Comparing microsatellite variation in north-east Atlantic cod (Gadus morhua L.) to genetic structuring as revealed by the pantophysin (Pan I) locus

Variation at seven microsatellite loci was compared with variation observed at Pan I, a single copy nuclear DNA gene coding for pantophysin, in 14 samples of Atlantic cod ( Gadus morhua ) stretching from Spitsbergen to the North Sea. Population structuring indicated by the two types of markers was concordant and in agreement with the traditional grouping of cod in the study area into three main populations: north-east Arctic cod (NEAC), Norwegian coastal cod (NCC) and North Sea cod (NSC). Microsatellites, however, revealed genetic heterogeneity not only within NCC, as did Pan I, but also within NEAC and NSC, which appeared to be homogenous when analysed for Pan I. Moreover, microsatellites displayed lower levels of differentiation than Pan I between NEAC and two other groups. Differences in the magnitude of differentiation for the two types of markers may be attributable to higher levels of polymorphism and alleged selective neutrality of microsatellites. Isolation by distance was clearly apparent for microsatellites but was less evident for Pan I, indicating that environmentally induced selection appears to shape the patterns of genetic differentiation for this marker. Even though the population structure of north-east Atlantic cod, as revealed by microsatellites and Pan I, appears to be maintained largely by restricted gene flow, selection acting on a recent historical time scale probably contributes to the observed geographic pattern of Pan I frequencies.     

Two adjacent inversions maintain genomic differentiation between migratory and stationary ecotypes of Atlantic cod

Atlantic cod is composed of multiple migratory and stationary populations widely distributed in the North Atlantic Ocean. The Northeast Arctic cod (NEAC) population in the Barents Sea undertakes annual spawning migrations to the northern Norwegian coast. Although spawning occurs sympatrically with the stationary Norwegian coastal cod (NCC), phenotypic and genetic differences between NEAC and NCC are maintained. In this study, we resolve the enigma by revealing the mechanisms underlying these differences. Extended linkage disequilibrium (LD) and population divergence were demonstrated in a 17.4‐Mb region on linkage group 1 (LG1) based on genotypes of 494 SNPs from 192 parents of farmed families of NEAC, NCC or NEACxNCC crosses. Linkage analyses revealed two adjacent inversions within this region that repress meiotic recombination in NEACxNCC crosses. We identified a NEAC‐specific haplotype consisting of 186 SNPs that was fixed in NEAC sampled from the Barents Sea, but segregating under Hardy–Weinberg equilibrium in eight NCC stocks. Comparative genomic analyses determine the NEAC configuration of the inversions to be the derived state and date it to ~1.6–2.0 Mya. The haplotype block harbours 763 genes, including candidates regulating swim bladder pressure, haem synthesis and skeletal muscle organization conferring adaptation to long‐distance migrations and vertical movements down to large depths. Our results suggest that the migratory ecotype experiences strong directional selection for the two adjacent inversions on LG1. Despite interbreeding between NEAC and NCC, the inversions are maintaining genetic differentiation, and we hypothesize the co‐occurrence of multiple adaptive alleles forming a ‘supergene’ in the NEAC population.     

Trophically transmitted parasites in wild Atlantic salmon post‐smolts from Norwegian fjords

The community structure of trophically transmitted intestinal helminths of Atlantic salmon Salmo salar post‐smolts was highly variable among four fjords in Norway. There were no severely pathogenic parasite species. Post‐smolts from the southernmost Trondheimsfjord had a higher diversity of freshwater parasite species compared to the three northern fjords (Tanafjord, Altafjord and Malangen). In contrast, the highest diversity and proportion of marine species was found in the three northern fjords. Post‐smolts were generally more infected with marine parasites in the outer rather than inner parts of all of the fjords. The prevalence of the acanthocephalan Echinorynchus gadi (range: 13–42%) and marine trematodes (range: 14–47%) was higher in post‐smolts in outer zones of the northern fjords than in fish from Trondheimsfjord (0 and 6%, respectively). The within‐fjord variability and north‐south geographical gradient in parasite infection patterns reflected differences in marine feeding of the post‐smolts on potential intermediate hosts such as amphipods ( E. gadi ) and fish larvae (trematodes), which were higher in the northern fjords (range: 27–28 and 67–85%, respectively) than in Trondheimsfjord (5 and 19%, respectively). High intensities of marine parasites suggest that some post‐smolts from northern fjords may have a prolonged fjord‐feeding compared to those from Trondheimsfjord. Parasites of both freshwater and marine origin appear to be suitable as bio‐indicators of feeding and migratory pattern of Atlantic salmon post‐smolts and preadults during their seaward migration.     

