Macroalgal eukaryotic microbiome composition indicates novel phylogenetic diversity and broad host spectrum of oomycete pathogens.

Seaweeds are important components of marine ecosystems with emerging potential in aquaculture and as sources of biofuel, food products and pharmacological compounds. However, an increasingly recognised threat to natural and industrial seaweed populations is infection with parasitic single-celled eukaryotes from the relatively understudied oomycete lineage. Here we examine the eukaryomes of diverse brown, red and green marine macroalgae collected from polar (Baffin Island), cold-temperate (Falkland Islands) and tropical (Ascension Island) locations, with a focus on oomycete and closely related diatom taxa. Using 18S rRNA gene amplicon sequencing, we show unexpected genetic and taxonomic diversity of the eukaryomes, a strong broad-brush association between eukaryome composition and geographic location, and some evidence of association between eukaryome structure and macroalgal phylogenetic relationships (phylosymbiosis). However, the oomycete fraction of the eukaryome showed disparate patterns of diversity and structure, highlighting much weaker association with geography and no evidence of phylosymbiosis. We present several novel haplotypes of the most common oomycete Eurychasma dicksonii and report for the first time a cosmopolitan distribution and absence of host specificity of this important pathogen. This indicates rich diversity in macroalgal oomycete pathogens and highlights that these pathogens may be generalist and highly adaptable to diverse environmental conditions.

A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs.

Spliced leader trans-splicing of pre-mRNAs is a critical step in the gene expression of many eukaryotes. How the spliced leader RNA and its target transcripts are brought together to form the trans-spliceosome remains an important unanswered question. Using immunoprecipitation followed by protein analysis via mass spectrometry and RIP-Seq, we show that the nematode-specific proteins, SNA-3 and SUT-1, form a complex with a set of enigmatic non-coding RNAs, the SmY RNAs. Our work redefines the SmY snRNP and shows for the first time that it is essential for nematode viability and is involved in spliced leader trans-splicing. SNA-3 and SUT-1 are associated with the 5' ends of most, if not all, nascent capped RNA polymerase II transcripts, and they also interact with components of the major nematode spliced leader (SL1) snRNP. We show that depletion of SNA-3 impairs the co-immunoprecipitation between one of the SL1 snRNP components, SNA-2, and several core spliceosomal proteins. We thus propose that the SmY snRNP recruits the SL1 snRNP to the 5' ends of nascent pre-mRNAs, an instrumental step in the assembly of the trans-spliceosome.

Large effective population size masks population genetic structure in Hirondellea amphipods within the deepest marine ecosystem, the Mariana Trench.

The examination of genetic structure in the deep-ocean hadal zone has focused on divergence between tectonic trenches to understand how environment and geography may drive species divergence and promote endemism. There has been little attempt to examine localized genetic structure within trenches, partly because of logistical challenges associated with sampling at an appropriate scale, and the large effective population sizes of species that can be sampled adequately may mask underlying genetic structure. Here we examine genetic structure in the superabundant amphipod Hirondellea gigas in the Mariana Trench at depths of 8126-10,545 m. RAD sequencing was used to identify 3182 loci containing 43,408 single nucleotide polymorphisms (SNPs) across individuals after stringent pruning of loci to prevent paralogous multicopy genomic regions being erroneously merged. Principal components analysis of SNP genotypes resolved no genetic structure between sampling locations, consistent with a signature of panmixia. However, discriminant analysis of principal components identified divergence between all sites driven by 301 outlier SNPs in 169 loci and significantly associated with latitude and depth. Functional annotation of loci identified differences between singleton loci used in analysis and paralogous loci pruned from the data set and also between outlier and nonoutlier loci, all consistent with hypotheses explaining the role of transposable elements driving genome dynamics. This study challenges the traditional perspective that highly abundant amphipods within a trench form a single panmictic population. We discuss the findings in relation to eco-evolutionary and ontogenetic processes operating in the deep sea, and highlight key challenges associated with population genetic analysis in nonmodel systems with inherent large effective population sizes and genomes.

The transcriptomic signature of responses to larval crowding in Drosophila melanogaster.

