Molecular Tuning of Rhodopsins for High Visual Sensitivity in Different Light Environments : Variation in Absorbance Spectrum and Opsin Sequence within and between Species

Visual pigments of different animal species must have evolved at some stage to match the prevailing light environments, since all visual functions depend on their ability to absorb available photons and transduce the event into a reliable neural signal. There is a large literature on correlation between the light environment and spectral sensitivity between different fish species. However, little work has been done on evolutionary adaptation between separated populations within species. More generally, little is known about the rate of evolutionary adaptation to changing spectral environments. The objective of this thesis is to illuminate the constraints under which the evolutionary tuning of visual pigments works as evident in: scope, tempo, available molecular routes, and signal/noise trade-offs. Aquatic environments offer Nature s own laboratories for research on visual pigment properties, as naturally occurring light environments offer an enormous range of variation in both spectral composition and intensity. The present thesis focuses on the visual pigments that serve dim-light vision in two groups of model species, teleost fishes and mysid crustaceans. The geographical emphasis is in the brackish Baltic Sea area with its well-known postglacial isolation history and its aquatic fauna of both marine and fresh-water origin. The absorbance spectrum of the (single) dim-light visual pigment were recorded by microspectrophotometry (MSP) in single rods of 26 fish species and single rhabdoms of 8 opossum shrimp populations of the genus Mysis inhabiting marine, brackish or freshwater environments. Additionally, spectral sensitivity was determined from six Mysis populations by electroretinogram (ERG) recording. The rod opsin gene was sequenced in individuals of four allopatric populations of the sand goby (Pomatoschistus minutus). Rod opsins of two other goby species were investigated as outgroups for comparison. Rod absorbance spectra of the Baltic subspecies or populations of the primarily marine species herring (Clupea harengus membras), sand goby (P. minutus), and flounder (Platichthys flesus) were long-wavelength-shifted compared to their marine populations. The spectral shifts are consistent with adaptation for improved quantum catch (QC) as well as improved signal-to-noise ratio (SNR) of vision in the Baltic light environment. Since the chromophore of the pigment was pure A1 in all cases, this has apparently been achieved by evolutionary tuning of the opsin visual pigment. By contrast, no opsin-based differences were evident between lake and sea populations of species of fresh-water origin, which can tune their pigment by varying chromophore ratios. A more detailed analysis of differences in absorbance spectra and opsin sequence between and within populations was conducted using the sand goby as model species. Four allopatric populations from the Baltic Sea (B), Swedish west coast (S), English Channel (E), and Adriatic Sea (A) were examined. Rod absorbance spectra, characterized by the wavelength of maximum absorbance (λmax), differed between populations and correlated with differences in the spectral light transmission of the respective water bodies. The greatest λmax shift as well as the greatest opsin sequence difference was between the Baltic and the Adriatic populations. The significant within-population variation of the Baltic λmax values (506-511 nm) was analyzed on the level of individuals and was shown to correlate well with opsin sequence substitutions. The sequences of individuals with λmax at shorter wavelengths were identical to that of the Swedish population, whereas those with λmax at longer wavelengths additionally had substitution F261F/Y in the sixth transmembrane helix of the protein. This substitution (Y261) was also present in the Baltic common gobies and is known to redshift spectra. The tuning mechanism of the long-wavelength type Baltic sand gobies is assumed to be the co-expression of F261 and Y261 in all rods to produce ≈ 5 nm redshift. The polymorphism of the Baltic sand goby population possibly indicates ambiguous selection pressures in the Baltic Sea. The visual pigments of all lake populations of the opossum shrimp (Mysis relicta) were red-shifted by 25 nm compared with all Baltic Sea populations. This is calculated to confer a significant advantage in both QC and SNR in many humus-rich lakes with reddish water. Since only A2 chromophore was present, the differences obviously reflect evolutionary tuning of the visual protein, the opsin. The changes have occurred within the ca. 9000 years that the lakes have been isolated from the Sea after the most recent glaciation. At present, it seems that the mechanism explaining the spectral differences between lake and sea populations is not an amino acid substitution at any other conventional tuning site, but the mechanism is yet to be found.