The development of haddock and Atlantic cod sperm cryopreservation techniques and the effect of sperm age on cryopreservation success

Three cryoprotectants [dimethyl sulphoxide (DMSO), propylene glycol (PG) and glycerol], two diluents (sucrose‐ and saline‐based), two sperm collection times, two freezing rates and three times between thaw and activation (0, 30 and 60 min) were tested in order to develop a protocol for the cryopreservation of sperm of haddock Melanogrammus aeglefinus and Atlantic cod Gadus morhua . The faster freezing rate resulted in extremely low post‐thaw motility in comparison to the slower freezing rate, which was successful for sperm from both gadids. In both cases, the use of PG resulted in significantly higher post‐thaw sperm motility‐recovery indices than with DMSO or glycerol, which did not differ significantly from one another. Diluent had no effect on post‐thaw sperm motility for Atlantic cod or haddock. Sperm collected at the end of the spawning season tended to have reduced post‐thaw motility compared to that collected 2 weeks after the start of spawning. A 30 min delay between thaw and activation of haddock and Atlantic cod sperm resulted in a significant decrease in sperm motility. When PG was used as cryoprotectant, sperm motility continued to decrease between 30 and 60 min post‐thaw. With DMSO or glycerol as cryoprotectant, motilities were already very low after 30 min post‐thaw and did not decrease any further after 60 min. Cryoprotectant, diluent and time between thaw and activation had no effect on mean or maximum sperm swimming speeds for either Atlantic cod or haddock sperm. Fertilization success for haddock eggs, like sperm motility, was higher with PG‐frozen sperm than DMSO‐ or glycerol‐frozen sperm. These results constitute the first reported successful cryopreservation of haddock sperm and improve on previous methods used to cryopreserve sperm from Atlantic cod.     

Genetic divergence at the synaptophysin (Syp I) locus among Norwegian coastal and north-east Arctic populations of Atlantic cod

Several nuclear RFLP loci have been discovered recently that exhibit extensive allele frequency variation among Norwegian coastal and north-east Arctic populations of Atlantic cod Gadus morhua. One of these polymorphisms was detected by hybridizing an anonymous cDNA clone (GM798) against genomic DNA digested with the restriction enzyme DraI. This cDNA clone has now been sequenced and identified as synaptophysin (Syp I), an integral synaptic vesicle membrane protein. Primers were constructed that amplify an intron of the Syp I gene that is polymorphic for the DraI site, thus making it possible to use a PCR-based assay to score the polymorphism. A total of 965 individuals sampled from the Barents Sea, coastal areas and fjords in northern Norway have been analysed for this polymorphism. The results confirm that highly significant differences exist between NE Arctic and coastal cod at the Syp I locus. A cluster analysis revealed a deep split between coastal and Arctic populations and hierarchical F-statistics indicated that about 40% of the total variation was attributable to differences between Arctic and coastal groups. The temporal stability of allele frequencies was assessed by comparing Syp I allele frequencies among samples of juveniles (0 group) captured at specific locations in fjords in consecutive years and among samples of adults and juveniles collected from the same fjord. Samples of juveniles collected in 1994 and 1995 in Malangen were genetically indistinguishable whereas juveniles sampled from Dønnesfjord and Ullsfjorden over the same 2-year period exhibited significant differences. Adults and 0-group individuals collected from the same fjord were found to be genetically indistinguishable in Malangen, but not in Balsfjorden. In addition to detecting large differences among Arctic and coastal groups, the Syp I locus suggests that genetic heterogeneity exists among resident populations of cod in different fjords and that gene flow among populations throughout northern Norway may be considerably lower than previously believed.     

Genotyping of pantophysin I (Pan I) of Atlantic cod (Gadus morhua L.) by allele‐specific PCR

The two main allelic variants of the Atlantic cod (Gadus morhua L.) pantophysin I (Pan I) locus have different frequencies within different cod stocks. The Dra I polymorphism which distinguishes the two alleles can thus be used for discrimination of coastal and offshore cod populations. We present a new method for Pan I genotyping using fluorescent allele‐specific duplex polymerase chain reaction (PCR). This method is more rapid, reliable and cost‐effective than the previously published method and it is not affected by DNA source and quality. This improvement is important for studies demanding high throughput and accuracy of Pan I genotyping     

Survival and behaviour of post‐smolt Atlantic salmon in coastal habitat with extreme tides

