Experimental data from the development of Lymnaea stagnalis embryo test for chemicals hazard assessment.

This study aimed to contribute to the development of an embryo-test using the gastropod Lymnaea stagnalis, identified by the Organization for Economic Co-operation and Development (OECD) as a potential invertebrate test animal model. Together with the Potamopyrgus antipodarum, were the first mollusc models to be included in the organization testing guidelines. The focus was on validating an embryo toxicity test to cover the sensitive embryogenesis phase and on obtaining testing information on all of the model life cycle stages, contributing to close an identified gap within this context. Adhering to OECD guidelines, namely the L. stagnalis reproductive test, the study examined mortality rates, abnormality rates, development, growth, hatching rates, hearth rates, and pre-testing media suitability, during the embryogenesis, and the obtained dataset made available for further studies. Cadmium was chosen as the positive test compound due to its well-studied nature and the model's proven sensitivity to the compound, working as a reference compound for the test development. The data were collected in two 12-day assays under consistent conditions, each using 144 L. stagnalis embryos (<24 h old) from 6 egg masses (288 embryos total). Six 48-well microplates were utilized per assay, accommodating five different cadmium concentrations (32, 70, 155, 341, 750 µg/L) and a control group. Recorded parameters encompassed developmental stage, embryo position within the chorion, developmental abnormalities, hatchings, and mortality. Data analysis involved classifying embryos based on developmental stage and position, taking an exploratory approach to define the relevance of the different parameters in the compound hazard assessment during the embryogenesis. Measurements considered embryo area, perimeter, length, height, width, interocular distance, and heart rate. This dataset does not provide treated information but the raw data obtained during the proposed metodological development and toxicity testing process. The purpose of this article is to make the obtained raw data available, clearly defining the acquisition methodology to provide a comparison basis for future or existent works within this context.

Deep-sea mining: using hyperbaric conditions to study the impact of sediment plumes in the subtidal clam Spisula solida.

With the growing interest to exploit mineral resources in the deep-sea, there is the need to establish guidelines and frameworks to support hazard and risk assessment schemes. The present study used a subtidal species of filter-feeding bivalve, the clam Spisula solida, as a proxy to better understand the impacts of sediment plumes in marine organisms under hyperbaric conditions. Four concentrations of suspended sediments (0 g/L, 1 g/L, 2 g/L, and 4 g/L) were used in a mixture with different grain sizes at 4 Bar for 96 h. Functional (filtration rate-FR) and biochemical endpoints (catalase-CAT, glutathione s-transferase-GST, and lipid peroxidation-LPO) were analyzed in the gonads, digestive gland, and gills of S. solida after a 96-h exposure at 4 Bar (the natural limit of the species vertical distribution). The FR showed a decreasing trend with the increasing sediment concentrations (significant effects at 2 and 4 g/L). Additionally, significant changes were observed for some of the tested oxidative stress biomarkers, which were concentration and tissue-dependent, i.e., CAT activity was significantly elevated in gills (1 g/L treatment), and GST was decreased in digestive gland (1 g/L treatment). Overall, the results show that suspended sediments, at 2 and 4 g/L, have negative functional impacts in the bivalve S. solida providing additional insights to improve hazard assessment of deep-sea mining. These findings represent a step forward to ensure the mitigation of the potential negative effects of deep-sea resource exploitation.

A Multiplex Molecular Cell-Based Sensor to Detect Ligands of PPARs: An Optimized Tool for Drug Discovery in Cyanobacteria.

