Polychlorinated biphenyl (PCB) load, lipid reserves and biotransformation activity in migrating Atlantic salmon from River Mörrum, Sweden.

Atlantic salmon accumulate high levels of contaminants such as polychlorinated biphenyls (PCBs) in their lipids during the adult growth phase spent at sea. The lipids are later utilized during migration for swimming and biological adaptations. We hypothesize that migrating salmons' biotransformation processes are affected by the high levels of built-up PCBs compared to salmon that in a pre-migrational stage. For these analyses we sampled adult Atlantic salmon during migration in the Swedish River Mörrum and measured the 21 most common PCB congeners ( summation operatorPCB) and lipid levels in muscle tissue, aryl hydrocarbon receptor (AHR2) and cytochrome P4501A1 (CYP1A1) transcript levels as well as ethoxyresorufin-O-deethylase activity (EROD) in liver. We also determined which AHR2 genotypes the salmon carried. We show that EROD activity is correlated to CYP1A1 level but not to summation operatorPCB concentration. summation operatorPCB concentration does not predict levels of neither the AHR2 nor CYP1A1 genes. We find no associations between specific AHR2 transcription levels and AHR2 genotypes or a correlation between AHR2 and CYP1A1 transcription levels, which is in direct contrast to pre-migrational adult salmon from the Baltic Sea. When we compare River Mörrum to salmon we have previously sampled in the Baltic Sea we show that migrating salmon have significantly lower lipid levels in their muscles; higher muscle concentrations of summation operatorPCB on a lipid basis; and significantly lower CYP1A1 and EROD levels compared to salmon from the Baltic Sea. Also, transcript levels of three out of four AHR2 genes are significantly different. In conclusion, migrating Swedish Atlantic salmon carry higher concentrations of PCBs in their lipids compared to salmon in the Baltic Sea, but have lower activation of biotransformation genes and enzymes. Our results indicate that accumulated pollutants from the Baltic Sea are deactivated inside the migrating salmon's lipid tissues and increase in concentration when migration is initiated thereby limiting their impact on biotransformation processes.

TECHNICAL ADVANCES: A microarray for large-scale genomic and transcriptional analyses of the zebra finch (Taeniopygia guttata) and other passerines.

The microarray technology has revolutionized biological research in the last decade. By monitoring the expression of many genes simultaneously, microarrays can elucidate gene function, as well as scan entire genomes for candidate genes encoding complex traits. However, because of high costs of sequencing and design, microarrays have largely been restricted to a few model species. Cross-species microarray (CSM) analyses, where microarrays are used for other species than the one they were designed for, have had varied success. We have conducted a CSM analysis by hybridizing genomic DNA from the common whitethroat (Sylvia communis) on a newly developed Affymetrix array designed for the zebra finch (Taeniopygia guttata), the Lund-zf array. The results indicate a very high potential for the zebra finch array to act as a CSM utility in other passerine birds. When hybridizing zebra finch genomic DNA, 98% of the gene representatives had higher signal intensities than the background cut-off, and for the common whitethroat, we found the equivalent proportion to be as high as 96%. This was surprising given the fact that finches and warblers diverged 25-50 million years ago, but may be explained by a relatively low sequence divergence between passerines (89-93%). Passerine birds are widely used in studies of ecology and evolution, and a zebra finch array that can be used for many species may have a large impact on future research directions.

Polychlorinated biphenyl load, aryl hydrocarbon receptor, and cytochrome P4501A1 induction in a wild population of Atlantic salmon (Salmo salar) from the Baltic Sea.

The toxicity induced by several environmental pollutants is mediated by the aryl hydrocarbon receptor (AHR), which controls the expression of many biotransformation genes, such as cytochrome P4501A1 (CYP1A1). Previous studies have indicated that fish populations can evolve tolerance to persistent chlorinated pollutants by down-regulating the AHR pathway. Here, we measure to what extent tissue loads of polychlorinated biphenyl (PCB) congeners and AHR genotypes contribute to biotransformation capacity in wild, foraging Atlantic salmon (Salmo salar L.) from the Baltic Sea. In muscle, the sum of the 21 most common PCB congeners (ZPCB) was correlated with three extracted AHR agonists (PCBs 77/110, 118/123/149, and 105/132/153). Both the AHR agonists as well as sigmaPCB were correlated with lipid content. The sigmaPCB, controlled for the effects of sex and lipid content in muscle tissue, did not predict mRNA transcript levels of the measured AHRs (AHR2alpha, AHR2gamma, and AHR2delta) or CYP1A1 in liver. However, all AHR2 mRNA transcript levels were positively correlated with CYP1A1 level. In turn, the CYP1A1 level was negatively correlated with concentration of the muscle-tissue antioxidant astaxanthin, suggesting that astaxanthin is depleted when biotransformation processes (CYP1A1) are activated. No correlation was found between ethoxyresorufin-O-deethylase activity and sigmaPCB, CYP1A1, or antioxidant levels. In 5'-flanking regions of the AHR2 genes, we identified multiple allelic variants that were used for genotyping. The mRNA transcript level of AHR2alpha was significantly associated with the AHR2alpha 5'-flanking region genotype and with the interaction of the genotype and individual PCB level. These results suggest that in wild Atlantic salmon from the Baltic Sea, active production of AHR2 mRNA by means of PCB exposure may be affected by genetic polymorphisms at the AHR2 loci.