The early marine migration of 55 Atlantic salmon post‐smolts tagged with acoustic transmitters was automatically monitored using 13 to 25 km long arrays of receivers deployed inside the Bay of Fundy, a coastal system on the east coast of Canada. The survival of post‐smolts from groups with short‐ and long‐term transmitters in coastal habitat up to 10 km from the river was 92 to 100%, indicating a successful transition to salt water and departure. Migration for 68 to 77% of post‐smolts followed a direct route and it was rapid (transit time usually <12 h). Post‐smolts initially migrated in a south to south‐west direction ( i.e . orientation towards the mouth of the bay) and they were aggregated near the coast. Post‐smolts with long‐term transmitters were monitored 20 km from the river where they continued to be aggregated, moving near the coast through a 'common corridor', and their survival to that point was at least 84%. Post‐smolts from both groups travelled heading out of the coastal system during ebb tides. Flood tides interrupted migration, and they caused changes in travel direction and delays in departure for post‐smolts not leaving by a direct route. Monitoring of coastal habitat inside the Bay of Fundy intercepted 62% of migrating post‐smolts with long‐term transmitters returning after an initial absence of 2 to 22 days. Returning post‐smolts displayed a resident behaviour, using the habitat monitored inside the Bay of Fundy during July and August.     

Recolonization history and large-scale dispersal in the open sea: the case study of the North Atlantic cod, Gadus morhua L.

Most studies of the genetic structure of Atlantic cod have focused on small geographical scales. In the present study, the genetic structure of cod sampled on spawning grounds in the North Atlantic was examined using eight microsatellite loci and the Pan I locus. A total of 954 cod was collected from nine different regions: the Baltic Sea, the North Sea, the Celtic Sea, the Irish Sea and Icelandic waters during spring 2002 and spring 2003, from Norwegian waters and the Faroe Islands (North and West spawning grounds) in spring 2003, and from Canadian waters in 1998. Temporal stability among spawning grounds was observed in Icelandic waters and the Celtic Sea, and no significant difference was observed between the samples from the Baltic Sea and between the samples from Faroese waters. F -statistics showed significant differences between most populations and a pattern of isolation-by-distance was described with microsatellite loci. The Pan I locus revealed the presence of two genetically distinguishable basins, the North-west Atlantic composed of the Icelandic and Canadian samples and the North-east Atlantic composed of all other samples. Permutation of allele sizes at each microsatellite locus among allelic states supported a mutational component to the genetic differentiation, indicating a historical origin of the observed variation. Estimation of the time of divergence was approximately 3000 generations, which places the origin of current genetic pattern of cod in the North Atlantic in the late Weichselian (Wisconsinian period), at last glacial maximum.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 315–329.     

Geographical differences in marine feeding of Atlantic salmon post‐smolts in Norwegian fjords

Stomach content analyses were conducted on Atlantic salmon Salmo salar post‐smolt (average size, 119–154 mm fork length, L _F) caught in eight large Norwegian fjord systems along a north–south geographical axis during 1998–2001. In general, post‐smolts from southern Norway showed low feeding intensity in the fjords, whereas extensive feeding was observed in fjords in the northern and middle parts of Norway. The marine diet mainly included different crustaceans and in particular marine pelagic fish larvae (sand-eels Ammodytes spp., herring Clupea harengus and gadoids), but with a substantial spatial and annual variation in prey diversity and feeding intensity. Insects were most frequently taken in the estuary, although fishes often made a large contribution in mass. In contrast, fishes, and to some extent various crustaceans (particularly Hyperiidae, Gammaridae, Euphausiacea and Copepoda) dominated the diet in the middle and outer parts of the fjords, where post‐smolts also fed more extensively than in the inner part. The results indicate that extensive feeding immediately after sea entrance may be more common for post‐smolts in the northern and middle parts of Norway, than in the southern fjords. The observed differences in post‐smolt feeding may be due to spatial and temporal differences in prey availability within and between the different types of fjord systems, and this might influence post‐smolt growth and survival.     

