Gene expression and biochemical patterns in the digestive gland of the mussel Mytilus galloprovincialis (Lamarck, 1819) exposed to 17α-ethinylestradiol.

Contaminants of emerging concern (CECs) are a class of chemicals that can spread throughout the environment and may cause adverse biological and ecological effects. While there are many different classes of CECs, one of the most well documented in the aquatic environment are pharmaceutical drugs, such as natural and synthetic estrogens. In particular, the widespread presence of the synthetic estrogen 17 α-Ethinylestradiol (EE2) in water may lead to bioaccumulation in sediment and biota. EE2 is the primary component in contraceptive pills, and is a derivative of the natural hormone estradiol (E2). In this study, the mussel Mytilus galloprovincialis was exposed to EE2 in a semi-static and time-dependent experiment, for a total exposure period of 28 days. Biochemical and transcriptomics analyses were performed on mussel digestive glands after exposure for 14 (T14) and 28 (T28) days. Metabolic and DNA impairments, as well as activation of antioxidant and biotransformation enzymes activation, were detected in T28 exposed mussels. RNA-Seq analysis showed significant differential expression of 160 (T14 compared to controls), 33 (T28 compared to controls) and 79 (T14 compared to T28) genes. Signs of stress after EE2 treatment included up-regulation of gene/proteins involved with immune function, lipid transport, and metabolic and antibacterial properties. This study elucidates the underlying mechanisms of EE2 in a filter feeding organisms to elucidate the effects of this human pharmaceutical on aquatic biota.

A multidisciplinary integrated approach using Pachygrapsus marmoratus to assess the impact of port activities on mediterranean marine protected areas.

The establishment of marine protected areas is considered the main global strategy to halt the loss of marine biodiversity. Since most of marine areas are open systems, this form of habitat protection cannot prevent their contamination due to human activities performed outside of their borders. Innovative approaches to assess the health status of protected marine habitats are therefore needed. Here we developed a multidisciplinary approach that combines ecological characteristics, bioaccumulation of inorganic and organic pollutants, cell damage (micronuclei frequency, nuclear alterations and LPO) and enzymatic (AChE, CAT, IDH, LDH, GST and CAT) markers focused on an intertidal brachyuran crab, Pachygrapsus marmoratus, to assess the impacts of contaminant exposure on Mediterranean coastal habitats. As study sites we selected two protected areas and two sites within industrial ports of the Ligurian Sea. Our results showed that the selected crab species is an excellent bioindicator. Individuals collected in sites with the highest levels of heavy metal pollution showed the highest signals of stress responses at both cellular and enzymatic levels, coupled with a high incidence of the parasite Sacculina carcini, a signal of impairment of their standard development and reproduction cycle. We could also prove that one of the selected marine protected areas showed the same intensity of impact as its adjacent port site. Our multidisciplinary approach proved to be a valuable tool to assess the environmental quality and health of protected and disturbed Mediterranean coastal environments and to inform efficient management and protection schemes for such habitats.

Aliens Coming by Ships: Distribution and Origins of the Ocellated Skink Populations in Peninsular Italy.

The ocellated skink (Chalcides ocellatus) is a widespread lizard, naturally distributed between the Maghreb and coastal Pakistan, with few insular populations in the Mediterranean coastal area. Some populations of this species have also been recorded in peninsular Italy, Campania and Southern Tuscany due to accidental introductions via touristic and commercial routes. In this work, we conducted genetic analyses on mitochondrial DNA COXI, cytb and 16S mtDNA genes on a sample of Italian insular and peninsular populations. Differently from what previously suggested, the nucleus in Portici (Southern Italy) may have originated from Sardinia. The intense trade and touristic traffic between Sardinia and Southern Tuscany may have been responsible for the introduction of this lizard also to Central Italy.

Differential gene expression and chemical patterns of an intertidal crab inhabiting a polluted port and an adjacent marine protected area.

