Trace element levels in the muscles of three tern species (Aves: Laridae) from the western Arabian Gulf: environmental assessment and implications for conservation.

In the Arabian Gulf (called also Persian Gulf; hereafter 'the Gulf'), Jana and Karan Islands are recognized as one of the most Important Bird Areas in the region. Many migratory breeding seabirds, like the Greater Crested Tern Thalasseus bergii, White-cheeked Tern Sterna repressa and Bridled Tern Onychoprion anaethetus, depend on these islands during the breeding season. However, these aquatic wildlife species are suffering from intensified urban and industrial coastal development and various contamination events including wars and related oil spills. In this study, we used these three piscivorous top predator birds to analyse the levels of 19 trace elements (TEs; i.e. Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Pb, Sr, V and Zn) in 15 muscular tissue samples from Jana and Karan Islands. PERMANOVA analysis showed no difference in contamination profile between sites nor between species probably due to their spatial and ecological proximity and therefore similar levels of exposure to TEs. Comparing these levels with existing literature, our results showed no particular concern for all elements, except for Al (maximum values recorded = 116.5 µg g-1 d.w.) and, in two samples, Ba (33.67 µg g-1 d.w.) and Pb (5.6 µg g-1 d.w.). The results can be considered as an initial step for supplementary evaluations with a larger number of samples and specified time intervals for the collection of specimens. This study provided baseline information on the pollution status of these two ecologically important sites which require a continuous biomonitoring programme.

Systematics of European coastal anchovies (genus Engraulis Cuvier).

Reports of morphological differences between European anchovy (Engraulis cf. encrasicolus) from coastal and marine habitats have long existed in the ichthyologic literature and have given rise to a long-standing debate on their taxonomic status. More recently, molecular studies have confirmed the existence of genetic differentiation between the two anchovy ecotypes. Using ancestry-informative markers, we show that coastal anchovies throughout the Mediterranean share a common ancestry and that substantial genetic differentiation persists in different pairs of coastal/marine populations despite the presence of limited gene flow. On the basis of genetic and ecological arguments, we propose that coastal anchovies deserve a species status of their own (E. maeoticus) and argue that a unified taxonomical framework is critical for future research and management.

DNA Taxonomy Confirms the Identity of the Widely-Disjunct Mediterranean and Atlantic Populations of the Tufted Ghost Crab Ocypode cursor (Crustacea: Decapoda: Ocypodidae).

The distribution area of the tufted ghost crab Ocypode cursor includes two widely separate sub-areas, i.e. the tropical and subtropical Atlantic coasts of Africa and Macaronesia, and the central-eastern Mediterranean basin. The current disjunct distribution of the species is possibly the remnant of a previous wider and continuous distribution area that was fragmented during the Pleistocene, with the disappearance of the species from the temperate Atlantic Ocean and the western Mediterranean basin, and its survival in the warmer areas of the eastern Atlantic and the Mediterranean Sea. Such disjunction is thus compatible with an ancient isolation between the Mediterranean and Atlantic populations of the species, which could in fact constitute two well-characterized independent evolutionary lineages, or even two cryptic species. Unexpectedly, the sequencing of a fragment of the mtDNA COI gene from Mediterranean and Atlantic Ocypode cursor allopatric populations showed the cohesion of the species throughout its distribution range, and the nesting of Mediterranean populations within the single Atlantic population studied. This pattern is hereby tentatively ascribed to an incomplete lineage sorting due to the large population sizes of both the Atlantic and Mediterranean subpopulations of the species. The current westward expansion of the species in the Mediterranean Sea originating from the Levantine basin, due to ongoing regional sea warming, follows a typical phalanx dispersal mode.

The DNA barcoding of Mediterranean combtooth blennies suggests the paraphyly of some taxa (Perciformes, Blenniidae).

A dataset including novel and publicly available mtDNA COI sequences of 14 Mediterranean combtooth blenny species belonging to nine genera was assembled in order to provide a reference dataset for DNA barcoding studies. Some inconsistencies in the current taxonomy of some genera were observed. In particular, the monophyly of the genera Parablennius and Salaria is not supported by the present dataset and the absence of reciprocal monophyly between the morphospecies Salaria basilisca and S. pavo questions their status and stresses the need of a revision of the genus Salaria.