Trenngitter - eine Alternative zu den Trichternetzen in der Garnelenfischerei

Experimental fishing trials were conducted in the Elbe Estuary using an experimental 3 m-standard beamtrawl. To avoid the by-catch of fish, a sorting grid was used. The elliptical grid was constructed of 6 mm diameter stainless steel bars with a spacing of 13 mm between the bars and housed in a cylindrical frame of 400 mm diameter. It was installed in the extension piece just in front of the codend angled at 45°, with a fish outlet at the top. A series of 10 tows of 15 minutes duration at a towing speed of 3 kn was done. The catch of the main codend was compared with the catch separated by the sorting grid. This achieved a reduction of 56 % of plaice (Pleuronectes platessa), 75 % of flounder (Platichthys flesus), 99 % of whiting (Merlangius merlangus), 94 % of cod (Gadus morhua) and 49 % of smelt (Osmerus eperlanus) with a mean loss of 43 % of shrimps (Crangon crangon). English grid trials in the Humber estuary using a flapper set or guiding funnel in front of the sorting grid device demonstrated reasonable lower escapement rates for fish and shrimps.

Effects of stress on the immune system of fish

The effects of stress on the immune system of various fish species including dab Limanda limanda, flounder Platichthys flesus, sea bass Dicentrarchus labrax and gobies Zosterisessor ophiocephalus, were investigated from laboratory and field experiments, using various assays to measure immunocompetence, correlated with histological and ultrastructural observations. Modulation of the immune system was demonstrated at tissue, cellular and biochemical levels following exposure to various stressors. The spleen somatic index was depressed in dab stressed in the laboratory and gobies collected from polluted sites in the Venice Lagoon. Differential blood cell counts consistently showed an increase in phagocytes and decrease in thrombocytes in fish exposed to various stressors. Phagocytic activity from spleen and kidney adherent cells was stimulated in dab stressed by transportation but depressed in fish exposed to chemical pollutants. Respiratory burst activity in phagocytic cells was also stimulated in stressed dab but depressed in sea bass exposed to cadmium. The results are discussed in relation to current concepts on stress in fish and the regulation of the immune system.

Mugilid Fish Are Sentinels of Exposure to Endocrine Disrupting Compounds in Coastal and Estuarine Environments

Effects on fish reproduction can result from a variety of toxicity mechanisms first operating at the molecular level. Notably, the presence in the environment of some compounds termed endocrine disrupting chemicals (EDCs) can cause adverse effects on reproduction by interfering with the endocrine system. In some cases, exposure to EDCs leads to the animal feminization and male fish may develop oocytes in testis (intersex condition). Mugilid fish are well suited sentinel organisms to study the effects of reproductive EDCs in the monitoring of estuarine/marine environments. Up-regulation of aromatases and vitellogenins in males and juveniles and the presence of intersex individuals have been described in a wide array of mullet species worldwide. There is a need to develop new molecular markers to identify early feminization responses and intersex condition in fish populations, studying mechanisms that regulate gonad differentiation under exposure to xenoestrogens. Interestingly, an electrophoresis of gonad RNA, shows a strong expression of 5S rRNA in oocytes, indicating the potential of 5S rRNA and its regulating proteins to become useful molecular makers of oocyte presence in testis. Therefore, the use of these oocyte markers to sex and identify intersex mullets could constitute powerful molecular biomarkers to assess xenoestrogenicity in field conditions.

Internationaler BMB/ICES-Workshop "Fischkankheiten und Parasiten in der Ostsee"

From 25 November until 8 Deeember 1994, a sea-going workshop with the above title was carried out on board RV "Walther Herwig IlI" under the umbrella of the Baltie Marine Biologists (BMlJ) and the International Council for the Exploration of the Sea (ICES). Under the co-convenorship of the first two authors of this paper, 11 seientists from 8 of the 9 countries bordering the Baltic Sea (except Sweden) actively involved in research monitoring into fish diseases/parasites participated. The main reason for holding the workshop was that, although fish disease monitoring programmes on the abundance and spatial distribution of fish diseases/parasites are being carried out by some of the countries bordering the Baltic Sea, there is still a striking lack of seientific information as compared to other seas, such as the North Sea. Moreover, since new fish disease monitoring programmes have recently been started (mainly by the new Baltic republics Latvia and Estonia, which recently became ICES-member countries) or are planned for the near future (Lithuania), BMB as weil as ICES felt the need for a interealibration and standardization of methodologies applied during fish disease/parasite surveys for the specific conditions in the Baltic Sea. Therefore, the major objectives of the workshop were -to obtain information about the disease/parasite prevalence in Baltic (Platichthys flesus) and other abundant fish species along a transect from the western (Mecklenhurg Bight) to the eastern (Gulf of Finland) Baltic Sea to be used as a baseline for future studies, -to intercalibrate methodologies applied for sampling, diagnosis of diseases/parasites, reporting and analyses of disease data, -if necessary, to recommend standard methodologies to be used for fish disease/parasite monitoring programmes under the specific conditions in the Baltic Sea. Although the data have yet not been completely analyzed, there is indication for some pronounced spatial trends regarding the prevalence of certain diseases/parasites, mainly for Iymphocystis and skin ulcerations in flounder but also for diseases and parasites of cod (Gadus morhua) which can be seen in figures 2 and 4. Recommendations for standard methodologies will later be published elsewhere.

