Transcriptome of the dead: characterisation of immune genes and marker development from necropsy samples in a free-ranging marine mammal

Background Transcriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism. However, because many genes have tissue-specific expression patterns, developing a complete transcriptome usually requires a 'discovery pool' of individuals to be sacrificed in order to harvest mRNA from as many different types of tissue as possible. This hinders transcriptome development in large, charismatic and endangered species, many of which stand the most to gain from such approaches. To circumvent this problem in a model pinniped species, we 454 sequenced cDNA from testis, heart, spleen, intestine, kidney and lung tissues obtained from nine adult male Antarctic fur seals (Arctocephalus gazella) that died of natural causes at Bird Island, South Georgia. Results After applying stringent quality control criteria based on length and annotation, we obtained 12,397 contigs which, in combination with 454 data previously obtained from skin, gave a total of 23,096 unique contigs. Homology was found to 77.0% of dog (Canis lupus familiaris) transcripts, suggesting that the combined assembly represents a substantial proportion of this species' transcriptome. Moreover, only 0.5% of transcripts revealed sequence similarity to bacteria, implying minimal contamination, and the percentage of transcripts involved in cell death was low at 2.6%. Transcripts with immune-related annotations were almost five-fold enriched relative to skin and represented 13.2% of all spleen-specific contigs. By reference to the dog, we also identified transcripts revealing homology to five class I, ten class II and three class III genes of the Major Histocompatibility Complex and derived the putative genomic distribution of 17,121 contigs, 2,119 in silico mined microsatellites and 9,382 single nucleotide polymorphisms. Conclusions Our findings suggest that transcriptome development based on samples collected post mortem may greatly facilitate genomic studies, not only of marine mammals but also more generally of species that are of conservation concern

Avaluació de la població de llagostes (Palinurus elephas) de les Illes Medes. Exercici 1995

Podeu consultar l'Informe complet a: http://hdl.handle.net/2445/2466

Experimental validation of in silico predicted RAD locus frequencies using genomic resources and short read data from a model marine mammal

Vendrami D, Forcada J, Hoffman J. Experimental validation of in silico predicted RAD locus frequencies using genomic resources and short read data from a model marine mammal. BMC Genomics. 2019;20(1): 72

Long term movements and activity patterns of an Antarctic marine apex predator: the Leopard Seal

Leopard seals are an important Antarctic apex predator that can affect marine ecosystems through local predation. Here we report on the successful use of micro geolocation logging sensor tags to track the movements, and activity, of four leopard seals for trips of between 142–446 days including one individual in two separate years. Whilst the sample size is small the results represent an advance in our limited knowledge of leopard seals. We show the longest periods of tracking of leopard seals’ migratory behaviour between the pack ice, close to the Antarctic continent, and the sub-Antarctic island of South Georgia. It appears that these tracked animals migrate in a directed manner towards Bird Island and, during their residency, use this as a central place for foraging trips as well as exploiting the local penguin and seal populations. Movements to the South Orkney Islands were also recorded, similar to those observed in other predators in the region including the krill fishery. Analysis of habitat associations, taking into account location errors, indicated the tracked seals had an affinity for shallow shelf water and regions of sea ice. Wet and dry sensors revealed that seals hauled out for between 22 and 31% of the time with maximum of 74 hours and a median of between 9 and 11 hours. The longest period a seal remained in the water was between 13 and 25 days. Fitting GAMMs showed that haul out rates changed throughout the year with the highest values occurring during the summer which has implications for visual surveys. Peak haul out occurred around midday for the months between October and April but was more evenly spread across the day between May and September. The seals’ movements between, and behaviour within, areas important to breeding populations of birds and other seals, coupled with the dynamics of the region’s fisheries, shows an understanding of leopard seal ecology is vital in the management of the Southern Ocean resources

A bioenergetics model for estimating prey consumption by an Adélie penguin population in East Antarctica

Quantifying prey consumption by top predators is a crucial component of ecosystem-based management in the Southern Ocean. In this study, we developed a bioenergetics model to estimate prey consumption by a top predator, the Adélie penguin Pygoscelis adeliae. Our model predicts prey consumption throughout the breeding season and incorporates uncertainty in model parameters using Monte Carlo simulation. The model was parameterized with data obtained at Béchervaise Island, the site of a long-term monitoring program in East Antarctica. We parameterized the model (1) using 13 yr of penguin population data, (2) for a year in which penguins successfully reared their chicks (2001-2002) and (3) for a year with low breeding success (1998-1999). Daily per capita energy consumption during the breeding season averaged 4269 kJ d-1 (95% CI: 4187-4352 kJ d–1) and 4684 kJ d-1 (95% CI: 4596-4771 kJ d–1) for males and females, respectively. Over the entire breeding season a male breeder consumes 470 MJ (95% CI: 461-479 MJ) compared to 515 MJ (95% CI: 506-525 MJ) for a female. On average, the Béchervaise Island population of 1836 breeding pairs consumes 16447 MJ d-1 which amounts to 1809224 MJ during the breeding season. On the basis of variable breeding success and the proportion of krill and fish in their diet, we estimate that this population consumes 78 to 406 t of krill and 4 to 46 t of fish each breeding season. Our results demonstrate clear periods of peak consumption associated with the penguins’ breeding cycle

An 85K SNP Array Uncovers Inbreeding and Cryptic Relatedness in an Antarctic Fur Seal Breeding Colony

