des-AP-CavTK-I and desAPTA-CavTK-I, showing that cockroach DPP does not only liberate Xaa-Pro, but also Xaa-Ala dipeptides. The fragment desAPTA-CavTK-I was completely inactive on the cockroach hindgut. To compare, LemTRP-3 and CavTK-II, which lack a Pro(2), were not cleaved by DPP IV. Enzyme histochemistry for DPP IV was performed on cryostat sections of brain and intestine with Gly-Pro-4-methoxy-2-naphthylamide as the substrate and Fast Blue B as the chromogen. Strong histochemical labeling was seen in specific neuropils of the brain such as the calyces of the mushroom bodies, the antennal glomeruli, and the central body. Also, the inner lining of the midgut (the peritrophic membrane) and the malpighian tubules were strongly labeled by reaction product. In both the brain and intestine, the enzyme-histochemical reaction was inhibited by diprotin A.