Gamma irradiation-induced offspring masculinization is associated with epigenetic changes in female zebrafish.

Sex ratio variation is a key topic in ecology, because of its direct effects on population dynamics and thus, on animal conservation strategies. Among factors affecting sex ratio, types of sex determination systems have a central role, since some species could have a sex determined by genetic factors, environmental factors or a mix of those two. Yet, most studies on the factors affecting sex determination have focused on temperature or endocrine-disrupting chemicals (EDCs), and much less is known regarding other factors. Exposure to gamma irradiation was found to trigger offspring masculinization in zebrafish. Here we aimed at deciphering the potential mechanisms involved, by focusing on stress (i.e. cortisol) and epigenetic regulation of key genes involved in sex differentiation in fish. Cortisol levels in exposed and control (F0) zebrafish females' gonads were similar. However, irradiation increased the DNA methylation level of foxl2a and cyp19a1a in females of the F0 and F1 generation, respectively, while no effects were detected in testis. Overall, our results suggest that parental exposure could alter offspring sex ratio, at least in part by inducing methylation changes in ovaries.

Trematode genetic patterns at host individual and population scales provide insights about infection mechanisms.

Multiple parasites can infect a single host, creating a dynamic environment where each parasite must compete over host resources. Such interactions can cause greater harm to the host than single infections and can also have negative consequences for the parasites themselves. In their first intermediate hosts, trematodes multiply asexually and can eventually reach up to 20% of the host's biomass. In most species, it is unclear whether this biomass results from a single infection or co-infection by 2 or more infective stages (miracidia), the latter being more likely a priori in areas where prevalence of infection is high. Using as model system the trematode Bucephalus minimus and its first intermediate host cockles, we examined the genetic diversity of the cytochrome c oxidase subunit I region in B. minimus from 3 distinct geographical areas and performed a phylogeographic study of B. minimus populations along the Northeast Atlantic coast. Within localities, the high genetic variability found across trematodes infecting different individual cockles, compared to the absence of variability within the same host, suggests that infections could be generally originating from a single miracidium. On a large spatial scale, we uncovered significant population structure of B. minimus, specifically between the north and south of Bay of Biscay. Although other explanations are possible, we suggest this pattern may be driven by the population structure of the final host.

Sex-specific DNA methylation and transcription of zbtb38 and effects of gene-environment interactions on its natural antisense transcript in zebrafish.

There is increasing evidence for the involvement of epigenetics in sex determination, maintenance, and plasticity, from plants to humans. In our previous work, we reported a transgenerational feminization of a zebrafish population for which the first generation was exposed to cadmium, a metal with endocrine disrupting effects. In this study, starting from the previously performed whole methylome analysis, we focused on the zbtb38 gene and hypothesized that it could be involved in sex differentiation and Cd-induced offspring feminization. We observed sex-specific patterns of both DNA methylation and RNA transcription levels of zbtb38. We also discovered that the non-coding exon 3 of zbtb38 encodes for a natural antisense transcript (NAT). The activity of this NAT was found to be influenced by both genetic and environmental factors. Furthermore, increasing transcription levels of this NAT in parental gametes was highly correlated with offspring sex ratios. Since zbtb38 itself encodes for a transcription factor that binds methylated DNA, our results support a non-negligible role of zbtb38 not only in orchestrating the sex-specific transcriptome (i.e., sex differentiation) but also, via its NAT, offspring sex ratios.

Molecular confirmation of pearl formation in arctic mussels (Mytilus edulis) caused by Gymnophallus bursicola (Odhner 1900) metacercariae.

In recent field studies, suspected gymnophallid metacercariae were histologically located in the mantle of mussels from the Norwegian Sea. Mussels from the sites in which that infection was detected also presented abnormally high pearl numbers. It has been previously described that gymnophallid metacercariae could cause pearl formation processes in mussels, as a host reaction to encapsulate these metacercariae. Given the pathological host reaction these parasites elicit, a study was performed to identify gymnophallid metacercariae found in mussels collected from Tromsø at morphological and molecular level and to assess, by the use of molecular tools, the relationship between the parasite and the biological material inside the pearls. As a result, Gymnophallus bursicola metacercariae infecting Norwegian Mytilus edulis were identified according to morphological characters, along with the first 18S rDNA and COI sequences for this trematode species. In addition, parasite DNA from the core of the pearls was extracted and amplified for the first time, confirming the parasitological origin of these pearls. This procedure could allow identifying different parasitic organisms responsible for the generation of pearls in bivalves.

