Tomas Bollner

17α-Ethinyl estradiol affects anxiety and shoaling behavior in adult male zebra fish (Danio rerio)

Ethinyl estradiol is a potent endocrine disrupting compound in fish and ubiquitously present in the aquatic environment. In this study, we exposed adult zebra fish (Danio rerio) males to 0, 5 or 25 ng Ethinyl estradiol/L for 14 days and analyzed the effects on non-reproductive behavior. Effects of treatment of the exposed males was shown by vitellogenin induction, while brain aromatase (CYP 19B) activity was not significantly altered. Both concentrations of Ethinyl estradiol significantly altered the behavior in the Novel tank test, where anxiety is determined as the tendency to stay at the bottom when introduced into an unfamiliar environment. The effects were, however, opposite for the two concentrations. Fish that were exposed to 5 ng/L had longer latency before upswim, fewer transitions to the upper half and shorter total time spent in the upper half compared with control fish, while 25 ng Ethinyl estradiol treatment resulted in shorter latency and more and longer visits to the upper half. The swimming activity of 25, but not 5 ng-exposed fish were slightly but significantly reduced, and these fish tended to spend a lot of time at the surface. We also studied the shoaling behavior as the tendency to leave a shoal of littermates trapped behind a Plexiglas barrier at one end of the test tank. The fish treated with Ethinyl estradiol had significantly longer latency before leaving shoal mates and left the shoal fewer times. Further, the fish exposed to 5 ng/L also spent significantly less time away from shoal than control fish. Fertilization frequency was higher in males exposed to 5 ng/L Ethinyl estradiol when compared with control males, while no spawning was observed after treatment with 25 ng/L. The testes from both treatment groups contained a normal distribution of spermatogenesis stages, and no abnormality in testis morphology could be observed. In conclusion, we have observed effects on two behaviors not related to reproduction in zebra fish males after treatment with Ethinyl estradiol, adding to the ecological consequences of contamination of aquatic environments with estrogenic substances.

Proline-specific dipeptidyl peptidase activity in the cockroach brain and intestine : Partial characterization, distribution, and inactivation of tachykinin-related peptides

Proline‐specific dipeptidyl peptidase (DPP IV) is an established enzyme known to degrade neuropeptides and peptide hormones in vertebrate tissues. DPP IV cleaves peptides at the Pro2 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 Pro2, 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. J. Comp. Neurol. 418:81–92, 2000.

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.

Regeneration of the Central Nervous System of the Ascidian Ciona Intestinalis

Regeneration of the neural complex of the protochordate Ciona intestinalis is morphologically described. The time needed to regenerate the central ganglion was found to be approximately 10–14 days at 18°C. Differentiated cells were characterized by staining for argyrophilia.