Diencephalic asymmetries: morphological, immunohistochemical and ultrastructural analysis of habenular nuclei in elasmobranchs and teleosts

The habenular nuclei are diencephalic structures present in Vertebrates and they form, with the associated fiber systems, a part of the system that connects the telencephalon to the ventral mesencephalon (Concha M. L. and Wilson S. W., 2001). In representative species of almost all classes of Vertebrates the habenular nuclei are asymmetric, both in terms of size and of neuronal and neurochemical organization, although different types of asymmetry follow different evolutionary courses. Previous studies have analyzed the spread and diversity of the asymmetry in species for which data are not clear (Kemali M. et al., 1980). Notwithstanding that, it’s still not totally understood the evolution of the phenomenon, and the ontogenetic mechanisms that have led to the habenular asymmetry development are not clear (Smeets W.J. et al., 1983). For the present study 14 species of Elasmobranchs and 15 species of Teleostean have been used. Brains removed from the animals have been fixed using 4% paraformaldehyde in phosphate buffer; brains have been analyzed with different tecniques, and I used histological, immunohistochemical and ultrastructural analysis to describe this asymmetry. My results confirm data previously obtained studying other Elasmobranchs species, in which the left habenula is larger than the right one; the Teleostean show some slightly differences regarding the size of the habenular ganglia, in some species, in which the left habenular nucleus is larger than the right. In the course of studies, a correlation between the habits of life and the diencephalic asymmetry seems to emerge: among the Teleostean analyzed, the species with benthic life (like Lepidorhombus boscii, Platichthys flesus, Solea vulgaris) seem to possess a slight asymmetry, analogous to the one of the Elasmobranchs, while in the other species (like Liza aurata, Anguilla anguilla, Trisopterus minutus) the habenulae are symmetrical. However, various aspects of the neuroanatomical asymmetries of the epithalamus have not been deepened in order to obtain a complete picture of the evolution of this phenomenon, and new searches are needed to examine the species without clear asymmetry, in order to understand the spread and the diversity of the asymmetry among the habenulae between the Vertebrates.

Development of polymorphic microsatellite markers suitable for genetic linkage mapping of olive flounder Paralichthys olivaceus

Microsatellite markers are important for gene mapping and for marker-assisted selection. Sixty-five polymorphic microsatellite markers were developed with an enriched partial genomic library from olive flounder Paralichthys olivaceus an important commercial fish species in Korea. The variability of these markers was tested in 30 individuals collected from the East Sea (Korea). The number of alleles for each locus ranged from 2 to 33 (mean, 17.1). Observed and expected heterozygosity as well as polymorphism information content varied from 0.313 to 1.000 (mean, 0.788), from 0.323 to 0.977 (mean, 0.820), and from 0.277 to 0.960 (mean, 0.787), respectively. Nine loci showed significant deviation from the Hardy-Weinberg equilibrium after sequential Bonferroni correction. Analysis with MICROCHECKER suggested the presence of null alleles at five of these loci with estimated null allele frequencies of 0.126-0.285. These new microsatellite markers from genomic libraries will be useful for constructing a P. olivaceus linkage map.

Spawning, egg development, and early life history dynamics of arrowtooth flounder (Atheresthes stomias) in the Gulf of Alaska