Natural selection and the genetic differentiation of coastal and Arctic populations of the Atlantic cod in northern Norway: a test involving nucleotide sequence variation at the pantophysin (PanI) locus

To examine the role of contemporary selection in maintaining significant allele frequency differences at the pantophysin (PanI) locus among populations of the Atlantic cod, Gadus morhua, in northern Norway, we sequenced 127 PanIA alleles sampled from six coastal and two Barents Sea populations. The distributions of variable sites segregating within the PanIA allelic class were then compared among the populations. Significant differences were detected in the overall frequencies of PanIA alleles among populations within coastal and Arctic regions that was similar in magnitude to heterogeneity in the distributions of polymorphic sites segregating within the PanIA allelic class. The differentiation observed at silent sites in the PanIA allelic class contradicts the predicted effects of widescale gene flow and suggests that postsettlement selection acting on cohorts cannot be responsible for the genetic differences described between coastal and Arctic populations. Our results suggest that the marked differences observed between coastal and Arctic populations of G. morhua in northern Norway at the PanI locus reflect the action of recent diversifying selection and that populations throughout the region may be more independent than suggested by previous studies.     

Long-term studies on genetic interaction between wild and ranched cod Gadus morhua by use of a genetic marked strain

Releases of farmed fish, whether accidental from commercial aquaculture facilities or intentional as part of stock enhancement/ranching activities, are considered to pose a risk to native gene pools. Stock enhancement studies of Atlantic cod, Gadus morhua , based on artificially produced juveniles, were initiated in 1984 in western Norway, and genetic aspects were incorporated. In order to investigate potential interbreeding between released and wild cod, a genetically marked cod strain was developed, being homozygotic for a rare allele ( GPI‐1*30 ) expressed in white muscle tissue. In the period from 1990 to 1994, juveniles from the genetic marked strain were released in large quantities in three locations (Masfjord, Øygarden, Heimarkspollen), giving a significant increase of the marker allele in the local wild cod populations. Recently, studies have been conducted in the same areas to estimate the extent of interbreeding between the wild and released cod. The results, however, revealed no permanent increase of the frequency of the marker allele and/or GPI‐1*30 heterozygotes as would be expected from interbreeding. The recent data are compared with comprehensive genetic data of the cod populations in the areas before the actual releases, covering the full period from 1994 to 2003. The present results are also discussed in relation to fishing pressure on coastal cod, migration information and reproductive success of released, genetically marked cod.     

Micropogonias undulatus(L.), another exotic arrival in European waters

Two living specimens of the Atlantic croaker Micropogonias undulatus have been caught during recent years in Belgian waters (Southern Bight of the North Sea): one in August 1998 as a by‐catch of the commercial shrimp fishery in coastal waters, another in October 2001 on a tidal flat in the brackish part of the Scheldt Estuary. These represent the first records of this north‐west Atlantic species in European waters, and their occurrence is likely to be due to transportation in ship ballast water.     

A review of the genus Malakichthys Döderlein (Perciformes: Acropomatidae) with the description of a new species

Malakichthys Döderlein, 1883, comprises seven species: type species Malakichthys griseus Döderlein 1883, from southern Japan to north‐western Australia; Malakichthys barbatus Yamanoue & Yoseda, 2001 , from southern Japan, the South China Sea, north‐western Australia and the Coral Sea; Malakichthys elegans Matsubara & Yamaguti, 1943 , from southern Japan south to the Arafura Sea; Malakichthys levis Yamanoue & Matsuura, 2001 , from northern Australia; Malakichthys mochizuki Yamanoue & Matsuura, 2001 , from the Coral Sea; Malakichthys similis sp. nov. from the Bohol Sea, Philippines; Malakichthys wakiyae Jordan & Hubbs, 1925 , from southern Japan and Taiwan. Species of Malakichthys are very similar to each other in general appearance but are distinguishable by spination on the lower jaw, features of the proximal‐middle radial of the first anal‐fin pterygiophore, the number of anal‐fin soft rays and body depth.     

Feeding ecology of silver hake larvae on the Western Bank, Scotian Shelf, and comparison with Atlantic Cod

The feeding ecology of the larvae of silver hake Merluccius bilinearis was examined during two time periods (October 1998 and December 1992) on the Western Bank, Scotian Shelf, north‐west Atlantic, and compared with the feeding ecology of Atlantic cod Gadus morhua larvae collected in the same samples in December 1992. During both time periods silver hake exhibited strong selection for late stage copepodids and adult copepods at a small size (>3·5 mm total length, L _T). The niche width measured as the diet breadth index ( I _DB) of silver hake declined rapidly as they increased in size and remained relatively constant from 3 to 11 mm L _T, during each time period. Atlantic cod larvae exhibited a broader niche width that was curvilinear over the same L _T. Atlantic cod were also less selective than silver hake, incorporating both naupliar and early stage copepodids in their diets throughout the length classes examined. Simple isometric relationships did not explain the differences in diet, as Atlantic cod larvae continued to feed on early stages of copepods at large size, while silver hake larvae quickly switched to large prey items. The strong selection and narrow I _DB observed for silver hake probably reflects adaptation to spawning during the periods between major secondary production peaks in temperate waters.     