Intraspecific competition at the larval stage is an important ecological factor affecting life-history, adaptation and evolutionary trajectory in holometabolous insects. However, the molecular pathways underpinning these ecological processes are poorly characterized. We reared Drosophila melanogaster at three egg densities (5, 60, and 300 eggs/mL) and sequenced the transcriptomes of pooled third-instar larvae. We also examined emergence time, egg-to-adult viability, adult mass, and adult sex-ratio at each density. Medium crowding had minor detrimental effects on adult phenotypes compared to low density and yielded 24 differentially expressed genes (DEGs), including several chitinase enzymes. In contrast, high crowding had substantial detrimental effects on adult phenotypes and yielded 2107 DEGs. Among these, upregulated gene sets were enriched in sugar, steroid and amino acid metabolism as well as DNA replication pathways, whereas downregulated gene sets were enriched in ABC transporters, taurine, Toll/Imd signaling, and P450 xenobiotics metabolism pathways. Overall, our findings show that larval crowding has a large consistent effect on several molecular pathways (i.e., core responses) with few pathways displaying density-specific regulation (i.e., idiosyncratic responses). This provides important insights into how holometabolous insects respond to intraspecific competition during development.

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex.

Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5' untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

Monodopsis and Vischeria Genomes Shed New Light on the Biology of Eustigmatophyte Algae.

Members of eustigmatophyte algae, especially Nannochloropsis and Microchloropsis, have been tapped for biofuel production owing to their exceptionally high lipid content. Although extensive genomic, transcriptomic, and synthetic biology toolkits have been made available for Nannochloropsis and Microchloropsis, very little is known about other eustigmatophytes. Here we present three near-chromosomal and gapless genome assemblies of Monodopsis strains C73 and C141 (60 Mb) and Vischeria strain C74 (106 Mb), which are the sister groups to Nannochloropsis and Microchloropsis in the order Eustigmatales. These genomes contain unusually high percentages of simple repeats, ranging from 12% to 21% of the total assembly size. Unlike Nannochloropsis and Microchloropsis, long interspersed nuclear element repeats are abundant in Monodopsis and Vischeria and might constitute the centromeric regions. We found that both mevalonate and nonmevalonate pathways for terpenoid biosynthesis are present in Monodopsis and Vischeria, which is different from Nannochloropsis and Microchloropsis that have only the latter. Our analysis further revealed extensive spliced leader trans-splicing in Monodopsis and Vischeria at 36-61% of genes. Altogether, the high-quality genomes of Monodopsis and Vischeria not only serve as the much-needed outgroups to advance Nannochloropsis and Microchloropsis research, but also shed new light on the biology and evolution of eustigmatophyte algae.

SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data.

BACKGROUND: Spliced leader (SL) trans-splicing replaces the 5' end of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from elsewhere in the genome. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons may have independently evolved multiple times throughout Eukarya, yet our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes. RESULTS: Here we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5' read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogating corresponding SL RNA genes for sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5' SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that (1) SLIDR correctly detects expected SLs and often discovers novel SL variants; (2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons. CONCLUSIONS: SLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya and improve gene discovery and annotation for a wide range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be retrieved from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

Comparative genomics of cetartiodactyla: energy metabolism underpins the transition to an aquatic lifestyle.

Foraging disruption caused by human activities is emerging as a key issue in cetacean conservation because it can affect nutrient levels and the amount of energy available to individuals to invest into reproduction. Our ability to predict how anthropogenic stressors affect these ecological processes and ultimately population trajectory depends crucially on our understanding of the complex physiological mechanisms that detect nutrient availability and regulate energy metabolism, foraging behavior and life-history decisions. These physiological mechanisms are likely to differ considerably from terrestrial mammalian model systems. Here, we examine nucleotide substitution rates in cetacean and other artiodactyl genomes to identify signatures of selection in genes associated with nutrient sensing pathways. We also estimated the likely physiological consequences of adaptive amino acid substitutions for pathway functions. Our results highlight that genes involved in the insulin, mTOR and NF-ĸB pathways are subject to significant positive selection in cetaceans compared to terrestrial artiodactyla. These genes may have been positively selected to enable cetaceans to adapt to a glucose-poor diet, to overcome deleterious effects caused by hypoxia during diving (e.g. oxidative stress and inflammation) and to modify fat-depot signaling functions in a manner different to terrestrial mammals. We thus show that adaptation in cetaceans to an aquatic lifestyle significantly affected functions in nutrient sensing pathways. The use of fat stores as a condition index in cetaceans may be confounded by the multiple and critical roles fat has in regulating cetacean metabolism, foraging behavior and diving physiology.

Resolution of polycistronic RNA by SL2 trans-splicing is a widely conserved nematode trait.

