The complete mitochondrial genome of the zebra seabream Diplodus cervinus (Perciformes, Sparidae) from the Mediterranean Sea.

The complete nucleotide sequence of the mitochondrial (mt) genome of the demersal zebra seabream Diplodus cervinus (Lowe, 1838) was determined for the first time. The double stranded circular molecule is 16,559 base pairs (bp) in length and encodes for the typical 37 metazoan mitochondrial genes, and 2 non-coding regions (D-loop and L-origin). The gene arrangement of the D. cervinus mt genome follows the usual one for fishes. The nucleotide sequences of the mt protein coding and ribosomal genes of D. cervinus mt genome were aligned with orthologous sequences from representatives of the Sparidae family and phylogenetic relationships were inferred. Maximum likelihood analyses placed D. cervinus as a sister species of Diplodus sargus (Linnaeus, 1758).

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.

A Comparative Analysis of Mucus Immunomodulatory Properties from Seven Marine Gastropods from the Mediterranean Sea.

The treatment of inflammatory and immune-related diseases due to dysfunctioning of the immune system necessitates modulation of the immune response through immunomodulatory compounds. Marine environments are considered as a new frontier for health benefit product implementations. Marine biodiversity is still a low explored resource, despite it is expected to represent an important platform for chemical bioactive compounds. Within the phylum Mollusca, gastropods are known to synthetize mucus, the latter presenting relevant bioactive properties, e.g., related to immunomodulant molecules able to activate the innate and acquired immune system. This study proposes a bioprospecting of the immunomodulant activity of mucus isolated from seven common gastropod species from the Gulf of Naples (Mediterranean Sea). Results showed that not all mucus displayed a significant cytotoxic activity on the two human cancer cell lines A549 and A2058. On the other hand, the mucus from Bolinus brandaris was strongly bioactive and was therefore thoroughly investigated at cellular, molecular, and protein levels on the human monocytes U937 line. It can conclusively induce monocyte differentiation in vitro and significantly stimulate natural immunity response.

Benthic litter in the continental slope of the Gulf of Naples (central-western Mediterranean Sea) hosts limited fouling communities but facilitates molluscan spawning.

Seafloor pollution by benthic litter is an emerging phenomenon, although debris colonization by biota remains largely unexplored. We characterized the litter of the continental slope (~400-600 m) of the Gulf of Naples (Mediterranean) and investigated its fouling biota through integrative taxonomic approaches. Plastic pieces (82 %) with land-based origin (96 %) and limited sizes (10-20 cm) were the items most commonly encountered, suggesting a transfer to deep waters through floating and sinking. The majority of the items were not fouled, and the debris hosted an impoverished biota, leading to hypothesize that benthic litter supports wide communities only in shallow waters. Higher colonization rates were observed for gastropod and cephalopod eggs with no preference for materials and sizes, suggesting that even small pieces of soft plastic provide a spawning habitat for molluscs and affect species' connectivity in the deep-sea ecosystem. Holistic approaches are necessary to evaluate interactions between litter and biota.

The complete mitochondrial genome of the white seabream Diplodus sargus (Perciformes: Sparidae) from the Tyrrhenian sea.

The white seabream Diplodus sargus (Linnaeus, 1758) is a species of interest for commercial fisheries throughout its range of distribution and it is also reared using aquaculture techniques. Herein, we present the first complete sequence and annotation of the mitochondrial genome of this species. The D. sargus mitogenome is 16,515 base pairs in length and contains 13 protein-coding genes, 2 rRNA, 22 tRNA, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.3% A, 28.9% C, 26.8% T, and 17.0% G. Maximum likelihood analyses placed D. sargus as a sister species of Diplodus puntazzo. This study provides valuable information for further studying identification methods and evolutionary relationships of Sparidae species.

Aedes albopictus diversity and relationships in south-western Europe and Brazil by rDNA/mtDNA and phenotypic analyses: ITS-2, a useful marker for spread studies.

