The enigmatic RNase MRP of kinetoplastids.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

From tides to nucleotides: Genomic signatures of adaptation to environmental heterogeneity in barnacles.

The northern acorn barnacle (Semibalanus balanoides) is a robust system to study the genetic basis of adaptations to highly heterogeneous environments. Adult barnacles may be exposed to highly dissimilar levels of thermal stress depending on where they settle in the intertidal (i.e., closer to the upper or lower tidal boundary). For instance, barnacles near the upper tidal limit experience episodic summer temperatures above recorded heat coma levels. This differential stress at the microhabitat level is also dependent on the aspect of sun exposure. In the present study, we used pool-seq approaches to conduct a genome wide screen for loci responding to intertidal zonation across the North Atlantic basin (Maine, Rhode Island, and Norway). Our analysis discovered 382 genomic regions containing SNPs which are consistently zonated (i.e., SNPs whose frequencies vary depending on their position in the rocky intertidal) across all surveyed habitats. Notably, most zonated SNPs are young and private to the North Atlantic. These regions show high levels of genetic differentiation across ecologically extreme microhabitats concomitant with elevated levels of genetic variation and Tajima's D, suggesting the action of non-neutral processes. Overall, these findings support the hypothesis that spatially heterogeneous selection is a general and repeatable feature for this species, and that natural selection can maintain functional genetic variation in heterogeneous environments.

Genomic Characterization of the Barnacle Balanus improvisus Reveals Extreme Nucleotide Diversity in Coding Regions.

Barnacles are key marine crustaceans in several habitats, and they constitute a common practical problem by causing biofouling on man-made marine constructions and ships. Despite causing considerable ecological and economic impacts, there is a surprising void of basic genomic knowledge, and a barnacle reference genome is lacking. We here set out to characterize the genome of the bay barnacle Balanus improvisus (= Amphibalanus improvisus) based on short-read whole-genome sequencing and experimental genome size estimation. We show both experimentally (DNA staining and flow cytometry) and computationally (k-mer analysis) that B. improvisus has a haploid genome size of ~ 740 Mbp. A pilot genome assembly rendered a total assembly size of ~ 600 Mbp and was highly fragmented with an N50 of only 2.2 kbp. Further assembly-based and assembly-free analyses revealed that the very limited assembly contiguity is due to the B. improvisus genome having an extremely high nucleotide diversity (π) in coding regions (average π ≈ 5% and average π in fourfold degenerate sites ≈ 20%), and an overall high repeat content (at least 40%). We also report on high variation in the α-octopamine receptor OctA (average π = 3.6%), which might increase the risk that barnacle populations evolve resistance toward antifouling agents. The genomic features described here can help in planning for a future high-quality reference genome, which is urgently needed to properly explore and understand proteins of interest in barnacle biology and marine biotechnology and for developing better antifouling strategies.

The round goby genome provides insights into mechanisms that may facilitate biological invasions.

BACKGROUND: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. RESULTS: We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. CONCLUSIONS: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish.

Sensory receptor repertoire in cyprid antennules of the barnacle Balanus improvisus.

Barnacle settlement involves sensing of a variety of exogenous cues. A pair of antennules is the main sensory organ that the cyprid larva uses to explore the surface. Antennules are equipped with a number of setae that have both chemo- and mechanosensing function. The current study explores the repertoire of sensory receptors in Balanus improvisus cyprid antennules with the goal to better understand sensory systems involved in the settling behavior of this species. We carried out transcriptome sequencing of dissected B. improvisus cyprid antennules. The generated transcriptome assembly was used to search for sensory receptors using HMM models. Among potential chemosensory genes, we identified the ionotropic receptors IR25a, IR8a and IR93a, and several divergent IR candidates to be expressed in the cyprid antennules. We found one gustatory-like receptor but no odorant receptors, chemosensory or odorant-binding proteins. Apart from chemosensory receptors, we also identified 13 potential mechanosensory genes represented by several transient receptor potential channels (TRP) subfamilies. Furthermore, we analyzed changes in expression profiles of IRs and TRPs during the B. improvisus settling process. Several of the sensory genes were differentially expressed during the course of larval settlement. This study gives expanded knowledge about the sensory systems present in barnacles, a taxonomic group for which only limited information about receptors is currently available. It furthermore serves as a starting point for more in depth studies of how sensory signaling affects settling behavior in barnacles with implications for preventing biofouling.