Cyanobacteria produce a wealth of secondary metabolites. Since these organisms attach fatty acids into molecules in unprecedented ways, cyanobacteria can serve as a novel source for bioactive compounds acting as ligands for Peroxisome Proliferator-Activated Receptors (PPAR). PPARs (PPARα, PPARß/δ and PPARγ) are ligand-activated nuclear receptors, involved in the regulation of various metabolic and cellular processes, thus serving as potential drug targets for a variety of pathologies. Yet, given that PPARs' agonists can have pan-, dual- or isoform-specific action, some controversy has been raised over currently approved drugs and their side effects, highlighting the need for novel molecules. Here, we expand and validate a cell-based PPAR transactivation activity biosensor, and test it in a screening campaign to guide drug discovery. Biosensor upgrades included the use of different reporter genes to increase signal intensity and stability, a different promoter to modulate reporter gene expression, and multiplexing to improve efficiency. Sensor's limit of detection (LOD) ranged from 0.36-0.89 nM in uniplex and 0.89-1.35 nM in multiplex mode. In triplex mode, the sensor's feature screening, a total of 848 fractions of 96 cyanobacteria extracts were screened. Hits were confirmed in multiplex mode and in uniplex mode, yielding one strain detected to have action on PPARα and three strains to have dual action on PPARα and -ß.

The Preservation of PPARγ Genome Duplicates in Some Teleost Lineages: Insights into Lipid Metabolism and Xenobiotic Exploitation.

Three peroxisome proliferator-activated receptor paralogues (PPARα, -ß and -γ) are currently recognized in vertebrate genomes. PPARγ is known to modulate nutrition, adipogenesis and immunity in vertebrates. Natural ligands of PPARγ have been proposed; however, the receptor also binds synthetic ligands such as endocrine disruptors. Two paralogues of PPARα and PPARß have been documented in teleost species, a consequence of the 3R WGD. Recently, two PPARγ paralogue genes were also identified in Astyanax mexicanus. We aimed to determine whether the presence of two PPARγ paralogues is prevalent in other teleost genomes, through genomic and phylogenetic analysis. Our results showed that besides Characiformes, two PPARγ paralogous genes were also identified in other teleost taxa, coinciding with the teleost-specific, whole-genome duplication and with the retention of both genes prior to the separation of the Clupeocephala. To functionally characterize these genes, we used the European sardine (Sardina pilchardus) as a model. PPARγA and PPARγB display a different tissue distribution, despite the similarity of their functional profiles: they are unresponsive to tested fatty acids and other human PPARγ ligands yet yield a transcriptional response in the presence of tributyltin (TBT). This observation puts forward the relevance of comparative analysis to decipher alternative binding architectures and broadens the disruptive potential of man-made chemicals for aquatic species.

Brain and testis: more alike than previously thought?

Several strands of evidence indicate the presence of marked similarities between human brain and testis. Understanding these similarities and their implications has become a topic of interest among the scientific community. Indeed, an association of intelligence with some semen quality parameters has been reported and a relation between dysfunctions of the human brain and testis has also been evident. Numerous common molecular features are evident when these tissues are compared, which is reflected in the huge number of common proteins. At the functional level, human neurons and sperm share a number of characteristics, including the importance of the exocytotic process and the presence of similar receptors and signalling pathways. The common proteins are mainly involved in exocytosis, tissue development and neuron/brain-associated biological processes. With this analysis, we conclude that human brain and testis share several biochemical characteristics which, in addition to their involvement in the speciation process, could, at least in part, be responsible for the expression of a huge number of common proteins. Nonetheless, this is an underexplored topic, and the connection between these tissues needs to be clarified, which could help to understand the dysfunctions affecting brain and testis, as well as to develop improved therapeutic strategies.

Proteogenomic Characterization of the Cement and Adhesive Gland of the Pelagic Gooseneck Barnacle Lepas anatifera.

We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.

Transcriptomic data on the transgenerational exposure of the keystone amphipod Gammarus locusta to simvastatin.