The acquisition of data to safeguard marine protected areas located close to ports is important in order to develop plans that allow effective protection from pollution as well as sustainable development of the port. The area Secche della Meloria is a Marine Protected Area (MPA-MEL) three miles from Livorno Harbour (LH), which is characterized by a long history of pollution. Here we studied the bioaccumulation and transcriptomic patterns of the marbled crab, Pachygrapsus marmoratus (Fabricius, 1787) (Crustacea; Brachyura, Grapsidae), inhabiting the two selected sites. Results showed that the two crab populations are significantly different in their chemical composition of trace elements and Polyciclic Aromatic Hydrocarbons (PAHs), and gene expression patterns (1280 DEGs). Enrichment analysis indicated that crabs at LH had the highest stress response genes, and they were associated with higher levels of bioaccumulation detected in body tissues. We are confident that the significant differential gene expression profiles observed between crabs, characterized by significant chemical differences, is associated with responses to contaminant exposure.

De novo transcriptome assembly for Pachygrapsus marmoratus, an intertidal brachyuran crab.

The marble crab Pachygrapsus marmoratus inhabits the rocky shores of the Mediterranean Sea, Black Sea and East Atlantic Ocean. As other intertidal species, it is considered a model species to study the effects of environmental stressors on natural populations. In this study, we performed Illumina next-generation sequencing on eleven P. marmoratus specimens with the aims to (i) reconstruct their whole transcriptome, (ii) perform a functional annotation of the assembled transcriptome and (iii) develop gene-based markers for future genetic and genomic studies on this as well as other brachyuran species. We obtained a transcriptome assembly constituted by 56,308 unigenes and covering about 60.3 Mbp. We detected 43,915 Simple Sequence Repeats (SSRs) and 192,631 high-quality Single Nucleotide Polymorphisms (SNPs). Due to the scarcity of genomic resources in decapods, and crabs in particular, our results constitute a valuable resource for future studies on brachyuran crabs. The present data also represent a sound resource to investigate biological responses to pollution in intertidal and marine populations.

Relationship between wild-caught organisms for bioassays and sampling areas: Widespread serpulid early-development comparison between two distinct populations after trace element exposure.

Previous studies suggested the suitability of the brackish-water serpulid (Ficopomatus enigmaticus) to be used as model organism for both marine and brackish waters monitoring, by the performance of sperm toxicity and larval development assays. The present study focused on larval development after the exposure of two F. enigmaticus populations (Mediterranean and Atlantic, collected in Italy and Portugal, respectively) to different trace elements (copper, mercury, arsenic, cadmium, and lead) at different concentrations. Results of larval development assays were presented as the percentage of abnormal developed larvae. The effect, measured in terms of EC50 for all toxicants tested, showed that mercury was the most toxic metal for larvae of both populations. Specifically, the tested trace elements may be racked in the following order from the highest to the lowest toxicity: Mediterranean: mercury > copper > lead > arsenic > cadmium; Atlantic: mercury > copper > cadmium > arsenic > lead. Responses of both populations were similar for arsenic. Lead was the least toxic element for the Atlantic population, while cadmium showed the least toxicity for the Mediterranean population. These preliminary results demonstrate the sensitivity and suitability of the organisms to be used in ecotoxicological bioassays and monitoring protocols. Moreover, chemical analyses on soft tissues and calcareous tubes of collected test organisms and their sampling site water were performed, to identify and quantify the concentration of the tested trace elements in these 3 matrices. Populations exhibited less sensitivity to a certain element together with a relevantly higher concentration of the same element in soft tissues. This may indicate a certain resistance to particular contaminant toxic effects by organisms that tend to accumulate the same toxicant. This highlights the potential correlation between wild-caught test organisms' responses and a deep characterization of the sampling site to identify putative abnormalities or differences in model organism response during bioassay execution.