BACKGROUND: Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread. METHODS: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape. RESULTS: Specimens from seven populations from Spain, France and Brazil provided 12 cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2-7 intra-individual (mean 4.7) and 16-34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil. CONCLUSIONS: ITS-2 proves to be a useful marker to assess Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe.

Molecular taxonomy confirms that the northeastern Atlantic and Mediterranean Sea harbor a single lancelet, Branchiostoma lanceolatum (Pallas, 1774) (Cephalochordata: Leptocardii: Branchiostomatidae).

Branchiostomatidae (lancelets or amphioxus) comprises about 30 species, several of which are well-established models in evolutionary development. Our zoological and ecological knowledge of the family is nonetheless limited. Despite evident differences can be found among known populations, the taxonomy of Branchiostoma lanceolatum (type species of the genus Branchiostoma) has never been investigated with modern methods through its range in the northeastern Atlantic and Mediterranean Sea. We address this via a multilocus molecular approach and comparing specimens collected from different European populations. Results obtained here confirm the presence of a single species inhabiting the range between the topotypical localities of B. lanceolatum (Atlantic Ocean) and of its junior synonym B. lubricum (Mediterranean Sea), without evincing geographical structure between populations. This suggests that environment most likely drives the characteristics observed in different geographic areas. The long larval phase and the slow mutation rate in lancelets may have played a key role in the evolutionary history of this iconic species.

Insights into the role of deep-sea squids of the genus Histioteuthis (Histioteuthidae) in the life cycle of ascaridoid parasites in the Central Mediterranean Sea waters.

Ascaridoid nematodes comprise a wide range of heteroxenous parasites infecting top fish predators and marine mammals as definitive hosts, with crustaceans, squids, and fishes acting as intermediate/paratenic hosts. Limited data exist on the species and role of several intermediate and paratenic hosts in the life cycle of these parasites. In the aim of adding knowledge on the role of squid species in their life cycle, we have here investigated the larval ascaridoid nematodes collected from the deep-sea umbrella squid Histioteuthis bonnelli and the reverse jewel squid Histioteuthis reversa captured in the Central Mediterranean Sea (Tyrrhenian Sea). Morphological study and sequence analysis of the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) and the mitochondrial cytochrome c oxidase subunit 2 (mtDNA cox2) gene locus revealed the occurrence of Anisakis physeteris and of an unidentified species of the genus Lappetascaris. Sequence analysis revealed that specimens of Lappetascaris from both squid species matched at 100% sequences previously deposited in GenBank from larval ascaridoids collected in octopuses of the genus Eledone of the Mediterranean Sea. The Bayesian inference tree topology obtained from the analysis of the fragments amplified showed that Lappetascaris specimens were included in a major clade comprising Hysterothylacium species collected in fishes of the families Xiphiidae and Istiophoridae. As regards the site of infection in the squid host species, A. physeteris larvae predominated (60.7%) in the gonads, while those of Lappetascaris (76.3%) were found infecting the mantle musculature. The overall high values of parasitic load suggest both squid species as transmitting hosts of third stage larvae of Lappetascaris to top predator fishes, as well as the umbrella squid as an intermediate/paratenic host in the life cycle of A. physeteris in the Mediterranean Sea.

New Parasite Records for the Sunfish Mola mola in the Mediterranean Sea and Their Potential Use as Biological Tags for Long-Distance Host Migration.

Studies describing the parasite fauna of sunfish species from the Mediterranean Sea are to date limited, despite information gained through parasitological examination may reveal unknown ecological and biological aspects of both hosts and parasites. Moreover, recent molecular studies on sunfish taxonomy revealed the presence of two species belonging to the genus Mola in the Mediterranean basin, namely M. mola and M. alexandrini. These two fish taxa have long been synonymized or confused among them, which implies that the majority of the studies carried out so far reported the parasites infecting both species under a single host species, generally referred to as M. mola. We hereby investigated the parasite fauna of a 43 cm long M. mola specimen from the Mediterranean Sea, whose identification was confirmed by molecular tool, and provided the first evidence of the occurrence of the nematode Anisakis simplex (s.s.) and of the cestode Gymnorhynchus isuri in Mola species anywhere. The use of helminth species as biological tags for the sunfish is also discussed.

The Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Crustacea: Decapoda: Varunidae) reappears in the northern Adriatic Sea: Another intrusion attempt or the trace of an overlooked population?

Eriocheir sinensis is included among the 100 most invasive aquatic invertebrates due to its colonization history and the impacts to biodiversity and economy in the newly invaded environments. Despite of that, its attempts of colonization of the Mediterranean basin seem to have failed so far. In this paper, we summarize the status and the distribution of the species in the Mediterranean and report a further finding from the northern Adriatic Sea, confirmed through an integrative taxonomy approach. We finally discuss the vectors of introduction that may be at the basis of the present record, highlighting the unexplored possibility that an undetected population is already thriving in the area. Whatever of the hypotheses done here is true, the Adriatic Sea could be a new perfect house for this invader, suggesting that more attention should be paid by stakeholders in preventing rather than in remediating possible impacts.

Genetic uniformity, geographical spread and anthropogenic habitat modifications of lymnaeid vectors found in a One Health initiative in the highest human fascioliasis hyperendemic of the Bolivian Altiplano.

BACKGROUND: Fascioliasis is a snail-borne zoonotic trematodiasis emerging due to climate changes, anthropogenic environment modifications, and livestock movements. Many areas where Fasciola hepatica is endemic in humans have been described in Latin America altitude areas. Highest prevalences and intensities were reported from four provinces of the northern Bolivian Altiplano, where preventive chemotherapy is ongoing. New strategies are now incorporated to decrease infection/re-infection risk, assessment of human infection sources to enable efficient prevention measures, and additionally a One Health initiative in a selected zone. Subsequent extension of these pilot interventions to the remaining Altiplano is key. METHODS: To verify reproducibility throughout, 133 specimens from 25 lymnaeid populations representative of the whole Altiplano, and 11 used for population dynamics studies, were analyzed by rDNA ITS2 and ITS1 and mtDNA cox1 and 16S sequencing to assess their classification, variability and geographical spread. RESULTS: Lymnaeid populations proved to belong to a monomorphic group, Galba truncatula. Only a single cox1 mutation was found in a local population. Two cox1 haplotypes were new. Comparisons of transmission foci data from the 1990's with those of 2018 demonstrated an endemic area expansion. Altitudinal, northward and southward expansions suggest movements of livestock transporting G. truncatula snails, with increasing temperatures transforming previously unsuitable habitats into suitable transmission areas. Transmission foci appear to be stable when compared to past field observations, except for those modified by human activities, including construction of new roads or control measures undertaken in relation to fascioliasis. CONCLUSIONS: For a One Health initiative, the control of only one Fasciola species and snail vector species simplifies efforts because of the lower transmission complexity. Vector monomorphism suggests uniformity of vector population responses after control measure implementation. Hyperendemic area outer boundary instability suggests a climate change impact. All populations outside previously known boundaries were close to villages, human dwellings and/or schools, and should therefore be considered during disease control planning. The remarkable southward expansion implies that a fifth province, Aroma, should now be included within preventive chemotherapy programmes. This study highlights the need for lymnaeid molecular identification, transmission foci stability monitoring, and potential vector spread assessment.

The complete mitochondrial genome of the sharpsnout seabream Diplodus puntazzo (Perciformes: Sparidae).

The sharpsnout seabream Diplodus puntazzo Walbaum, 1792 is a target species of small-scale fishery activities and is cage-cultured for human consumption. Nonetheless, genetic information on this species is limited. We here first sequence its complete mitochondrial genome. The sequence is composed of 16,638 base pairs, accounting for 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.4% A, 28.9% C, 26.9% T, and 16.8% G. Maximum likelihood analyses placed D. puntazzo close to Acanthopagrus and some Pagellus species.

Lymnaeid Snail Vectors of Fascioliasis, Including the First Finding of Lymnaea neotropica in Ecuador, Assessed by Ribosomal DNA Sequencing in the Southern Zone Close to the Peru Border.