The use of transcriptomics data brings new insights and works as a powerful tool to explore the molecular mode of action (MoA) of transgenerational inheritance effects of contaminants of emerging concern. Therefore, in this dataset, we present the transcriptomic data of the transgenerational effects of environmentally relevant simvastatin levels, one of the most prescribed human pharmaceuticals, in the keystone amphipod species Gammarus locusta. In summary, G. locusta juveniles were maintained under simvastatin exposure up to adulthood (exposed group - F0E) and the offspring of F0E were transferred to control water for the three subsequent generations (transgenerational group - F1T, F2T and F3T). To gain insights into the biological functions and canonical pathways transgenerationally disrupted by simvastatin, a G. locusta de novo transcriptome assembly was produced and the transcriptomic profiles of three individual G. locusta females, per group, over the four generations (F0 to F3) - solvent control groups (F0.C, F1.C, F2.C and F3.C), F0 320 ng/L simvastatin exposed group (F0.320E) and F1 to F3 320 transgenerational group (F1.320T; F2.320T and F3.320T) - were analyzed. Briefly, Illumina HiSeq™ 2500 platform was used to perform RNA sequencing, and due to the unavailability of G. locusta genome, the RNA-seq datasets were assembled de novo using Trinity and annotated with Trinotate software. After assembly and post-processing steps, 106093 transcripts with N50 of 2371 bp and mean sequence length of 1343.98 bp was produced. BUSCO analyses showed a transcriptome with gene completeness of 97.5 % Arthropoda library profile. The Bowtie2, RSEM and edgeR tools were used for the differential gene expression (DEGs) analyses that allowed the identification of a high quantity of genes differentially expressed in all generations. Finally, to identify the main metabolic pathways affected by the transgenerational effects of SIM across all generations, the DGEs genes were blasted onto KEGG pathways database using the KAAS webserver. The data furnished in this article allows a better molecular understanding of the transgenerational effects produced by simvastatin in the keystone amphipod G. locusta and has major implications for hazard and risk assessment of pharmaceuticals and other emerging contaminants. This article is related to the research article entitled "Transgenerational inheritance of chemical-induced signature: a case study with simvastatin [1].

The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes.

Adhesive secretion has a fundamental role in barnacles' survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.

Data collection on the use of embryo bioassays with aquatic animals for toxicity testing and hazard assessment of emerging pollutants.

A thorough bibliographic survey on the use of embryo-tests with aquatic animals for toxicity testing was performed. The data regarding to the compounds sensitivity (NOEC, LOEC, EC50 and LC50), the available resources for the different animal models (knowledge on the life-cycle, amenability for laboratory breeding, number of embryos produced and reproductive strategy, genomic and transcriptomic resources), together with the European pieces of legislation regarding to animal testing and the available testing guidelines of national and international agencies (OECD, EPA, ISO, ASTM, ICES) were gathered, aiming to the standardization of new embryo-test model species for toxicity testing of new and existing compounds. The data contained in this Data in Brief article is presented and discussed in the review article with the title Embryo bioassays with aquatic animals for toxicity testing and hazard assessment of emerging pollutants: a review [1]. The dataset is provided with this article as a supplementary file.

Embryo bioassays with aquatic animals for toxicity testing and hazard assessment of emerging pollutants: A review.

This review article gathers the available information on the use of embryo-tests as high-throughput tools for toxicity screening, hazard assessment and prioritization of new and existing chemical compounds. The approach is contextualized considering the new legal trends for animal experimentation, fostering the 3R policy, with reduction of experimental animals, addressing the potential of embryo-tests as high-throughput toxicity screening and prioritizing tools. Further, the current test guidelines, such as the ones provided by OECD and EPA, focus mainly in a limited number of animal lineages, particularly vertebrates and arthropods. To extrapolate hazard assessment to the ecosystem scale, a larger diversity of taxa should be tested. The use of new experimental animal models in toxicity testing, from a representative set of taxa, was thoroughly revised and discussed in this review. Here, we critically review current tools and the main advantages and drawbacks of different animal models and set researcher priorities.

Obesogens in the aquatic environment: an evolutionary and toxicological perspective.