Altered ovarian transcriptome is linked to early mortality and abnormalities in zebrafish embryos after maternal exposure to gamma irradiation.

Recent laboratory studies focusing on multigenerational approach demonstrated drastic phenotypic effects after chronic fish irradiation exposure. No irradiation effect at phenotypic scale was observed for F0 (reproductive performances) while early mortality and malformations were observed in F1 offspring whether they were irradiated or not. The objective was to study molecular mechanisms likely to be involved in these phenotypic effects induced by parental irradiation. Thus, F0 adult zebrafish were irradiated for ten days until reproduction and maternal involvement in offspring development was assessed. Levels of maternal provided cortisol and vitellogenin, needed for embryo development, were not impacted by irradiation. However, maternal transcriptome highlighted irradiation effect on processes involved in oocyte development, as well as on essential maternal factors needed for offspring development. Therefore, this study highlighted the importance of parental exposure on offspring fate and of the importance of multigenerational exposure in risk assessment.

A New Polyclad Flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Platyhelminthes, Polycladida) from France. Can this Foreign Flatworm be Responsible for the Deterioration of Oyster and Mussel Farms?

A new species of polyclad flatworm, Idiostylochus tortuosus gen. nov., sp. nov. (Polycladida, Idioplanidae), from Arcachon Bay (France) is described. This description is based on a morphological analysis and a molecular analysis using partial sequences of the 28S and cytochrome Oxidase I (COI) genes. After the molecular analysis Idiostylochus gen. nov. appears to be the second genus of the Family Idioplanidae and closely related to the family Latocestidae as well as the genera Leptostylochus and Mirostylochus. The molecular data revealed that the new species may belong to an Indonesian or Indo-Pacific family, closely related to genera with origins in South Pacific Ocean waters. This species was found feeding on the oysters and mussels of the Arcachon farms.

Two new species of Marphysa (Annelida, Eunicidae) from southern Australia.

Two new species of Marphysa Quatrefages, 1866 are described from the southeast coast of Australia. With the presence of only compound spinigers and the branchiae present over many chaetigers, Marphysa baudini n. sp. belongs to the Sanguinea-group. This species has ventral cirri with an inflated base and digitiform tip and thick and wide anodont pectinate chaetae, with 3-5 internal long and thick teeth. With the presence of only compound falcigers, Marphysa davidattenboroughi n. sp., belongs to the "Aenea-group". This species is characterised by the presence of a bilobed prostomium, a single pair of pygidial cirri and by the presence of thick, and wide anodont pectinate chaetae with 4-6 long internal and thick teeth.

Transgenerational endocrine disruptor effects of cadmium in zebrafish and contribution of standing epigenetic variation to adaptation.

Evidence has emerged that environmentally-induced epigenetic changes can have long-lasting effects on gene transcription across generations. These recent findings highlight the need to investigate the transgenerational impacts of pollutants to assess their long term effects on populations. In this study, we investigated the transgenerational effect of cadmium on zebrafish across 4 generations. A first whole methylome approach carried out on fish of the first two generations led us to focus our investigations on the estradiol receptor alpha gene (esr1). We observed a sex-dependent transgenerational inheritance of Cd-induced DNA methylation changes up to the last generation. These changes were associated with single nucleotide polymorphisms (SNPs) that were themselves at the origin of the creation or deletion of methylation sites. Thus, Cd-induced genetic selection gave rise to DNA methylation changes. We also analyzed the transcription level of various sections of esr1 as well as estrogen responsive genes. While Cd triggered transgenerational disorders, Cd-induced epigenetic changes in esr1 contributed to the rapid transgenerational adaptation of fish to Cd. Our results provide insight into the processes underpinning rapid adaptation and highlight the need to maintain genetic diversity within natural populations to bolster the resilience of species faced with the global environmental changes.

Genetic and epigenetic interplay allows rapid transgenerational adaptation to metal pollution in zebrafish.