PURPOSE: Fascioliasis is a freshwater snail-borne zoonotic trematodiasis of high pathogenicity and wide veterinary repercussions. In South America, moreover, it causes serious public health problems, with high human infection rates in Andean countries. Ecuador offers a worrying risky scenario due to its physiography, including many human infection reports and animal endemicity throughout its Andean highlands. METHODS: Endemic areas with increasing animal fascioliasis were surveyed for lymnaeid snails in the province of Loja, southern Ecuador, close to the border of Peru, the country known to present the widest human fascioliasis endemic zone. The altitude of the sampling sites ranged between 150 and 1770 m a.s.l., and their location was close to human villages. Biotopes surveyed were characterized according to fascioliasis transmission needs. RESULTS: The species Lymnaea schirazensis and L. neotropica were identified by rDNA ITS-2 and ITS-1 sequencing. The non-transmitting L. schirazensis combined haplotype agreed with populations of this species previously reported from northern Ecuador. The finding of the efficient vector L. neotropica is reported for the first time in Ecuador and suggests a passive introduction from neighbouring Peru by uncontrolled livestock transport. CONCLUSIONS: Rice irrigation system implementation, lymnaeid finding on Taraxacum (dandelion) plants which are consumed fresh in salads by people, and Saccharum (sugarcane), whose bark is peeled off with the teeth, represent potential infection sources for humans. The closeness to the Cajamarca human hyperendemic area in northern Peru, where the same two lymnaeids have been also found and triclabendazole resistance reported, is an additional risk to be considered regarding the livestock transborder exchange.

Caenogastropod mitogenomics.

In order to further resolve the phylogenetic relationships within Caenogastropoda, the complete mitochondrial (mt) genomes of Cochlostoma hidalgoi (Cyclophoroidea), Naticarius hebraeus (Naticoidea), Galeodea echinophora (Tonnoidea), and Columbella adansoni (Buccinoidea), and the partial mt genome of Erosaria spurca (Cypraeoidea) were sequenced. All newly determined mt genomes conformed to the consensus gene order of caenogastropods, except that of C. hidalgoi, which differed in the relative positions of the trnD, trnQ, trnG, trnY, and trnT genes. Phylogenetic reconstruction of the caenogastropod tree was performed using probabilistic methods and based on the deduced amino acid sequences of concatenated mt protein coding genes. The reconstructed phylogeny recovered Architaenioglossa (superfamilies Cyclophoroidea, Ampullarioidea, and Viviparoidea) as a grade. The monophyly of Sorbeoconcha (all caenogastropods but Architaenioglossa) was supported by most but not all phylogenetic analyses (excluding Vermetoidea, which has a long branch). The relative phylogenetic position of Cerithioidea with respect to Hypsogastropoda remains unresolved. The monophyly of Hypsogastropoda (without Vermetoidea) is strongly supported. Within this clade, Littorinimorpha should be considered a grade. Several superfamilies (Abyssochrysoidea, Rissooidea, Truncatelloidea, and Naticoidea) branched off successively before a siphonate clade (Stromboidea, Cypraeoidea, Tonnoidea, Neogastropoda), which is strongly supported. The relative phylogenetic position of Vermetoidea could not be determined due to long-branch attraction artifacts. The superfamily Tonnoidea was recovered within Neogastropoda, which questions the monophyly of the latter as traditionally defined. The polyphyly of Muricoidea could be tentatively resolved excluding the families Volutidae and Babyloniidae, which would imply raising them to the rank of superfamilies.

The complete mitochondrial genome of Scutopus ventrolineatus (Mollusca: Chaetodermomorpha) supports the Aculifera hypothesis.