Spliced leader trans-splicing is essential for the processing and translation of polycistronic RNAs generated by eukaryotic operons. In C. elegans, a specialized spliced leader, SL2, provides the 5' end for uncapped pre-mRNAs derived from polycistronic RNAs. Studies of other nematodes suggested that SL2-type trans-splicing is a relatively recent innovation, confined to Rhabditina, the clade containing C. elegans and its close relatives. Here we conduct a survey of transcriptome-wide spliced leader trans-splicing in Trichinella spiralis, a distant relative of C. elegans with a particularly diverse repertoire of 15 spliced leaders. By systematically comparing the genomic context of trans-splicing events for each spliced leader, we identified a subset of T. spiralis spliced leaders that are specifically used to process polycistronic RNAs-the first examples of SL2-type spliced leaders outside of Rhabditina. These T. spiralis spliced leader RNAs possess a perfectly conserved stem-loop motif previously shown to be essential for SL2-type trans-splicing in C. elegans We show that genes trans-spliced to these SL2-type spliced leaders are organized in operonic fashion, with short intercistronic distances. A subset of T. spiralis operons show conservation of synteny with C. elegans operons. Our work substantially revises our understanding of nematode spliced leader trans-splicing, showing that SL2 trans-splicing is a major mechanism for nematode polycistronic RNA processing, which may have evolved prior to the radiation of the Nematoda. This work has important implications for the improvement of genome annotation pipelines in nematodes and other eukaryotes with operonic gene organization.

Author Correction: Signatures of balancing selection in toll-like receptor (TLRs) genes - novel insights from a free-living rodent.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Microbiome composition within a sympatric species complex of intertidal isopods (Jaera albifrons).

The increasingly recognised effects of microbiomes on the eco-evolutionary dynamics of their hosts are promoting a view of the "hologenome" as an integral host-symbiont evolutionary entity. For example, sex-ratio distorting reproductive parasites such as Wolbachia are well-studied pivotal drivers of invertebrate reproductive processes, and more recent work is highlighting novel effects of microbiome assemblages on host mating behaviour and developmental incompatibilities that underpin or reinforce reproductive isolation processes. However, examining the hologenome and its eco-evolutionary effects in natural populations is challenging because microbiome composition is considerably influenced by environmental factors. Here we illustrate these challenges in a sympatric species complex of intertidal isopods (Jaera albifrons spp.) with pervasive sex-ratio distortion and ecological and behavioural reproductive isolation mechanisms. We deep-sequence the bacterial 16S rRNA gene among males and females collected in spring and summer from two coasts in north-east Scotland, and examine microbiome composition with a particular focus on reproductive parasites. Microbiomes of all species were diverse (overall 3,317 unique sequences among 3.8 million reads) and comprised mainly Proteobacteria and Bacteroidetes taxa typical of the marine intertidal zone, in particular Vibrio spp. However, we found little evidence of the reproductive parasites Wolbachia, Rickettsia, Spiroplasma and Cardinium, suggesting alternative causes of sex-ratio distortion. Notwithstanding, a significant proportion of the variance in microbiome composition among samples was explained by sex (14.1 %), nested within geographic (26.9 %) and seasonal (39.6 %) variance components. The functional relevance of this sex signal was difficult to ascertain given the absence of reproductive parasites, the ephemeral nature of the species assemblages and substantial environmental variability. These results establish the Jaera albifrons species complex as an intriguing system for examining the effects of microbiomes on reproductive processes and speciation, and highlight the difficulties associated with snapshot assays of microbiome composition in dynamic and complex environments.

Signatures of balancing selection in toll-like receptor (TLRs) genes - novel insights from a free-living rodent.

Selective pressure from pathogens is considered a key selective force driving the evolution of components of the immune system. Since single components of the immune system may interact with many pathogens, and single pathogens may be recognized by multiple components of the immune system, gaining a better understanding of the mechanisms of parasite-driven selection requires the study of multiple genes and pathogens. Toll-like receptors (TLRs) are a large gene family that code for antigen-presenting components of the innate immune response. In the present paper we characterize polymorphism and signatures of selection in seven TLRs in free-living bank voles Myodes glareolus. We report the first evidence of balancing selection in several TLR genes, supported by positive values of Fu and Li's D* in TLR2 and TLR5, and positive values of Tajima's D in LRR regions within TLR1 and TLR2. We further found significant associations between amino-acid alleles of TLR1 and TLR5 and susceptibility to infection with the blood pathogen Bartonella. Interestingly, selection patterns in TLRs presenting virus-derived motifs (TLR7 and TLR9) differed considerably from those interacting with bacterial PAMPs. In contrast to the highly variable TLRs presenting bacterial motifs, TLR7 and TLR9 had low polymorphism and displayed signatures of directional selection. These findings suggest different functional responses across the TLR gene family and highlight the complexity of parasite-driven selection.

The role of parasite-driven selection in shaping landscape genomic structure in red grouse (Lagopus lagopus scotica).