The rise of obesity in humans is a major health concern of our times, affecting an increasing proportion of the population worldwide. It is now evident that this phenomenon is not only associated with the lack of exercise and a balanced diet, but also due to environmental factors, such as exposure to environmental chemicals that interfere with lipid homeostasis. These chemicals, also known as obesogens, are present in a wide range of products of our daily life, such as cosmetics, paints, plastics, food cans and pesticide-treated food, among others. A growing body of evidences indicates that their action is not limited to mammals. Obesogens also end up in the aquatic environment, potentially affecting its ecosystems. In fact, reports show that some environmental chemicals are able to alter lipid homeostasis, impacting weight, lipid profile, signaling pathways and/or protein activity, of several taxa of aquatic animals. Such perturbations may give rise to physiological disorders and disease. Although largely unexplored from a comparative perspective, the key molecular components implicated in lipid homeostasis have likely appeared early in animal evolution. Therefore, it is not surprising that the obesogen effects are found in other animal groups beyond mammals. Collectively, data indicates that suspected obesogens impact lipid metabolism across phyla that have diverged over 600 million years ago. Thus, a consistent link between environmental chemical exposure and the obesity epidemic has emerged. This review aims to summarize the available information on the effects of putative obesogens in aquatic organisms, considering the similarities and differences of lipid homeostasis pathways among metazoans, thus contributing to a better understanding of the etiology of obesity in human populations. Finally, we identify the knowledge gaps in this field and we set future research priorities.

Cloning and functional characterization of a retinoid X receptor orthologue in Platynereis dumerilii: An evolutionary and toxicological perspective.

In the present work we provide the first isolation and functional characterization of a RXR orthologue in an annelid species, the Platynereis dumerilii. Using an in vitro luciferase reporter assay we evaluated the annelid receptor ability to respond to ligand 9-cis-retinoic acid, TBT and TPT. Our results show that the annelid RXR responds to 9-cis-retinoic acid and to the organotins by activating reporter gene transcription. The findings suggest a conserved mode of action of the receptor in response to a common signaling molecule and modulation by organotin compounds between vertebrates and Lophotrochozoans.

Genome specific PPARαB duplicates in salmonids and insights into estrogenic regulation in brown trout.

Peroxisome proliferator-activated receptors (PPARs) are key regulators of many processes in vertebrates, such as carbohydrate and lipid metabolism. PPARα, a member of the PPAR nuclear receptor gene subfamily (NR1C1), is involved in fatty acid metabolism, namely in peroxisomal ß-oxidation. Two gene paralogues, pparαA and pparαB, were described in several teleost species with their origin dating back to the teleost-specific genome duplication (3R). Given the additional salmonid-specific genome duplication (4R), four genes could be theoretically anticipated for this gene subfamily. In this work, we examined the pparα gene repertoire in brown trout, Salmo trutta f. fario. Data disclosed two pparα-like sequences in brown trout. Phylogenetic analyses further revealed that the isolated genes are most likely genome pparαB duplicates, pparαBa and pparαBb, while pparαA is apparently absent in salmonids. Both genes showed a ubiquitous mRNA expression across a panel of 11 different organs. In vitro exposed primary brown trout hepatocytes strongly suggest that pparα gene paralogues are differently regulated by ethinylestradiol (EE2). PparαBb mRNA expression significantly decreased with dosage, reaching significance after exposure to 50µM EE2, while pparαBa mRNA increased, significant at 1µM EE2. The present data enhances the understanding of pparα function and evolution in teleost, and reinforces the evidence of a potential crosstalk between estrogenic and pparα signaling pathways.

Total substitution of dietary fish oil by vegetable oils stimulates muscle hypertrophic growth in Senegalese sole and the upregulation of fgf6.

The long term effects of fish oil (FO) substitution by increasing the levels of vegetable oils (VO), 0% (CTR), 50% (VO50) and 100% (VO100), in diets for Senegalese sole were evaluated in terms of skeletal muscle cellularity and expression of related genes. After 140 days of feeding, all fish had similar body weight and length. The inclusion of 50% VO did not result in differences in muscle cellularity, but dorsal muscle cross-sectional area and fast-twitch fibre diameter increased in fish fed total FO substitution, whilst fibre density was reduced (P < 0.05) in relation to CTR. The total number of fibres was similar in all treatments. FO substitution did not affect the transcript levels of myogenic genes (myf5, mrf4, myog, myod1, myod2), but resulted in a two-fold increase of fgf6 transcript levels compared to CTR (P < 0.05). The relative expression of igf-I was higher in VO100 than in VO50, but was similar to CTR. FO substitution resulted in cellularity changes related to the stimulation of muscle hypertrophic growth, but not hyperplastic growth, and associated with a nutritional modulation of fgf6 by dietary VO. This study indicates that 50% VO does not affect the muscle phenotype, but total FO substitution stimulates muscle hypertrophy.

