Developmental toxicity of pre-production plastic pellets affects a large swathe of invertebrate taxa.

Microplastics pose risks to marine organisms through ingestion, entanglement, and as carriers of toxic additives and environmental pollutants. Plastic pre-production pellet leachates have been shown to affect the development of sea urchins and, to some extent, mussels. The extent of those developmental effects on other animal phyla remains unknown. Here, we test the toxicity of environmental mixed nurdle samples and new PVC pellets for the embryonic development or asexual reproduction by regeneration of animals from all the major animal superphyla (Lophotrochozoa, Ecdysozoa, Deuterostomia and Cnidaria). Our results show diverse, concentration-dependent impacts in all the species sampled for new pellets, and for molluscs and deuterostomes for environmental samples. Embryo axial formation, cell specification and, specially, morphogenesis seem to be the main processes affected by plastic leachate exposure. Our study serves as a proof of principle for the potentially catastrophic effects that increasing plastic concentrations in the oceans and other ecosystems can have across animal populations from all major animal superphyla.

Reaping the benefits of liquid handlers for high-throughput gene expression profiling in a marine model invertebrate.

BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Nitric Oxide Function and Nitric Oxide Synthase Evolution in Aquatic Chordates.

Nitric oxide (NO) is a key signaling molecule in almost all organisms and is active in a variety of physiological and pathological processes. Our understanding of the peculiarities and functions of this simple gas has increased considerably by extending studies to non-mammal vertebrates and invertebrates. In this review, we report the nitric oxide synthase (Nos) genes so far characterized in chordates and provide an extensive, detailed, and comparative analysis of the function of NO in the aquatic chordates tunicates, cephalochordates, teleost fishes, and amphibians. This comprehensive set of data adds new elements to our understanding of Nos evolution, from the single gene commonly found in invertebrates to the three genes present in vertebrates.

Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development.

Nitric oxide (NO) is an ancestral key signalling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission and immunity. Although our knowledge of NO synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologues in fishes with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in this lineage, changed this perspective. This finding prompted us to explore nos gene evolution, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralogue in the gills of developing shark, bichir, sturgeon, and gar, but not in lamprey, suggests that nos expression in this organ may have arisen in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes' roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

Expression Pattern of nos1 in the Developing Nervous System of Ray-Finned Fish.

Fish have colonized nearly all aquatic niches, making them an invaluable resource to understand vertebrate adaptation and gene family evolution, including the evolution of complex neural networks and modulatory neurotransmitter pathways. Among ancient regulatory molecules, the gaseous messenger nitric oxide (NO) is involved in a wide range of biological processes. Because of its short half-life, the modulatory capability of NO is strictly related to the local activity of nitric oxide synthases (Nos), enzymes that synthesize NO from L-arginine, making the localization of Nos mRNAs a reliable indirect proxy for the location of NO action domains, targets, and effectors. Within the diversified actinopterygian nos paralogs, nos1 (alias nnos) is ubiquitously present as a single copy gene across the gnathostome lineage, making it an ideal candidate for comparative studies. To investigate variations in the NO system across ray-finned fish phylogeny, we compared nos1 expression patterns during the development of two well-established experimental teleosts (zebrafish and medaka) with an early branching holostean (spotted gar), an important evolutionary bridge between teleosts and tetrapods. Data reported here highlight both conserved expression domains and species-specific nos1 territories, confirming the ancestry of this signaling system and expanding the number of biological processes implicated in NO activities.

Mitochondrial genome sequencing of marine leukaemias reveals cancer contagion between clam species in the Seas of Southern Europe.

Clonally transmissible cancers are tumour lineages that are transmitted between individuals via the transfer of living cancer cells. In marine bivalves, leukaemia-like transmissible cancers, called hemic neoplasia (HN), have demonstrated the ability to infect individuals from different species. We performed whole-genome sequencing in eight warty venus clams that were diagnosed with HN, from two sampling points located more than 1000 nautical miles away in the Atlantic Ocean and the Mediterranean Sea Coasts of Spain. Mitochondrial genome sequencing analysis from neoplastic animals revealed the coexistence of haplotypes from two different clam species. Phylogenies estimated from mitochondrial and nuclear markers confirmed this leukaemia originated in striped venus clams and later transmitted to clams of the species warty venus, in which it survives as a contagious cancer. The analysis of mitochondrial and nuclear gene sequences supports all studied tumours belong to a single neoplastic lineage that spreads in the Seas of Southern Europe.