Landscape genomics promises to provide novel insights into how neutral and adaptive processes shape genome-wide variation within and among populations. However, there has been little emphasis on examining whether individual-based phenotype-genotype relationships derived from approaches such as genome-wide association (GWAS) manifest themselves as a population-level signature of selection in a landscape context. The two may prove irreconcilable as individual-level patterns become diluted by high levels of gene flow and complex phenotypic or environmental heterogeneity. We illustrate this issue with a case study that examines the role of the highly prevalent gastrointestinal nematode Trichostrongylus tenuis in shaping genomic signatures of selection in red grouse (Lagopus lagopus scotica). Individual-level GWAS involving 384 SNPs has previously identified five SNPs that explain variation in T. tenuis burden. Here, we examine whether these same SNPs display population-level relationships between T. tenuis burden and genetic structure across a small-scale landscape of 21 sites with heterogeneous parasite pressure. Moreover, we identify adaptive SNPs showing signatures of directional selection using F(ST) outlier analysis and relate population- and individual-level patterns of multilocus neutral and adaptive genetic structure to T. tenuis burden. The five candidate SNPs for parasite-driven selection were neither associated with T. tenuis burden on a population level, nor under directional selection. Similarly, there was no evidence of parasite-driven selection in SNPs identified as candidates for directional selection. We discuss these results in the context of red grouse ecology and highlight the broader consequences for the utility of landscape genomics approaches for identifying signatures of selection.

Genome-wide association and genome partitioning reveal novel genomic regions underlying variation in gastrointestinal nematode burden in a wild bird.

Identifying the genetic architecture underlying complex phenotypes is a notoriously difficult problem that often impedes progress in understanding adaptive eco-evolutionary processes in natural populations. Host-parasite interactions are fundamentally important drivers of evolutionary processes, but a lack of understanding of the genes involved in the host's response to chronic parasite insult makes it particularly difficult to understand the mechanisms of host life history trade-offs and the adaptive dynamics involved. Here, we examine the genetic basis of gastrointestinal nematode (Trichostrongylus tenuis) burden in 695 red grouse (Lagopus lagopus scotica) individuals genotyped at 384 genome-wide SNPs. We first use genome-wide association to identify individual SNPs associated with nematode burden. We then partition genome-wide heritability to identify chromosomes with greater heritability than expected from gene content, due to harbouring a multitude of additive SNPs with individually undetectable effects. We identified five SNPs on five chromosomes that accounted for differences of up to 556 worms per bird, but together explained at best 4.9% of the phenotypic variance. These SNPs were closely linked to genes representing a range of physiological processes including the immune system, protein degradation and energy metabolism. Genome partitioning indicated genome-wide heritability of up to 29% and three chromosomes with excess heritability of up to 4.3% (total 8.9%). These results implicate SNPs and novel genomic regions underlying nematode burden in this system and suggest that this phenotype is somewhere between being based on few large-effect genes (oligogenic) and based on a large number of genes with small individual but large combined effects (polygenic).

Fine-scale population epigenetic structure in relation to gastrointestinal parasite load in red grouse (Lagopus lagopus scotica).

Epigenetic modification of cytosine methylation states can be elicited by environmental stresses and may be a key process affecting phenotypic plasticity and adaptation. Parasites are potent stressors with profound physiological and ecological effects on their host, but there is little understanding in how parasites may influence host methylation states. Here, we estimate epigenetic diversity and differentiation among 21 populations of red grouse (Lagopus lagopus scotica) in north-east Scotland and test for association of gastrointestinal parasite load (caecal nematode Trichostrongylus tenuis) with hepatic genome-wide and locus-specific methylation states. Following methylation-sensitive AFLP (MSAP), 129 bands, representing 73 methylation-susceptible and 56 nonmethylated epiloci, were scored across 234 individuals. The populations differed significantly in genome-wide methylation levels and were also significantly epigenetically (F(SC) = 0.0227; P < 0.001) and genetically (F(SC) = 0.0058; P < 0.001) differentiated. Parasite load was not associated with either genome-wide methylation levels or epigenetic differentiation. Instead, we found eight disproportionately differentiated epilocus-specific methylation states (F(ST) outliers) using bayescan software and significant positive and negative association of 35 methylation states with parasite load from bespoke generalized estimating equations (GEE), simple logistic regression (sam) and Bayesian environmental analysis (bayenv2). Following Sanger sequencing, genome mapping and geneontology (go) annotation, some of these epiloci were linked to genes involved in regulation of cell cycle, signalling, metabolism, immune system and notably rRNA methylation, histone acetylation and small RNAs. These findings demonstrate an epigenetic signature of parasite load in populations of a wild bird and suggest intriguing physiological effects of parasite-associated cytosine methylation.