Nucleotide polymorphism and natural selection at the pantophysin (Pan I) locus in the Atlantic cod, Gadus morhua (L.)

Molecular studies of nucleotide sequence variation have rarely attempted to test hypotheses related to geographically varying patterns of natural selection. The present study tested the role of spatially varying selection in producing significant linkage disequilibrium and large differences in the frequencies of two common alleles at the pantophysin (Pan I) locus among five populations of the Atlantic cod, Gadus morhua. Nucleotide sequences of 124 Pan I alleles showed strong evidence for an unusual mix of balancing and directional selection but no evidence of stable geographically varying selection. The alleles were highly divergent at both the nucleotide level (differing on average by 19 mutations) and at amino acid level (each having experienced three amino acid substitutions since diverging from a common ancestral allele). All six amino acid substitutions occurred in a 56-residue intravesicular loop (IV1 domain) of the vesicle protein and each involved a radical change. An analysis of molecular variation revealed significant heterogeneity in the frequencies of recently derived mutations segregating within both allelic classes, suggesting that two selective sweeps may be presently occurring among populations. The dynamic nature of the Pan I polymorphism in G. morhua and clear departure from equilibrium conditions invalidate a simple model of spatially varying selection.     

Genetic comparison of salmon from the White Sea and north-western Atlantic Ocean

Samples of salmon Salmo salar from the River Kachkovka and the River Nilma in northern Russia were analysed by starch gel electrophoresis and compared to three Norwegian stocks, the Neiden river in northern Norway and Øyreselv and Hopselv rivers on the west coast. The comparison included the following polymorphic loci: AAT-4 *, IDDH-2 *, IDHP-3 *, MDH- 3,4 *, MEP-2 *, ESTD * as well as the newly discovered polymorphic loci FBALD-3 * and TPI-3 *. Samples were run side by side on gels, and the alleles found in the Russian stocks were the same as those found in the Norwegian stocks, although the electrophoretic methods used lead to differences in designations of alleles. A polymorphism in ESTD * which involves a slow allele was commonly observed in the three northern populations of the Nilma, Kachkovka and Neiden rivers. This allele was absent in the other Norwegian stocks and in a major brood stock of farmed salmon in Norway. The IDHP-3 * 116 allele was found in unusually high frequencies in the northern populations. Thus, the variability observed at these two loci indicates a barrier to gene flow between the northern salmon stocks and the more southern stocks in the East Atlantic area.     

A first record of Isistius plutodus in the north‐eastern Atlantic

One specimen of the largetooth cookiecutter shark Isistius plutodus was caught in the north‐eastern Atlantic at 43°58' N; 28°32' W. This is the first record of this rare species in the north‐eastern Atlantic and the northernmost point of its known distribution.     

Validation of daily otolith increment formation in the larval myctophid fish Diaphus slender‐type spp.

The marginal increment growth in sagittal otoliths of the myctophid larvae Diaphus slender‐type spp. collected in the north‐eastern East China Sea was examined based on specimens collected at different times of the day. The calcification of the incremental zone began after midnight and took place throughout the day, and the discontinuous zone was formed only around midnight. These results indicate that otolith increments are formed daily.     

Transport of North Sea cod larvae into the Skagerrak coastal populations

The Atlantic cod (Gadus morhua) is economically one of the world's most important marine species--a species presently suffering from heavy overexploitation throughout its range of distribution. Although not fully understood, the Atlantic cod is believed to be structured into populations in a rather complex manner, whereby both highly migratory and more confined ocean-spawning stocks coexist with stationary coastal populations. Owing to the complex population structure, little is presently known about how overexploitation of offshore stocks may affect other segments of the species. Here, we use microsatellite DNA analyses of coastal and offshore cod in combination with oceanographic modelling to investigate the population structure of Atlantic cod in the North Sea-Skagerrak area and evaluate the potential for larval transport into coastal populations. Our results suggest an extensive but temporally variable drift of offshore cod larvae into coastal populations. In a year (2001) with high inflow of North Sea waters into the Skagerrak we find that juvenile cod caught along the Skagerrak coast are predominantly of North Sea origin, whereas in a year (2000) with low inflow juveniles appear to be of local origin. These findings indicate that offshore cod may influence coastal cod populations over large distances.