In humans and other animals, cancer cells divide excessively, forming tumours or flooding the blood, but they rarely spread to other individuals. However, some animals, including dogs, Tasmanian devils and bivalve molluscs like clams, cockles and mussels, can develop cancers that are transmitted from one individual to another. Despite these cancers being contagious, each one originates in a single animal, meaning that even when the cancer has spread to many individuals, its origins can be traced through its DNA. Cancer contagion is rare, but transmissible cancers seem to be particularly common in the oceans. In fact, 7 types of contagious cancer have been described in bivalve species so far. These cancers are known as 'hemic neoplasias', and are characterized by the uncontrolled division of blood-like cells, which can be released by the host they developed in, and survive in ocean water. When these cells encounter individuals from the same species, they can infect them, causing them to develop hemic neoplasia too There are still many unanswered questions about contagious cancers in bivalves. For example, how many species do the cancers affect, and which species do the cancers originate in? To address these questions, Garcia-Souto, Bruzos, Díaz et al. gathered over 400 specimens of a species of clam called the warty venus clam from the coastlines of Europe and examined them for signs of cancer. Clams collected in two regions of Spain showed signs of hemic neoplasia: one of the populations was from the Balearic Islands in the Mediterranean Sea, while the other came from the Atlantic coast of northwestern Spain. Analyzing the genomes of the tumours from each population showed that the cancer cells from both regions had likely originated in the same animal, indicating that the cancer is contagious and had spread through different populations. The analysis also revealed that the cancer did not originally develop in warty venus clams: the cancer cells contained DNA from both warty venus clams and another species called striped venus clams. These two species live close together in the Mediterranean Sea, suggesting that the cancer started in a striped venus clam and then spread to a warty venus clam. To determine whether the cancer still affected both species, Garcia-Souto, Bruzos, Díaz et al. screened 200 striped venus clams from the same areas, but no signs of cancer were found in these clams. This suggests that currently the cancer only affects the warty venus clam. These findings confirm that contagious cancers can jump between clam species, which could be threat to the marine environment. The fact that the cancer was so similar in clams from the Atlantic coast and from the Mediterranean Sea, however, suggests that it may have emerged very recently, or that human activity helped it to spread from one place to another. If the latter is the case, it may be possible to prevent further spread of these sea-borne cancers through human intervention.

Crosstalk between nitric oxide and retinoic acid pathways is essential for amphioxus pharynx development.

During animal ontogenesis, body axis patterning is finely regulated by complex interactions among several signaling pathways. Nitric oxide (NO) and retinoic acid (RA) are potent morphogens that play a pivotal role in vertebrate development. Their involvement in axial patterning of the head and pharynx shows conserved features in the chordate phylum. Indeed, in the cephalochordate amphioxus, NO and RA are crucial for the correct development of pharyngeal structures. Here, we demonstrate the functional cooperation between NO and RA that occurs during amphioxus embryogenesis. During neurulation, NO modulates RA production through the transcriptional regulation of Aldh1a.2 that irreversibly converts retinaldehyde into RA. On the other hand, RA directly or indirectly regulates the transcription of Nos genes. This reciprocal regulation of NO and RA pathways is essential for the normal pharyngeal development in amphioxus and it could be conserved in vertebrates.

Lactose Intolerance Assessed by Analysis of Genetic Polymorphism, Breath Test and Symptoms in Patients with Inflammatory Bowel Disease.

Many patients with inflammatory bowel disease (IBD) restrict dairy products to control their symptoms. The aim of the study was to investigate the prevalence of lactose intolerance assessed with hydrogen breath test (H-BT) in IBD patients in clinical remission compared to a sex, age and BMI matched control population. We further detected the prevalence of three single nucleotide polymorphisms of the lactase (LCT) gene: the lactase non persistence LCT-13910 CC (wildtype) and the intermediate phenotype LCT-22018 CT and LCT-13910 AG; finally, we assess the correlation between genotype and H-BT. A total of 54 IBD patients and 69 control who underwent clinical evaluation, H-BT and genetic test were enrolled. H-BT was positive in 64.8% IBD patients and 62.3% control (p = 0.3). The wild-type genotype was found in 85.2% IBD patients while CT-22018, AG-13910 and CT-22018/AG-13910 polymorphisms were found in 9.3%, 1.8% and 3.7%. In the control group, the wild-type genotype, CT-22018, AG-13910 and CT-22018/AG-13910 polymorphisms were found in 87%, 5.8%, 5.8% and 1.4% of cases, respectively. Therefore, the wild-type and polymorphisms' prevalence did not differ between IBD population and control group (85.2% vs. 87%, p = 0.1) (14.8% vs. 13%, p = 0.7). The correlation between positive H-BT and genetic analysis showed that the wild-type genotype was associated with higher rate of lactose intolerance in the total population (OR 5.31, 95%CI 1.73-16.29, p = 0.003) and in the IBD (OR 7.61, 95%CI 1.36-42.7, p = 0.02). The prevalence of lactose intolerance in IBD patients did not differ from that of control. Despite suggestive symptoms, about 1/3 of IBD patients are not lactose intolerant, thus not needing "a priori" elimination diet. This may encourage a rationale and balanced dietary management in IBD.