BACKGROUND: With more than 100000 living species, mollusks are the second most diverse metazoan phylum. The current taxonomic classification of mollusks recognizes eight classes (Neomeniomorpha, Chaetodermomorpha, Polyplacophora, Monoplacophora, Cephalopoda, Gastropoda, Bivalvia, and Scaphopoda) that exhibit very distinct body plans. In the past, phylogenetic relationships among mollusk classes have been contentious due to the lack of indisputable morphological synapomorphies. Fortunately, recent phylogenetic analyses based on multi-gene data sets are rendering promising results. In this regard, mitochondrial genomes have been widely used to reconstruct deep phylogenies. For mollusks, complete mitochondrial genomes are mostly available for gastropods, bivalves, and cephalopods, whereas other less-diverse lineages have few or none reported. RESULTS: The complete DNA sequence (14662 bp) of the mitochondrial genome of the chaetodermomorph Scutopus ventrolineatus Salvini-Plawen, 1968 was determined. Compared with other mollusks, the relative position of protein-coding genes in the mitochondrial genome of S. ventrolineatus is very similar to those reported for Polyplacophora, Cephalopoda and early-diverging lineages of Bivalvia and Gastropoda (Vetigastropoda and Neritimorpha; but not Patellogastropoda). The reconstructed phylogenetic tree based on combined mitochondrial and nuclear sequence data recovered monophyletic Aplacophora, Aculifera, and Conchifera. Within the latter, Cephalopoda was the sister group of Gastropoda and Bivalvia + Scaphopoda. CONCLUSIONS: Phylogenetic analyses of mitochondrial sequences showed strong among-lineage rate heterogeneity that produced long-branch attraction biases. Removal of long branches (namely those of bivalves and patellogastropods) ameliorated but not fully resolved the problem. Best results in terms of statistical support were achieved when mitochondrial and nuclear sequence data were concatenated.

The mitochondrial genome of Ifremeria nautilei and the phylogenetic position of the enigmatic deep-sea Abyssochrysoidea (Mollusca: Gastropoda).

The complete nucleotide sequence of the mitochondrial (mt) genome of the deep-sea vent snail Ifremeria nautilei (Gastropoda: Abyssochrysoidea) was determined. The double stranded circular molecule is 15,664 pb in length and encodes for the typical 37 metazoan mitochondrial genes. The gene arrangement of the Ifremeria mt genome is most similar to genome organization of caenogastropods and differs only on the relative position of the trnW gene. The deduced amino acid sequences of the mt protein coding genes of Ifremeria mt genome were aligned with orthologous sequences from representatives of the main lineages of gastropods and phylogenetic relationships were inferred. The reconstructed phylogeny supports that Ifremeria belongs to Caenogastropoda and that it is closely related to hypsogastropod superfamilies. Results were compared with a reconstructed nuclear-based phylogeny. Moreover, a relaxed molecular-clock timetree calibrated with fossils dated the divergence of Abyssochrysoidea in the Late Jurassic-Early Cretaceous indicating a relatively modern colonization of deep-sea environments by these snails.

State of knowledge of the Acotylea (Polycladida, Platyhelminthes) from the Mediterranean coasts of Spain: new records and new species.

Along the Mediterranean coast of the Iberian Peninsula, great species diversity is thought to exist, but our knowledge of Iberian polyclads is, in fact, very limited. This study contributes to the Polycladida (Platyhelminthes) of the Iberian Peninsula, in particular those of the Mediterranean coast. Nine species, mainly new species or new records, are described. Imogine stellae sp. nov. from Mar Menor (Murcia, Spain) is described, while I. mediterranea Galleni, 1976 is recorded for the first time in Spain. The genus Planocera Blainville, 1828 within the Mediterranean basin is reviewed: Planocera graffi Lang, 1879 is redescribed, and its synonymisation with Planocera pellucida (Mertens, 1833) considered. Also, the genus Notoplanella Bock, 1931 is represented by two species in Spain, N. inarmata Bock, 1931 type species, from Formentera Island and N. estelae sp. nov., from Mar Menor. Trigonoporus cephalophtalmus Lang, 1884 is rediscovered after the description of Lang (1884). Stylochus neapolitanus (Delle Chiaje, 1841-1844) Lang, 1884 is recorded and S. pilidium (Goette, 1881) is also redescribed, and Leptoplana mediterranea (Bock, 1913) is newly recorded for the Iberian Peninsula.