Arrowtooth flounder (Atheresthes stomias) has the highest biomass of any groundfish species in the Gulf of Alaska, is a voracious predator of age 1 walleye pollock (Theragra chalcogramma), and is a major component in the diet of Steller sea lions (Eumetopias jubatus). Owing to its ecological importance in the Gulf of Alaska and the limited information available on its reproduction, interest has intensified in describing its spawning and early life history. A study was undertaken in late January–February 2001–2003 in the Gulf of Alaska to obtain information on adult spawning location, depth distribution, and sexual maturity, and to obtain fertilized eggs for laboratory studies. Adults were found 200–600 m deep east of Kodiak Island over the outer continental shelf and upper slope, and southwest along the shelf break to the Shumagin Islands. Most ripe females (oocytes extruded with light pressure) were found at 400 m and most ripe males (milt extruded with light pressure) were found at depths ≥450 m. Eggs were fertilized and incubated in the laboratory at 3.0°, 4.5°, and 6.0°C. Eggs were reared to hatching, but larvae did not survive long enough to complete yolk absorption and develop pigment. Eggs were staged according to morphological hallmarks and incubation data were used to produce a stage duration table and a regression model to estimate egg age based on water temperature and developmental stage. Arrowtooth flounder eggs (1.58–1.98 mm in diameter) were collected in ichthyoplankton surveys along the continental shelf edge, primarily at depths ≥400 m. Early-stage eggs were found in tows that sampled to depths of ≥450 m. Larvae, which hatch between 3.9 and 4.8 mm standard length, increased in abundance with depth. Observations on arrowtooth flounder eggs and early-stage larvae were used to complete the description of the published partial developmental series.(PDF file contains 34 pages.)

The Summer Flounder, Scup, and Black Sea Bass Fishery of the Middle Atlantic Bight and Southern New England Waters

Summer flounder, Paralichthys dentatus, scup, Stenotomus chrysops, and black sea bass, Centropristis striata, cooccur within the Middle Atlantic Bight and off southern New England and are important components of commercial and recreational fisheries. The commercial otter trawl fishery for these species is primarily a winter fishery, whereas the recreational fishery takes place between late spring and autumn. The otter trawl fishery generally targets summer flounder, and less frequently scup, while black sea bass occurs as bycatch. Trips in which all three species were present yielded highest aggregate landings per unit of effort (LPUE) levels and occurred more often than trips landing only one or two species. More than 50% of the trips in the trawl fishery landed at least two of the three species. In contrast, greater than 75% of the recreational landings of each species occurred as a result of trips landing only one species. Differences in the fisheries resulted from the interactions of seasonal changes in species distributions and gear selectivity. (PDF file contains 18 pages.)

Codend selection of winter flounder Pseudopleuronectes americanus

Codend selection of winter flounder (Pseudopleuronectes americanus) in 76-127 mm mesh codends was examined from experiments conducted in Long Island Sound during the spring of 1986-87. The results show a slightly larger size at selection than was found in earlier work as indicated by the selection factor, 2.31 in the present study compared with 2.2 and 2.24 from previous studies. Diamond mesh was found to have a length at 50% retention about 1 cm longer (Lso =22.6 cm), and a selection range (3.4 cm) about 1 cm narrower, than square mesh in 102-mm codends. Tow duration varied from 1 to 2 hours using 114-mm diamond mesh. As has been found in previous studies, tow duration and Lso are positively related, with I-hour tows averaging 24.6 cm and 2-hour tows averaging 26.6 cm. The importance of the slope of the selection curve was examined in yield-per-recruit analyses by comparing knife-edge and stepwise recruitment. In all mesh sizes, stepwise recruitment provides a more conservative estimate of yield in the presence of a minimum size limit. Differences in yield estimates between the two models were generally small (1-7%), except in the largest mesh size, 127 mm, where yield is overestimated by 10% when assuming knife-edge recruitment. (PDF file contains 16 pages.)

Untersuchung von chlorierten Kohlenwasserstoffen (CKW) an Flunder (Platichtbys flesus) und Scholle (Pleuronectes platessa) aus der Nordsee

Im Rahmen des Internationalen Rates für Meeresforschung werden seit 1972 Studien zur Kontamination von Meerestieren der Nordsee mit Schadstoffen erhoben. Eine Intensivierung dieses Unternehmens erfolgte durch die Schaffung eines gemeinsamen internationalen Überwachungsprogrammes (Joint Monitoring Program) durch die Kommission von Oslo und Paris. In diesem Programm sind die Überwchungsgebiete für die Bundesrepublik vorgeschrieben. Auch Species, Anzahl und Gewebe sind festgelegt.

The blackback flounder and its fishery in New England and New York

A decline in the abundance of blackback flounders, together with the withdrawal of vessels from this fishery, has resulted in a lowered catch in recent years compared to the peak period 1928 through 1931. Data obtained from U. S. Fish and Wildlife Service Hatchery catch records and from fishermen's log book records show a drop in abundance of 63 per cent from the early 1930's to the present in the Boothbay Harbor region and of 31 to 40 per cent in the area south of Cape Cod. Information on the early life history and distribution of young blackback flounders and the size and age composition and distribution of fish subject to the commercial and sport fisheries indicates that the young are the product of local spawning and that the sport and commercial fisheries draw on a resident stock of primarily adult fish.