DNA barcoding of marine fishes from Saudi Arabian waters of the Gulf.

We used the cytochrome oxidase subunit I (coI) gene DNA to barcode 117 endemic Gulf and cosmopolitan Indo-West Pacific fish species belonging to 54 families and 13 orders. Novel DNA barcodes were provided for 18 fish species (Trachinocephalus sp., Nematalosa sp., Herklotsichthys lossei, Upeneus doriae, Trachurus indicus, Apogonichthyoides taeniatus, Verulux cypselurus, Favonigobius sp., Suezichthus gracilis, Sillago sp., Brachirus orientalis, Pegusa sp., Lepidotrigla bispinosa, Lepidotrigla sp., Grammoplites suppositus, Hippichthys sp., Paramonacanthus sp. and Triacanthus sp.). The species delimitation analysis, conducted with Poisson tree processes- Bayesian PTP (PTP-bPTP) and nucleotide-divergence-threshold (NDT) models), found 137 and 119 entities respectively. Overall, NDT method, neighbour-joining species tree and the prior taxonomic assessment provided similar results. Among the 54 families considered, only 10 (Ariommatidae, Ephippidae, Leiognathidae, Nemipteridae, Plotosidae, Pomacanthidae, Pomacentridae, Priacanthidae and Rachycentridae) showed the occurrence of molecular diagnostic pure characters. The DNA barcoding database developed during this study will help ichthyologists to identify and resolve the taxonomic ambiguities they may encounter with the fishes occurring in The Gulf and throughout the region.

On the Occurrence of the Invasive Freshwater Limpet Ferrissia californica (Mollusca: Gastropoda: Planorbidae) in Japan.

Two members of the genus Ferrissia Walker, 1903, namely Ferrissia nipponica (Kuroda, 1949) and F. japonica Habe and Burch, 1965, have been reported to occur in Japan, but due to a lack of molecular data, doubts have been expressed as to their validity. Furthermore, the possible presence of allochthonous Ferrissia taxa has been stated under a variety of names, so that even now there is no consensus on their presence and identity. Recently, freshwater limpets belonging to the planorbid genus Ferrissia were collected in an irrigation trough on the Izu island of Hachijojima, Tokyo, Japan. Molecular identification of the collected specimens, based on a fragment of the large ribosomal subunit 16S (mtDNA), unequivocally proved that they belong to the allochthonous, invasive species Ferrissia californica (Rowell, 1863), the protagonist of a relentless cryptic invasion worldwide to which can Japan can now be added. The shells of the collected specimens show some scars, which bear testimony to the fragile nature of the shell and, possibly, to the paucity of calcium in the inland waters of Hachijojima.

The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa).

Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 µm in length and 6.4 ± 0.52 µm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.

Overlooked cryptic endemism in copepods: systematics and natural history of the calanoid subgenus Occidodiaptomus Borutzky 1991 (Copepoda, Calanoida, Diaptomidae).

Our comprehension of the phylogeny and diversity of most inland-water crustaceans is currently hampered by their pronounced morphological bradytely, which contributed to the affirmation of the "Cosmopolitanism Paradigm" of freshwater taxa. However, growing evidence of the existence of cryptic diversity and molecular regionalism is available for calanoid copepods, thus stressing the need for careful morphological and molecular studies in order to soundly investigate the systematics, diversity and distribution patterns of the group. Diaptomid copepods were here chosen as model taxa, and the morphological and molecular diversity of the species belonging to the west-Mediterranean diaptomid subgenus Occidodiaptomus were investigated with the aim of comparing the patterns of morphological and molecular evolution in freshwater copepods. Three species currently lumped under the binomen Hemidiaptomus (Occidodiaptomus) ingens and two highly divergent clades within H. (O.) roubaui were distinguished, thus showing an apparent discordance between the molecular distances recorded and Occidodiaptomus morphological homogeneity, and highlighting a noteworthy decoupling between the morphological and molecular diversity in the subgenus. Current Occidodiaptomus diversity pattern is ascribed to a combined effect of ancient vicariance and recent dispersal events. It is stressed that the lack of sound calibration points for the molecular clock makes it difficult to soundly temporally frame the diversification events of interest in the taxon studied, and thus to asses the role of morphological bradytely and of accelerated molecular evolutionary rates in shaping the current diversity of the group.