Humble E, Paijmans A, Forcada J, Hoffman J. An 85K SNP Array Uncovers Inbreeding and Cryptic Relatedness in an Antarctic Fur Seal Breeding Colony. G3: Genes, Genomes, Genetics. 2020;10(8):2787-2799.High density single nucleotide polymorphism (SNP) arrays allow large numbers of individuals to be rapidly and cost-effectively genotyped at large numbers of genetic markers. However, despite being widely used in studies of humans and domesticated plants and animals, SNP arrays are lacking for most wild organisms. We developed a custom 85K Affymetrix Axiom array for an intensively studied pinniped, the Antarctic fur seal (Arctocephalus gazella). SNPs were discovered from a combination of genomic and transcriptomic resources and filtered according to strict criteria. Out of a total of 85,359 SNPs tiled on the array, 75,601 (88.6%) successfully converted and were polymorphic in 270 animals from a breeding colony at Bird Island in South Georgia. Evidence was found for inbreeding, with three genomic inbreeding coefficients being strongly intercorrelated and the proportion of the genome in runs of homozygosity being non-zero in all individuals. Furthermore, analysis of genomic relatedness coefficients identified previously unknown first-degree relatives and multiple second-degree relatives among a sample of ostensibly unrelated individuals. Such “cryptic relatedness” within fur seal breeding colonies may increase the likelihood of consanguineous matings and could therefore have implications for understanding fitness variation and mate choice. Finally, we demonstrate the cross-amplification potential of the array in three related pinniped species. Overall, our SNP array will facilitate future studies of Antarctic fur seals and has the potential to serve as a more general resource for the wider pinniped research community

Transcriptomic SNP discovery for custom genotyping arrays: impacts of sequence data, SNP calling method and genotyping technology on the probability of validation success

Background Single nucleotide polymorphism (SNP) discovery is an important goal of many studies. However, the number of ‘putative’ SNPs discovered from a sequence resource may not provide a reliable indication of the number that will successfully validate with a given genotyping technology. For this it may be necessary to account for factors such as the method used for SNP discovery and the type of sequence data from which it originates, suitability of the SNP flanking sequences for probe design, and genomic context. To explore the relative importance of these and other factors, we used Illumina sequencing to augment an existing Roche 454 transcriptome assembly for the Antarctic fur seal (Arctocephalus gazella). We then mapped the raw Illumina reads to the new hybrid transcriptome using BWA and BOWTIE2 before calling SNPs with GATK. The resulting markers were pooled with two existing sets of SNPs called from the original 454 assembly using NEWBLER and SWAP454. Finally, we explored the extent to which SNPs discovered using these four methods overlapped and predicted the corresponding validation outcomes for both Illumina Infinium iSelect HD and Affymetrix Axiom arrays. Results Collating markers across all discovery methods resulted in a global list of 34,718 SNPs. However, concordance between the methods was surprisingly poor, with only 51.0 % of SNPs being discovered by more than one method and 13.5 % being called from both the 454 and Illumina datasets. Using a predictive modeling approach, we could also show that SNPs called from the Illumina data were on average more likely to successfully validate, as were SNPs called by more than one method. Above and beyond this pattern, predicted validation outcomes were also consistently better for Affymetrix Axiom arrays. Conclusions Our results suggest that focusing on SNPs called by more than one method could potentially improve validation outcomes. They also highlight possible differences between alternative genotyping technologies that could be explored in future studies of non-model organisms

Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals.

Litzke V, Ottensmann M, Forcada J, Heitzmann L, Hoffman J. Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals. Ecology and evolution. 2019;9(14):7985-7996.Numerous studies have reported correlations between the heterozygosity of genetic markers and fitness. These heterozygosity-fitness correlations (HFCs) play a central role in evolutionary and conservation biology, yet their mechanistic basis remains open to debate. For example, fitness associations have been widely reported at both neutral and functional loci, yet few studies have directly compared the two, making it difficult to gauge the relative contributions of genome-wide inbreeding and specific functional genes to fitness. Here, we compared the effects of neutral and immune gene heterozygosity on death from bacterial infection in Antarctic fur seal (Arctocephalus gazella) pups. We specifically developed a panel of 13 microsatellites from expressed immune genes and genotyped these together with 48 neutral loci in 234 individuals, comprising 39 pups that were classified at necropsy as having most likely died of bacterial infection together with a five times larger matched sample of healthy surviving pups. Identity disequilibrium quantified from the neutral markers was positive and significant, indicative of variance in inbreeding within the study population. However, multilocus heterozygosity did not differ significantly between healthy and infected pups at either class of marker, and little evidence was found for fitness associations at individual loci. These results support a previous study of Antarctic fur seals that found no effects of heterozygosity at nine neutral microsatellites on neonatal survival and thereby help to refine our understanding of how HFCs vary across the life cycle. Given that nonsignificant HFCs are underreported in the literature, we also hope that our study will contribute toward a more balanced understanding of the wider importance of this phenomenon

Complete mitochondrial genome of the Antarctic fur seal (Arctocephalus gazella)

Nagel R, Forcada J, Hoffman J. Complete mitochondrial genome of the Antarctic fur seal (Arctocephalus gazella). Mitochondrial DNA Part B. 2019;4(2):2936-2937.The Antarctic fur seal (Arctocephalus gazella) is an abundant Antarctic otariid. Here, we present the complete mitochondrial DNA sequence of this species, which includes 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and the control region for a total length of 16,156 bp. A phylogenetic analysis including all 25 publically available pinniped mitogenomes nested the Antarctic fur seal within the Otariid clade, which was clearly resolved from the Phocidae and Odobenidae

Complete mitochondrial genome of the Antarctic fur seal (Arctocephalus gazella).

The Antarctic fur seal (Arctocephalus gazella) is an abundant Antarctic otariid. Here, we present the complete mitochondrial DNA sequence of this species, which includes 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and the control region for a total length of 16,156 bp. A phylogenetic analysis including all 25 publically available pinniped mitogenomes nested the Antarctic fur seal within the Otariid clade, which was clearly resolved from the Phocidae and Odobenidae