Short-term exposure to the simple polyphenolic compound gallic acid induces neuronal hyperactivity in zebrafish larvae.

A growing body of evidence suggests that the biological effects of polyphenols are not restricted to antioxidant activity, but they exert a wide range of modulatory effects on metabolic pathways, cellular signaling and gene expression. In this study, we tested the minimum safe concentration of gallic acid (GA) in 72 hpf zebrafish larvae in order to evaluate the effects on the central nervous system and the behavioral response. We showed that a short exposure (30 min) induces the depletion of the two main excitatory and inhibitory neurotransmitters, Glu and GABA, respectively, in the larval nervous system. The acute impairment of GABAergic-glutamatergic balance was paralleled by an increase of the fosab neuronal activity marker in specific brain areas, such as the forebrain, olfactory bulbs, pallial area, ventral midbrain, tegmentum, and the medulla oblongata ventral area. The neuronal excitation was mirrored by the increased cumulative motor response. The inhibition of the olfactory epithelium with brief cadmium exposition suggests a direct involvement of olfaction in the larvae response to GA. Our results demonstrate that a brief exposure to GA induces motoneuronal hyperexcitability in zebrafish. The behavioral response was probably elicited through the activation of an odorous, or chemical, stimulus. The specificity of the activated neuronal territories suggests the involvement of additional signaling pathways. Although the underlying molecular mechanisms remain to be elucidated, our data support the hypothesis that GA acts as an excitatory molecule, capable of inducing a specific nerve response. These results offer a new vision on potential effects of GA.

Nitric Oxide regulates mouth development in amphioxus.

The development of the mouth in animals has fascinated researchers for decades, and a recent study proposed the modern view of recurrent evolution of protostomy and deuterostomy. Here we expanded our knowledge about conserved traits of mouth formation in chordates, testing the hypothesis that nitric oxide (NO) is a potential regulator of this process. In the present work we show for the first time that NO is an essential cell signaling molecule for cephalochordate mouth formation, as previously shown for vertebrates, indicating its conserved ancestral role in chordates. The experimental decrease of NO during early amphioxus Branchiostoma lanceolatum development impaired the formation of the mouth and gill slits, demonstrating that it is a prerequisite in pharyngeal morphogenesis. Our results represent the first step in the understanding of NO physiology in non-vertebrate chordates, opening new evolutionary perspectives into the ancestral importance of NO homeostasis and acquisition of novel biological roles during evolution.

Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective.

BACKGROUND: The regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the Rab family proteins. Among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. Specific Rab proteins, as Rab32 and Rab38, have been shown to play a key role in melanosome biogenesis. We analysed the Rab32 and Rab38 genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications. RESULTS: We studied the molecular evolution of Rab supergroup III in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. We discovered a novel amino acid stretch, named FALK, shared by three related classes belonging to Rab supergroup III: Rab7L1, Rab32LO and Rab32/Rab38. Among these, we demonstrated that the Rab32LO class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. Synteny shows that one zebrafish gene, Rab38a, which is expressed in pigmented cells, retained the linkage with tyrosinase, a protein essential for pigmentation. Moreover, the chromosomal linkage of Rab32 or Rab38 with a member of the glutamate receptor metabotropic (Grm) family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor. Expression patterns of Rab32 and Rab38 genes in zebrafish, and Rab32/38 in amphioxus, indicate their involvement in development of pigmented cells and notochord. CONCLUSIONS: Phylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate Rab32/38 gene giving rise to vertebrate Rab32 and Rab38. The expression patterns of Rab38 paralogues highlight sub-functionalization event. Finally, the discovery of a chromosomal linkage between the Rab32 or Rab38 gene with a Grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution.

Evolution of the notochord.

A notochord is characteristic of developing chordates (which comprise amphioxus, tunicates and vertebrates), and, more arguably, is also found in some other animals. Although notochords have been well reviewed from a developmental genetic point of view, there has heretofore been no adequate survey of the dozen or so scenarios accounting for their evolutionary origin. Advances in molecular phylogenetics and developmental genetics have, on the one hand, failed to support many of these ideas (although, it is not impossible that some of these rejects may yet, at least in part, return to favor). On the other hand, current molecular approaches have actually stimulated the revival of two of the old proposals: first that the notochord is a novelty that arose in the chordates, and second that it is derived from a homologous structure, the axochord, that was present in annelid-like ancestors. In the long term, choosing whether the notochord is a chordate novelty or a legacy from an ancient annelid (or perhaps an evolutionary derivative from precursors yet to be proposed) will probably require descriptions of gene regulatory networks involved in the development of notochords and notochord-like structures in a wide spectrum of animals. For now, one-way forward will be studies of all aspects of the biology of enteropneust hemichordates, a group widely thought to be the key to understanding the evolutionary origin of the chordates.