Controlled uptake of PFOA in adult specimens of Paracentrotus lividus and evaluation of gene expression in their gonads and embryos.

Perfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years ago it turned out to be a dangerous pollutant which is now of concern for terrestrial and aquatic environments. Here, we investigated the bioaccumulation of PFOA in the sea urchin Paracentrotus lividus after exposure to different concentrations of the pollutant for 28 days. We observed rapid uptake of PFOA in the coelomic fluid collected weekly during the exposure period and high bioaccumulation in gonads at the end of the experiment. Interestingly, animals were also able to fast depurate when relocated to a clean environment. In addition, to assess the effect of PFOA on sea urchins' physiological pathways, we analysed the expression profile of some marker genes both in the gonads and in the embryos obtained from parents exposed to PFOA. Our results suggest that PFOA is a persistent, bioaccumulative compound that adversely affects the health of the exposed organisms and their offspring by causing significant changes in the expression of some key target genes and the occurrence of developmental anomalies in the embryos.

Bioaccumulation of trace elements and hydrocarbons in chondrichthyans of the western Arabian Gulf: Environmental and human health risk assessment and implications for conservation.

This is a first attempt to assess the levels of trace elements, PAHs, and TPHs in six elasmobranch species (Carcharhinus dussumieri, C. sorrah, Chiloscyllium arabicum, Gymnura poecilura, Sphyrna lewini, S. mokarran) from the Arabian Gulf. The chemical analysis showed that the concentrations of contaminants differed significantly between liver and muscle samples with both pelagic and benthic species and all families. For all species, contaminant concentrations were significantly higher in the liver than in the muscle. While muscle contaminant concentrations differed significantly with respect to species' lifestyles and families, those of liver showed no significant differences between pelagic and benthic species, neither between the four families nor between the six species. None of the analysed contaminants exceeded the internationally recognized standards in all studied species. These results enrich the knowledge on the bioaccumulation of contaminants in elasmobranchs and allow to assess the environmental status of the Arabian Gulf.

No evidence of DUI in the Mediterranean alien species Brachidontes pharaonis (P. Fisher, 1870) despite mitochondrial heteroplasmy.

Two genetically different mitochondrial haplogroups of Brachidontes pharaonis (p-distance 6.8%) have been identified in the Mediterranean Sea. This hinted at a possible presence of doubly uniparental inheritance in this species. To ascertain this possibility, we sequenced two complete mitogenomes of Brachidontes pharaonis mussels and performed a qPCR analysis to measure the relative mitogenome copy numbers of both mtDNAs. Despite the presence of two very similar regions composed entirely of repetitive sequences in the two haplogroups, no recombination between mitogenomes was detected. In heteroplasmic individuals, both mitogenomes were present in the generative tissues of both sexes, which argues against the presence of doubly uniparental inheritance in this species.

Unveiling the egg microbiota of the loggerhead sea turtle Caretta caretta in nesting beaches of the Mediterranean Sea.

Microbes have central roles in the development and health of animals, being the introduction of specific microbial species a potential conservation strategy to protect animals from emerging diseases. Thus, insight into the microbiota of the species and their habitats is essential. In this manuscript, we report for the first time the bacterial composition of all the components (eggshells of hatched and unhatched eggs, internal content of unhatched eggs, intestinal content of hatchling and pipping sea turtles, and sand) of three nesting beaches of Caretta caretta along the Italian coasts of the Mediterranean Sea. The analysis of 26 amplicon samples was carried out using next-generation sequencing analysis, targeting V3-V4 regions of the bacterial 16S rRNA gene. Samples featured mainly Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, whose percentages depended on the sample type. Our results showed that, although from different sampling sites, the internal content of the unhatched eggs, intestinal content of hatchling and pipping sea turtles share the microbiota, which was yet different from that of eggshells and sand of the same nesting beach. This study suggests the maternal and environmental influence alongside a protective role of eggshells in shaping the egg microbiota of Caretta caretta sea turtles.

Haemolymphatic Parameters in Two Aquaculture Crustacean Species Cherax destructor (Clark, 1836) and Cherax quadricarinatus (Von Martens, 1868).