Despite still being a matter of debate, there is growing evidence that pollutant-induced epigenetic changes can be propagated across generations. Whereas such modifications could have long-lasting effects on organisms and even on population, environmentally relevant data from long-term exposure combined with follow-up through multiple generations remain scarce for non-mammalian species. We performed a transgenerational experiment comprising four successive generations of zebrafish. Only fish from the first generation were exposed to an environmentally realistic concentration of cadmium (Cd). Using a whole methylome analysis, we first identified the DNA regions that were differentially methylated in response to Cd exposure and common to fish of the first two generations. Among them, we then focused our investigations on the exon 3 (ex3) of the cep19 gene. We indeed recorded transgenerational growth disorders in Cd-exposed fish, and a mutation in this exon is known to cause morbid obesity in mammals. Its methylation level was thus determined in zebrafish from all the four generations by means of a targeted and base resolution method. We observed a transgenerational inheritance of Cd-induced DNA methylation changes up to the fourth generation. However, these changes were closely associated with genetic variations, mainly a single nucleotide polymorphism. This single nucleotide polymorphism was itself at the origin of the creation or deletion of a methylation site and deeply impacted the methylation level of neighboring methylation sites. Cd-induced epigenetic changes were associated with different mRNA transcripts and an improved condition of Cd fish. Our results emphasize a tight relationship between genetic and epigenetic mechanisms and suggest that their interplay and pre-existing diversity can allow rapid adaptation to anthropogenic environmental changes.

Impact of nickel mining in New Caledonia on marbled eels Anguilla marmorata.

New Caledonia is particularly affected by nickel open pit mining activities because of the presence of ultramafic soils rich in metals. The particles dispersed by atmospheric transport and soil erosion during the excavation of nickel end up by deposition or leaching in rivers where they may be bioaccumulated by organisms living downstream the mines. Despite alarming freshwater metals concentrations, no study investigated the level of their bioaccumulation in eels, and if high bioaccumulation levels occur, the potential consequences on their health. The aim of this study was to determine how eels Anguilla marmorata are impacted in situ by metals issued from mining activity by measuring: morphometric parameters; metal concentrations in tissues and organs and transcription levels of target genes encoding proteins involved in several metabolic key functions. Among organs, liver was found to be the most affected by mining with average nickel concentrations of 5.14 mg/kg versus 1.63 mg/kg for eels away from mines leading to dysregulation of numerous genes involved in oxidative stress, DNA repair, apoptosis, reproduction and both lipid and mitochondrial metabolisms. This study should allow us to define in an integrated way if metals released by mining activities influence metals bioaccumulation in eels and induce biological effects.

Cellular and molecular mechanisms of NiONPs toxicity on eel hepatocytes HEPA-E1: An illustration of the impact of Ni release from mining activity in New Caledonia.

Anthropic activities such as open pit mining, amplify the natural erosion of metals contained in the soils, particularly in New Caledonia, leading to atmospheric emission of nickel oxide nanoparticles (NiONPs). These particles are produced during extraction end up in aquatic ecosystems through deposition or leaching in the rivers. Despite alarming freshwater Ni concentrations, only few studies have focused on the cellular and molecular mechanisms of NiONPs toxicity on aquatic organisms and particularly on eels. Those fish are known to be sensitive to metal contamination, especially their liver, which is a key organ for lipid metabolism, detoxification and reproduction. The objective of this study was to assess in vitro the cytotoxic effects of NiONPs on Anguilla japonica hepatocytes, HEPA-E1. HEPA-E1 were exposed to NiONPs (0.5-5 µg/cm2) for 4 or 24 h. Several endpoints were studied: (i) viability, (ii) ROS production, SOD activity and selected anti-oxidant genes expression, (iii) inflammation, (iv) calcium signalling, (v) mitochondrial function and (vi) apoptosis. The results evidenced that NiONPs induce a decrease of cell viability and an increase in oxidative stress with a significant superoxide anion production. An increase of mitochondrial calcium concentration and a decrease of mitochondrial membrane potential were observed, leading to apoptosis. These results underline the potential toxic impact of NiONPs on eels living in mining areas. Therefore, eel exposure to NiONPs can affect their migration and reproduction in New Caledonia.

Three new deep-sea species of Marphysa (Annelida, Eunicida, Eunicidae) from Papua New Guinea (Bismarck and Solomon seas).