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation.

The mitochondrial genome sequences of the round goby and the sand goby reveal patterns of recent evolution in gobiid fish.

BACKGROUND: Vertebrate mitochondrial genomes are optimized for fast replication and low cost of RNA expression. Accordingly, they are devoid of introns, are transcribed as polycistrons and contain very little intergenic sequences. Usually, vertebrate mitochondrial genomes measure between 16.5 and 17 kilobases (kb). RESULTS: During genome sequencing projects for two novel vertebrate models, the invasive round goby and the sand goby, we found that the sand goby genome is exceptionally small (16.4 kb), while the mitochondrial genome of the round goby is much larger than expected for a vertebrate. It is 19 kb in size and is thus one of the largest fish and even vertebrate mitochondrial genomes known to date. The expansion is attributable to a sequence insertion downstream of the putative transcriptional start site. This insertion carries traces of repeats from the control region, but is mostly novel. To get more information about this phenomenon, we gathered all available mitochondrial genomes of Gobiidae and of nine gobioid species, performed phylogenetic analyses, analysed gene arrangements, and compared gobiid mitochondrial genome sizes, ecological information and other species characteristics with respect to the mitochondrial phylogeny. This allowed us amongst others to identify a unique arrangement of tRNAs among Ponto-Caspian gobies. CONCLUSIONS: Our results indicate that the round goby mitochondrial genome may contain novel features. Since mitochondrial genome organisation is tightly linked to energy metabolism, these features may be linked to its invasion success. Also, the unique tRNA arrangement among Ponto-Caspian gobies may be helpful in studying the evolution of this highly adaptive and invasive species group. Finally, we find that the phylogeny of gobiids can be further refined by the use of longer stretches of linked DNA sequence.

The Story of a Hitchhiker: Population Genetic Patterns in the Invasive Barnacle Balanus(Amphibalanus) improvisus Darwin 1854.

Understanding the ecological and evolutionary forces that determine the genetic structure and spread of invasive species is a key component of invasion biology. The bay barnacle, Balanus improvisus (= Amphibalanus improvisus), is one of the most successful aquatic invaders worldwide, and is characterised by broad environmental tolerance. Although the species can spread through natural larval dispersal, human-mediated transport through (primarily) shipping has almost certainly contributed to the current global distribution of this species. Despite its worldwide distribution, little is known about the phylogeography of this species. Here, we characterize the population genetic structure and model dispersal dynamics of the barnacle B. improvisus, and describe how human-mediated spreading via shipping as well as natural larval dispersal may have contributed to observed genetic variation. We used both mitochondrial DNA (cytochrome c oxidase subunit I: COI) and nuclear microsatellites to characterize the genetic structure in 14 populations of B. improvisus on a global and regional scale (Baltic Sea). Genetic diversity was high in most populations, and many haplotypes were shared among populations on a global scale, indicating that long-distance dispersal (presumably through shipping and other anthropogenic activities) has played an important role in shaping the population genetic structure of this cosmopolitan species. We could not clearly confirm prior claims that B. improvisus originates from the western margins of the Atlantic coasts; although there were indications that Argentina could be part of a native region. In addition to dispersal via shipping, we show that natural larval dispersal may play an important role for further colonisation following initial introduction.

Metagenomic sequencing of marine periphyton: taxonomic and functional insights into biofilm communities.