In the last few years, there has been a notable development in the breeding of freshwater shrimp (astaciculture), which involved various species and in particular, the two Australian Parastacidae species, Cherax destructor and Cherax quadricarinatus. Information about the haemolymphatic parameters of these two species is fragmentary, and filling these gaps becomes important given their importance in aquaculture. Cellular and biochemical parameters were analyzed in both species to create a reference baseline for these parameters to identify the state of welfare or suffering of these animals. The results showed that the total haemocyte count, haemocyte subpopulations, enzymatic activities and pH are similar between the two species, while total protein and osmolality are higher in C. destructor than C. quadricarinatus. Knowledge of these parameters could assist in evaluating the good health status of these species kept in aquaculture facilities.

The European Pine Marten Martes martes (Linnaeus, 1758) Is Autochthonous in Sicily and Constitutes a Well-Characterised Major Phylogroup within the Species (Carnivora, Mustelidae).

No molecular data are currently available for the Sicilian populations of the European pine marten Martes martes, thus preventing any sound inference about its native or non-native status on the island, as well as the local phylogeography of the species. In order to investigate these issues, we sequenced two mtDNA markers in road-killed specimens collected in Sicily. Both markers consistently demonstrated the existence of a well-characterised Sicilian clade of the species, which is endemic to the island and constitutes the sister group of a clade including the Mediterranean and Central-North European major phylogroups of the European pine marten. Such evidence supports the autochthony of Martes martes in Sicily and points to a natural Pleistocene colonisation of the island followed by isolation. The occurrence of a, to date undetected, major phylogroup of the species in Sicily calls for the dedicated monitoring of the Sicilian populations of the species in order to preserve this evolutionarily significant unit.

Genetic architecture of the marbled goby Pomatoschistus marmoratus (Perciformes, Gobiidae) in the Mediterranean Sea.

The marbled goby Pomatoschistus marmoratus, a species inhabiting coastal Mediterranean lagoons, has been studied by measuring its mitochondrial DNA variation. This analysis revealed a Mediterranean west vs east split and, subsequently, an eastern differentiation among the Libyan-Tunisian Gulf, the Adriatic Sea and the Aegean Sea. The high cohesion between the samples collected in the vast area of western Mediterranean contrasts with the genetic mosaic of the more sub-structured eastern Mediterranean. This western homogeneity can not yet be fully explained even if a human-mediated migratory flow, due to a maritime traffic, has been posited. The pattern in the eastern basin revealed a genetic architecture possibly due to the non-migratory habit of the gobid. Within this perspective, the role of the Mediterranean lagoon habitat should be related to how much it amplifies the effects of historical (e.g. past sea-level changes) and environmental (e.g. present-day hydrographic regime) processes as regards the genetic structure of the inhabiting species.

A Comparative Analysis of Aquatic and Polyethylene-Associated Antibiotic-Resistant Microbiota in the Mediterranean Sea.

In this study, we evaluated the microbiome and the resistome profile of water and fragments of polyethylene (PE) waste collected at the same time from a stream and the seawater in a coastal area of Northwestern Sicily. Although a core microbiome was determined by sequencing of the V3-V4 region of the bacterial 16S rDNA gene, quantitative differences were found among the microbial communities on PE waste and the corresponding water samples. Our findings indicated that PE waste contains a more abundant and increased core microbiome diversity than the corresponding water samples. Moreover, PCR analysis of specific antibiotic resistance genes (ARGs) showed that PE waste harbors more ARGs than the water samples. Thus, PE waste could act as a carrier of antibiotic-resistant microbiota, representing an increased danger for the marine environment and living organisms, as well.

Occurrence and Antimicrobial Resistance of Arcobacter spp. Recovered from Aquatic Environments.