Three new species of Marphysa Quatrefages, 1866, Marphysabanana sp. nov., Marphysapapuaensis sp. nov., and Marphysazanolae sp. nov. are described from deep-sea sunken vegetation off Papua New Guinea, using both morphology and molecular data (for two species). With the presence of compound spinigers only and the branchiae present over many chaetigers, Marphysabanana sp. nov. belongs to the group B2. This species is characterised by the presence of eyes, the presence of branchiae starting from chaetiger 20, and by the presence of three types of pectinate chaetae and bidentate subacicular hooks starting from chaetigers 13-52. With the presence of compound falcigers only and the branchiae restricted to a short anterior region, Marphysapapuaensis sp. nov. belongs to the group C1. This species has a bilobed prostomium but no eyes, has branchiae from chaetigers 7 to 14-16 with up to 16 filaments. Marphysapapuaensis sp. nov. is also characterised by the presence of bidentate subacicular hooks from chaetiger 20 and by a single type of pectinate chaetae. Finally, Marphysazanolae sp. nov. belongs to the group C2, with the presence of compound falcigers only and the branchiae present over many chaetigers. This species is characterised by the absence of eyes, by the presence of branchiae with a single long filament starting from chaetiger 31, by unidentate subacicular hooks starting from chaetiger 28 and finally by one type of pectinate chaetae with very long outer teeth.

Revision of the French Terebellidae sensu stricto (Annelida, Terebelliformia), with descriptions of nine new species.

This work is the last of four papers of the Spaghetti Project, aiming to revise the species of terebellids, a.k.a. spaghetti worms, present in the European French waters. In this last paper the Terebellidae, sensu stricto, from French waters are revised based, on material available in the French marine stations, type materials stored in the MNHN collection and newly collected specimens. Nine new species are described using both morphological and molecular tools: Eupolymnia gili n. sp., E. lacazei n. sp., E. meissnerae n. sp., Lanice kellyslateri n. sp., Paramphitrite dragovabeci n. sp., Pista labruneae n. sp., P. miosseci n. sp., P. sauriaui n. sp., and Terebella banksyi n. sp. European species of Eupolymnia are distinguished mainly by the shape of the lateral lobes and the size of the branchial stems. The two species belonging to Lanice genus are distinguished by the fusion of the first ventral shields, the shape of both noto- and neuropodia, and the pigmentation of the upper lip. The two species of Paramphitrite are distinguished by the presence or absence of a medial dorsal gap between the pairs of branchiae, by the shape of the lateral lobes and the presence or absence of a nephridial papilla on segment 4. The different species of Pista are distinguished by the number of pairs of branchiae, the shape of the lateral lobes and uncini. Finally, the two species of Terebella are distinguished by the number of segments with nephridial and genital papillae and the segments on which the branchiae occur. An identification key for European species of Terebellidae sensu stricto is also provided.

Transgenerational epigenetic sex determination: Environment experienced by female fish affects offspring sex ratio.

Sex determination is a complex process that can be influenced by environment in various taxa. Disturbed environments can affect population sex ratios and thus threaten their viability. Emerging evidences support a role of epigenetic mechanisms, notably DNA methylation, in environmental sex determination (ESD). In this work, using zebrafish as model and a transgenerational experiment comprising 4 successive generations, we report a strength link between the promotor methylation level of three genes in female gonads and population sex ratio. One generation of zebrafish was exposed throughout its lifetime to cadmium (Cd), a non-essential metal, at an environmentally relevant concentration. The subsequent generations were not exposed. At the first and the third generation a subset of individuals was exposed to an elevated temperature, a well-known masculinizing factor in zebrafish. While heat was associated to an increase in the methylation level of cyp19a1a gene and population masculinization, foxl2a/dmrt1 methylation levels appeared to be influenced by Cd and fish density leading to offspring feminization. Ancestral Cd exposure indeed led to a progressive feminization of the population over generations and affected the sex plastic response of zebrafish in response to heat. The effect of Cd on the methylation level of foxl2a was observed until the third generation, supporting potential transgenerational inheritance. Our results support (i) a key role of cyp19a1a methylation in SD in zebrafish in response to environmental cues and (ii) the fact that the environment experienced by parents, namely mothers in the present case, can affect their offspring sex ratio via environment-induced DNA methylation changes in gonads.

Mitochondrial genome of Sabella spallanzanii (Gmelin, 1791) (Sabellida: Sabellidae).

We report the mitochondrial genome of Sabella spallanzanii, an invasive Mediterranean sabellid introduced to Australia and New Zealand. The mitogenome is 15,581 bp long and consists of 38 genes, including 13 protein coding genes, two rRNA genes, and 23 tRNA genes. It shows deviations from the putative annelid ground pattern, such as gene order re-arrangements and regions encoding on the negative strand. It is, however, very different from the mitogenome of the closely related serpulid, Spirobranchus giganteus. Phylogenetic analyses of the mitochondrial genes support a sister relationship of Sabella spallanzanii and Spirobranchus giganteus.