Periphyton communities are complex phototrophic, multispecies biofilms that develop on surfaces in aquatic environments. These communities harbor a large diversity of organisms comprising viruses, bacteria, algae, fungi, protozoans, and metazoans. However, thus far the total biodiversity of periphyton has not been described. In this study, we use metagenomics to characterize periphyton communities from the marine environment of the Swedish west coast. Although we found approximately ten times more eukaryotic rRNA marker gene sequences compared to prokaryotic, the whole metagenome-based similarity searches showed that bacteria constitute the most abundant phyla in these biofilms. We show that marine periphyton encompass a range of heterotrophic and phototrophic organisms. Heterotrophic bacteria, including the majority of proteobacterial clades and Bacteroidetes, and eukaryotic macro-invertebrates were found to dominate periphyton. The phototrophic groups comprise Cyanobacteria and the alpha-proteobacterial genus Roseobacter, followed by different micro- and macro-algae. We also assess the metabolic pathways that predispose these communities to an attached lifestyle. Functional indicators of the biofilm form of life in periphyton involve genes coding for enzymes that catalyze the production and degradation of extracellular polymeric substances, mainly in the form of complex sugars such as starch and glycogen-like meshes together with chitin. Genes for 278 different transporter proteins were detected in the metagenome, constituting the most abundant protein complexes. Finally, genes encoding enzymes that participate in anaerobic pathways, such as denitrification and methanogenesis, were detected suggesting the presence of anaerobic or low-oxygen micro-zones within the biofilms.

Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities.

BACKGROUND: Environmental shotgun sequencing (metagenomics) provides a new way to study communities in microbial ecology. We here use sequence data from the Global Ocean Sampling (GOS) expedition to investigate toxicant selection pressures revealed by the presence of detoxification genes in marine bacteria. To capture a broad range of potential toxicants we selected detoxification protein families representing systems protecting microorganisms from a variety of stressors, such as metals, organic compounds, antibiotics and oxygen radicals. RESULTS: Using a bioinformatics procedure based on comparative analysis to finished bacterial genomes we found that the amount of detoxification genes present in marine microorganisms seems surprisingly small. The underrepresentation is particularly evident for toxicant transporters and proteins involved in detoxifying metals. Exceptions are enzymes involved in oxidative stress defense where peroxidase enzymes are more abundant in marine bacteria compared to bacteria in general. In contrast, catalases are almost completely absent from the open ocean environment, suggesting that peroxidases and peroxiredoxins constitute a core line of defense against reactive oxygen species (ROS) in the marine milieu. CONCLUSIONS: We found no indication that detoxification systems would be generally more abundant close to the coast compared to the open ocean. On the contrary, for several of the protein families that displayed a significant geographical distribution, like peroxidase, penicillin binding transpeptidase and divalent ion transport protein, the open ocean samples showed the highest abundance. Along the same lines, the abundance of most detoxification proteins did not increase with estimated pollution. The low level of detoxification systems in marine bacteria indicate that the majority of marine bacteria have a low capacity to adapt to increased pollution. Our study exemplifies the use of metagenomics data in ecotoxicology, and in particular how anthropogenic consequences on life in the sea can be examined.

Molecular characterization of the α-subunit of Na⁺/K⁺ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.

Evolutionary loss of 8-oxo-G repair components among eukaryotes.

BACKGROUND: We have examined the phylogenetic pattern among eukaryotes of homologues of the E. coli 7,8-dihydro-8-oxoguanine (8-oxo-G) repair enzymes MutY, MutM, and MutT. RESULTS: These DNA repair enzymes are present in all large phylogenetic groups, with MutM homologues being the most universally conserved. All chordates and echinoderms were found to possess all three 8-oxo-G repair components. Likewise, the red and green algae examined have all three repair enzymes, while all land-living plants have MutY and MutM homologues, but lack MutT. However, for some phyla, e.g. protostomes, a more patchy distribution was found. Nematodes provide a striking example, where Caenorhabditis is the only identified example of an organism group having none of the three repair enzymes, while the genome of another nematode, Trichinella spiralis, instead encodes all three. The most complex distribution exists in fungi, where many different patterns of retention or loss of the three repair components are found. In addition, we found sequence insertions near or within the catalytic sites of MutY, MutM, and MutT to be present in some subgroups of Ascomycetes. CONCLUSION: The 8-oxo-G repair enzymes are ancient in origin, and loss of individual 8-oxo-G repair components at several distinct points in evolution appears to be the most likely explanation for the phylogenetic pattern among eukaryotes.

Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The alpha(2)-adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOctalpha) belongs to the alpha-adrenoceptor-like subfamily, and the other four (BiOctbeta-R1, BiOctbeta-R2, BiOctbeta-R3, and BiOctbeta-R4) belong to the beta-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of beta-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.