Associations between malaria and MHC genes in a migratory songbird.

Malaria parasites are a widespread and species-rich group infecting many wild populations of mammals, birds and reptiles. Studies on humans have demonstrated that genetic factors play a key role in the susceptibility and outcome of malaria infections. Until the present study, it has not been examined whether genetic variation in hosts is important for the outcome of malaria infections in natural avian populations. We investigated associations between major histocompatibility complex (MHC) genes and prevalence of three different avian malaria parasites (Haemoproteus payevskyi (GRW1), Plasmodium sp. (GRW2) and Plasmodium sp. (GRW4)) in a long-term study of great reed warblers Acrocephalus arundinaceus. We hypothesized that the MHC genes could either give full protection against a malaria infection, or confer protection against lethal malaria and direct the infection towards being milder. We found a positive association between numbers of MHC class I alleles (a measure of level of heterozygosity) and prevalence of the GRW2 parasite, suggesting the latter scenario. There was also a positive association between a specific MHC allele (B4b), previously shown to be under frequency-dependent selection in the study population, and prevalence of GRW2. These associations suggest that individuals carrying either a large number of MHC alleles or a specific MHC allele are protected against lethal malaria infections.

MHC-based patterns of social and extra-pair mate choice in the Seychelles warbler.

The existence and nature of indirect genetic benefits to mate choice remain contentious. Major histocompatibility complex (MHC) genes, which play a vital role in determining pathogen resistance in vertebrates, may be the link between mate choice and the genetic inheritance of vigour in offspring. Studies have shown that MHC-dependent mate choice can occur in mammal and fish species, but little work has focused on the role of the MHC in birds. We tested for MHC-dependent mating patterns in the Seychelles warbler (Acrocephalus sechellensis). There was no influence of MHC class I exon 3 variation on the choice of social mate. However, females were more likely to obtain extra-pair paternity (EPP) when their social mate had low MHC diversity, and the MHC diversity of the extra-pair male was significantly higher than that of the cuckolded male. There was no evidence that females were mating disassortatively, or that they preferred males with an intermediate number of MHC bands. Overall, the results are consistent with the 'good genes' rather than the 'genetic compatibility' hypothesis. As female choice will result in offspring of higher MHC diversity, MHC-dependent EPP may provide indirect benefits in the Seychelles warbler if survival is positively linked to MHC diversity.

Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.).

Aryl hydrocarbon receptor (AhR) genes encode proteins involved in mediating the toxic responses induced by several environmental pollutants. Here, we describe the identification of the first two AhR1 (alpha and beta) genes and two additional AhR2 (alpha and beta) genes in the tetraploid species Atlantic salmon (Salmo salar L.) from a cosmid library screening. Cosmid clones containing genomic salmon AhR sequences were isolated using a cDNA clone containing the coding region of the Atlantic salmon AhR2gamma as a probe. Screening revealed 14 positive clones, from which four were chosen for further analyses. One of the cosmids contained genomic AhR sequences that were highly similar to the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes. SMART RACE amplified two complete, highly similar but not identical AhR type 2 sequences from salmon cDNA, which from phylogenetic analyses were determined as the rainbow trout AhR2alpha and beta orthologs. The salmon AhR2alpha and beta encode proteins of 1071 and 1058 residues, respectively, and encompass characteristic AhR sequence elements like a basic-helix-loop-helix (bHLH) and two PER-ARNT-SIM (PAS) domains. Both genes are transcribed in liver, spleen and muscle tissues of adult salmon. A second cosmid contained partial sequences, which were identical to the previously characterized AhR2gamma gene. The last two cosmids contained partial genomic AhR sequences, which were more similar to other AhR type 1 fish genes than the four characterized salmon AhR2 genes. However, attempts to amplify the corresponding complete cDNA sequences of the inserts proved very difficult, suggesting that these genes are non-functional or very weakly transcribed in the examined tissues. Phylogenetic analyses of the conserved regions did, however, clearly indicate that these two AhRs belong to the AhR type 1 clade and have been assigned as the Atlantic salmon AhR1alpha and AhR1beta genes. Taken together, these findings demonstrate that multiple AhR genes are present in Atlantic salmon genome, which likely is a consequence of previous genome duplications in the evolutionary past of salmonids. Plausible explanations for the high incidence of AhR genes in fish and more specifically in salmonids, like rapid divergences in specialized functions, are discussed.