Another blow to the conserved gene order in Annelida: Evidence from mitochondrial genomes of the calcareous tubeworm genus Hydroides.

Mitochondrial genomes are frequently applied in phylogenetic and evolutionary studies across metazoans, yet they are still poorly represented in many groups of invertebrates, including annelids. Here, we report ten mitochondrial genomes from the annelid genus Hydroides (Serpulidae) and compare them with all available annelid mitogenomes. We detected all 13 protein coding genes in Hydroides spp., including the atp8 which was reported as a missing gene in the Christmas Tree worm Spirobranchus giganteus, another annelid of the family Serpulidae. All available mitochondrial genomes of Hydroides show a highly positive GC skew combined with a highly negative AT skew - a feature consistent with that found only in the mitogenome of S. giganteus. In addition, amino acid sequences of the 13 protein-coding genes showed a high genetic distance between the Hydroides clade and S. giganteus, suggesting a fast rate of mitochondrial sequence evolution in Serpulidae. The gene order of protein-coding genes within Hydroides exhibited extensive rearrangements at species level, and were different from the arrangement patterns of other annelids, including S. giganteus. Phylogenetic analyses based on protein-coding genes recovered Hydroides as a monophyletic group sister to Spirobranchus with a long branch, and sister to the fan worm Sabellidae. Yet the Serpulidae + Sabellidae clade was unexpectedly grouped with Sipuncula, suggesting that mitochondrial genomes alone are insufficient to resolve the phylogenetic position of Serpulidae within Annelida due to its high base substitution rates. Overall, our study revealed a high variability in the gene order arrangement of mitochondrial genomes within Serpulidae, provided evidence to question the conserved pattern of the mitochondrial gene order in Annelida and called for caution when applying mitochondrial genes to infer their phylogenetic relationships.

Revision of the French Polycirridae (Annelida, Terebelliformia), with descriptions of eight new species.

Polycirridae from French waters are revised based on material available in French marine stations and newly collected specimens. This work is the third part of the Spaghetti Project aiming to revise French species of "Spaghetti" terebellid worms. It describes eight new species using both morphological (for all species) and molecular (for some species) tools: Amaeana gremarei n. sp., Polycirrus catalanensis n. sp., P. glasbyi n. sp., P. gujanensis n. sp., P. idex n. sp., P. nogueirai n. sp., P. pennarbedae n. sp. and P. readi n. sp., in addition to a previously described species. An identification key for European species of Polycirridae is also provided.

A revision of the French Telothelepodidae and Thelepodidae (Annelida, Terebelliformia), with descriptions of three species and first European record of a non-indigenous species.

Telothelepodidae and Thelepodidae from French waters are revised based on material available in French marine stations and newly collected specimens. This work is the second part of the Spaghetti Project aiming to revise French species of "Spaghetti" worms. It describes three new species using both morphological and molecular tools: Streblosoma cabiochi n. sp., Streblosoma lindsayae n. sp. and Thelepus corsicanus n. sp. This study also permitted us to detect the presence of an Asiatic species, Thelepus japonicus, in Arcachon Bay and Normandy, introduced via oysters transfers. An identification key for European species of both families is also provided.

A new species of Marphysa (Annelida: Eunicida: Eunicidae) from India, with notes on previously described or reported species from the region.

A new species of Marphysa is described from India using both morphology and molecular data. The new species, Marphysa madrasi, belonging to the Teretiuscula-group, is characterised by having antennae about 2x longer than the prostomium, maxillae II and IV with a relatively large number of teeth (Mx II with 8+9; Mx IV with 7+11), compound spinigers present on a large range of mid-body chaetigers (at least 83-159), and pectinate chaetae present from the first few anterior chaetigers. We discuss all the species of this genus lacking compound falcigers (i.e., those belonging to the Sanguinea-, Mossambica- and Teretiuscula-groups), which have been reported from India and compare them to our new species. Only two accepted species in these groups can be confirmed as occurring in India, M. madrasi n. sp. and M. gravelyi Southern, 1921. We provide a table that summarises the important characters useful to identify species in this group of Marphysa with compound spinigers.