Arcobacter spp. are emerging waterborne and foodborne zoonotic pathogens responsible for gastroenteritis in humans. In this work, we evaluated the occurrence and the antimicrobial resistance profile of Arcobacter isolates recovered from different aquatic sources. Besides, we searched for Arcobacter spp. in seaweeds and the corresponding seawater samples. Bacteriological and molecular methods applied to 100 samples led to the isolation of 28 Arcobacter isolates from 27 samples. The highest prevalence was detected in rivers followed by artificial ponds, streams, well waters, and spring waters. Seaweeds contained a higher percentage of Arcobacter than the corresponding seawater samples. The isolates were identified as Arcobacter butzleri (96.4%) and Arcobacter cryaerophilus (3.6%). All the isolates showed a multi-drug resistance profile, being resistant to at least three different classes of antibiotics. Molecular analysis of genetic determinants responsible for tetracycline resistance in nine randomly chosen isolates revealed the presence of tetO and/or tetW. This work confirms the occurrence and the continuous emergence of antibiotic-resistant Arcobacter strains in environmental samples; also, the presence of quinolone-resistant Arcobacter spp. in aquatic sources used for water supply and irrigation represents a potential risk for human health.

Helminth Infection of the Loggerhead Sea Turtle Caretta caretta along the Coasts of Sicily and the North West Adriatic Sea.

We provide new data on the presence of helminth parasites in 64 individual loggerhead sea turtles Caretta caretta stranded along the coasts of Sicily and the northwest Adriatic Sea between June 2014 and August 2016. The necropsy examination revealed 31 individuals (48.4%) positive for endoparasites, showing a greater prevalence of trematodes than nematodes. In particular, seven species and a single genus of Trematoda (Hapalotrema) and a single species and genus of Nematoda (Kathlania) were identified. Among the Digenea flukes the species with the highest prevalence of infection were Rhytidodes gelatinosus (34.6%) and Hapalotrema sp. (33.3%), while among the Nematoda they were Kathlania sp. (33.3%) and Sulcascaris sulcata (33.3%). Analysis of variance (ANOVA) was applied among the recovery sites of the stranded loggerhead sea turtles and prevalence of endoparasites was used to highlight any relationship between the parasites and the origin of the hosts. ANOVA showed significant differences (p < 0.001) among the data used.

Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring.

The first determination of presence and biodistribution of PFOA in ninety specimens of sea urchin Paracentrotus lividus from two differently contaminated sites along Palermo's coastline (Sicily) is reported. Analyses were performed on the sea urchins' coelomic fluids, coelomocytes, gonads or mixed organs, as well as on seawater and Posidonia oceanica leaves samples from the collection sites. PFOA concentration ranged between 1 and 13 ng/L in seawater and between 0 and 794 ng/g in P. oceanica. The analyses carried out on individuals of P. lividus from the least polluted site (A) showed PFOA median values equal to 0 in all the matrices (coelomic fluid, coelomocytes and gonads). Conversely, individuals collected from the most polluted site (B) showed median PFOA concentrations of 21 ng/g in coelomic fluid, 153 ng/g in coelomocytes, and 195 ng/g in gonads. Calculated bioconcentration factors of log10BCF > 3.7 confirmed the very bioaccumulative nature of PFOA. Significant correlations were found between the PFOA concentration of the coelomic fluid versus the total PFOA concentration of the entire sea urchin. PERMANOVA (p = 0.001) end Welch's t-test (p < 0.001) analyses showed a difference between specimens collected from the two sites highlighting the potential application of P. lividus as sentinel species for PFOA biomonitoring.

Can phthalates move into the eggs of the loggerhead sea turtle Caretta caretta? The case of the nests on the Linosa Island in the Mediterranean Sea.

During the monitoring of Caretta caretta nests on the island of Linosa, 30 unhatched eggs from four nests were collected to study the presence of phthalates in their three components (shell, yolk, and albumen). Four phthalates, namely diethyl (DEP), dibutyl (DBP), di-(2-ethylhexyl) (DEHP), and dioctyl (DOTP) phthalic acid esters (PAE), which are widely used as additives in plastics, were detected in all egg components. The most frequently found phthalate was DBP, followed by DEHP in eggshell and yolk. Dimethyl- (DMP) and butylbenzyl-phthalate (BBP) were below the limits of detection for all samples. The high total phthalate recorded in the yolk suggests that contamination could arise by vitellogenesis. PERMANOVA analysis (p = 0.01) confirmed significant differences in the PAEs contamination profiles in the eggs from the four nests. This study confirms the negative impact of plastic related compounds posing questions about the potential adverse effects on organisms and their conservation status.