Characterization of two distinct aryl hydrocarbon receptor (AhR2) genes in Atlantic salmon (Salmo salar) and evidence for multiple AhR2 gene lineages in salmonid fish.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of several environmental contaminants, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin, and other halogenated hydrocarbons in vertebrates. This receptor initiates the transcription of several biotransformation enzymes, which in turn are responsible for causing severe harm to biological tissue. Here we describe the isolation and complete characterization of the first two AhR genes from the teleost fish Atlantic salmon (Salmo salar). The predicted amino acid sequences contain regions characteristic of other vertebrate AhRs including basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains but show little similarity to other vertebrate AhRs across the C-terminal half. Furthermore, they do not contain distinct Q-rich domains as found in the mammalian AhR, which is in line with previously described fish AhR genes. The salmon cDNAs encode 1106 and 1107 putative residues, respectively, approximately 50 amino acids longer than previously characterized AhR genes. Phylogenetic analyses demonstrated that the two salmon AhR sequences cluster within the AhR subfamily of the bHLH-PAS family, in a clade containing fish AhR2 genes. Although the two AhR2 forms are 92% identical at the amino acid level, the distribution of sequence differences and the presence of both forms in 30 tested individuals suggest that they are not allelic but derived from separate loci. Interestingly, they are not orthologs of the rainbow trout (Oncorhynchus mykiss) AhR2 alpha and beta genes and the new salmon loci are therefore here designated AhR2 gamma and AhR2 delta. In line with this, PCR with DNA from rainbow trout revealed a new trout AhR locus that was more similar to the two salmon genes than to the trout AhR2 alpha and beta genes, suggesting that the rainbow trout possesses at least three distinct AhR2 genes. The presence of multiple AhR genes in these species is probably a consequence of the genome duplications that occurred in the early evolution of fish and later also specifically in the salmonid lineage. Reverse transcription-PCR analyses revealed that both AhR2 gamma and AhR2 delta are transcribed in the liver, spleen and muscles of adult salmon.

Experimental evidence for major histocompatibility complex-allele-specific resistance to a bacterial infection.

The extreme polymorphism found at some major histocompatibility complex (MHC) loci is believed to be maintained by balancing selection caused by infectious pathogens. Experimental support for this is inconclusive. We have studied the interaction between certain MHC alleles and the bacterium Aeromonas salmonicida, which causes the severe disease furunculosis, in Atlantic salmon (Salmo salar L.). We designed full-sibling broods consisting of combinations of homozygote and heterozygote genotypes with respect to resistance or susceptibility alleles. The juveniles were experimentally infected with A. salmonicida and their individual survival was monitored. By comparing full siblings carrying different MHC genotypes the effects on survival due to other segregating genes were minimized. We show that a pathogen has the potential to cause very intense selection pressure on particular MHC alleles; the relative fitness difference between individuals carrying different MHC alleles was as high as 0.5. A co-dominant pattern of disease resistance/susceptibility was found, indicative of qualitative difference in the immune response between individuals carrying the high- and low-resistance alleles. Rather unexpectedly, survival was not higher among heterozygous individuals as compared with homozygous ones.

GENETIC SIMILARITY BETWEEN PARENTS PREDICTS HATCHING FAILURE: NONINCESTUOUS INBREEDING IN THE GREAT REED WARBLER?

The DNA-fingerprinting technique was used to find the true pedigrees and to detect the overall genetic similarity between mates of great reed warblers (Acrocephalus arundinaceus) at an isolated breeding site in Sweden. The study covered 4 yr preceded by 3 yr when almost all adults and nestlings in the study area had been banded. DNA fingerprinting revealed that the putative father had sired 97% of the young (N = 455). The mate's genetic similarity, revealed as the proportion of bands shared in restriction fragment length polymorphism (RFLP) patterns, was high compared with other species of wild birds. Also, band sharing was higher between mates native to the area than between pairs in which the female was experimentally introduced from a distant breeding site. Hatching success of eggs was negatively correlated with the degree of genetic similarity between the mates, whereas pedigree data, up to the level of great-grandparents, clearly demonstrated an absence of close inbreeding. These are the first data showing a significant fitness cost associated with the choice of a mate that has high genetic similarity, even if it is not a close kin. This cost might be caused by generalized negative consequences of genomewide inbreeding in the present study, possibly accentuated by recent population bottlenecks.