Effect of ammonia on the gene expression levels of the freshwater cyclopoid Eucyclops serrulatus.

Ammonia pollution is a critical issue in Europe, since more than half of the European freshwater bodies actually fail to meet EU quality standards for this chemical. In this study, the response of stress-related genes to a sublethal ammonia concentration has been investigated in the adults of the freshwater cyclopoid Eucyclops serrulatus. Two short-term exposures (12h and 24h) at 12mg/L NH4+ have been tested. Results indicate that 12mg/L NH4+ causes a significant increase in the expression of some proteins, namely CAT, HSP90 and HSP40, suggesting an activation of the protecting antioxidant system after both 12h and 24h.

Genomic resources notes accepted 1 October 2014-30 November 2014.

This article documents the public availability of (i) RAD sequencing data and validated SNPs for the American mink Neovison vison and (ii) Transcriptome resources for two nonmodel freshwater crustacean species, the copepod Eucyclops serrulatus and the amphipod Echinogammarus veneris.

Effects of egg testosterone on female mate choice and male sexual behavior in the pheasant.

Evidence is accumulating that sex steroids in the eggs, besides affecting progeny phenotype and behavior in the short term, also have enduring effects until adulthood, when they may translate into differences in reproductive strategies and success. Maternal steroids transfer may therefore affect both agonistic behavior and mate choice decisions, either through the promotion of body size and condition or through a priming effect on the neuroendocrine system. However, owing to the prevalence of a short-term perspective, relevance of maternal transfer of sex steroids to sexual selection processes has been seldom studied. Here we investigate the effects of an experimental increase in egg testosterone on male dominance and copulation success in the ring-necked pheasant, Phasianus colchicus, a polygynous galliform with multiple male ornamental traits, in captivity. We found that females from testosterone (T) injected eggs copulated less than control females. Males from T-injected eggs obtained more copulations than control males, specifically with control females. The effect of male 'ordinary' and secondary sexual traits on either dominance or copulation frequency did not depend on early exposure to T, nor did T treatment affect male dominance. Present results demonstrate that variation in the early hormonal environment set up by mothers affects sexual behavior of the offspring, which might translate into fitness differences.

Are large wattles related to particular MHC genotypes in the male pheasant?

In sexually dimorphic species, partners can assess heritable mate quality by analyzing costly sexual ornaments in terms of their dimension and possibly of their symmetry. In vertebrates an important aspect of genetic quality is the efficiency of the immune system, and in particular the Major Histocompatibility Complex (MHC). If ornaments are honest advertisements of pathogen resistance (good genes), in line with the Hamilton-Zuk hypothesis, a correlation between ornament expression and MHC profiles should exist. We tested this hypothesis in the common pheasant Phasianus colchicus by comparing male ornament characteristics (wattle and spur size, and wattle fluctuating asymmetry) with a portion of exon 2 of the class IIB MHC genes containing 19 putative antigen recognition sites. A total of 8 new alleles was observed in the MHCPhco exon IIB. We found significant differences in the occurrence of MHC genotypes between males carrying large or small wattles. Homozygous genotypes predicted large wattle males more correctly than small wattle males. The association between the dimension of the spur and the occurrence of MHC genotypes was marginally significant, however, we did not find any significant association between MHC genotypes and asymmetry. Our results suggest that female pheasants may use the ornament size as a cue to evaluate male quality and thus choose males carrying particular MHC profiles.

Characterization of polymorphic microsatellite loci in the marine isopod Sphaeroma terebrans (Crustacea, Isopoda).

Nine polymorphic microsatellites were characterized in the marine isopod Sphaeroma terebrans (Isopoda, Sphaeromatidae) for phylogeographic and parentage analyses. The number of alleles ranged from 3 to 10. Observed heterozygosity ranged from 0.428 to 0.950, while expected heterozygosity ranged from 0.532 to 0.889. Heterozygote deficiency was detected for one locus, possibly the result of null alleles.