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect.

Imposex development in Hexaplex trunculus (Gastropoda: Caenogastropoda) involves changes in the transcription levels of the retinoid X receptor (RXR).

In order to further demonstrate that TBT-induced imposex through RXR signaling is not species-specific, Hexaplex trunculus was selected as an experimental model species. We first isolated RXR in H. trunculus, and determined gene transcription through quantitative real-time PCR in key tissues (e.g., penis/penis-forming area and central nervous system:- CNS), upon exposure to tributyltin (TBT) (5 and 50 ng TBTL(-1)). Two months of exposure to TBT induced imposex and led to a significant increase in the severity of the phenomenon in females and an increase in male penis lengths. Exposure to TBT altered RXR gene transcription in a tissue and sex-specific manner. In the CNS, there were no significant changes in RXR gene transcription between control and TBT-exposed females. A similar trend was observed in male CNS. On the contrary, in the penis-forming area/penis of females exposed to TBT, a significant increase in RXR gene transcription was observed in the 50 ng TBTL(-1) group. Furthermore, a positive correlation was observed between overall female penis lengths and RXR gene transcription. In males, although a trend towards an increase in RXR gene transcription in penis was observed, differences did not reach significance. Overall, the results of the present study give further support to a local role of RXR in the penis-forming area during the development of imposex by TBT, thus suggesting a conserved function of RXR in penis formation at least within prosobranch gastropods.

The evolution of pepsinogen C genes in vertebrates: duplication, loss and functional diversification.

BACKGROUND: Aspartic proteases comprise a large group of enzymes involved in peptide proteolysis. This collection includes prominent enzymes globally categorized as pepsins, which are derived from pepsinogen precursors. Pepsins are involved in gastric digestion, a hallmark of vertebrate physiology. An important member among the pepsinogens is pepsinogen C (Pgc). A particular aspect of Pgc is its apparent single copy status, which contrasts with the numerous gene copies found for example in pepsinogen A (Pga). Although gene sequences with similarity to Pgc have been described in some vertebrate groups, no exhaustive evolutionary framework has been considered so far. METHODOLOGY/PRINCIPAL FINDINGS: By combining phylogenetics and genomic analysis, we find an unexpected Pgc diversity in the vertebrate sub-phylum. We were able to reconstruct gene duplication timings relative to the divergence of major vertebrate clades. Before tetrapod divergence, a single Pgc gene tandemly expanded to produce two gene lineages (Pgbc and Pgc2). These have been differentially retained in various classes. Accordingly, we find Pgc2 in sauropsids, amphibians and marsupials, but not in eutherian mammals. Pgbc was retained in amphibians, but duplicated in the ancestor of amniotes giving rise to Pgb and Pgc1. The latter was retained in mammals and probably in reptiles and marsupials but not in birds. Pgb was kept in all of the amniote clade with independent episodes of loss in some mammalian species. Lineage specific expansions of Pgc2 and Pgbc have also occurred in marsupials and amphibians respectively. We find that teleost and tetrapod Pgc genes reside in distinct genomic regions hinting at a possible translocation. CONCLUSIONS: We conclude that the repertoire of Pgc genes is larger than previously reported, and that tandem duplications have modelled the history of Pgc genes. We hypothesize that gene expansion lead to functional divergence in tetrapods, coincident with the invasion of terrestrial habitats.

Functional desaturase Fads1 (Δ5) and Fads2 (Δ6) orthologues evolved before the origin of jawed vertebrates.

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

Fluorescent in situ hybridisation to amphioxus chromosomes.

We describe an efficient protocol for mapping genes and other DNA sequences to amphioxus chromosomes using fluorescent in situ hybridisation. We apply this method to identify the number and location of ribosomal DNA gene clusters and telomere sequences in metaphase spreads of Branchiostoma floridae. We also describe how the locations of two single copy genes can be mapped relative to each other, and demonstrate this by mapping an amphioxus Pax gene relative to a homologue of the Notch gene. These methods have great potential for performing comparative genomics between amphioxus and vertebrates.