Ilaria Guarniero

Nematodes are abundant and ubiquitous animals which are poorly known at intraspecific level. This work represents the first attempt to fill the gap on basic knowledge of genetic variability and differentiation in Protostrongylus oryctolagi, a nematode parasite of lagomorphs. 68 cox1 sequences were obtained from brown hares collected in five locations in Northern and Central Italy, highlighting the presence of a high amount of genetic variation inside this species. The eleven haplotypes identified (Haplotype diversity equal to 0.702) were split into two lineages: lineage A (comprising six different haplotypes, A1-A6) and lineage B (B1-B5). The mean intra-lineage amount of genetic variation was 0.3%, whereas the inter-lineage percentage of variation was ten-fold higher (3%). These two lineages were non-randomly distributed in the investigated areas. Lineage A showed a preference for Central Italy (Tuscany) even if it was sporadically found also in northern territories (Emilia-Romagna), while B-haplotypes were present exclusively in Emilia-Romagna. The analysis of molecular variance identified two main barriers to gene flow: (i) a strong major one which separate samples of Central Italy (PIA and GR7) from the northern ones (RE1, RE3 and MO1; Φ_ST = 0.750, P = 0.00); (ii) a secondary faint barrier which separates Pianosa island from Grosseto (Φ_ST = 0.133, P = 0.00). Any difference was found among northern samples (Φ_ST = 0.009, P = 0.00). The observed data may be explained by several factors ranging from the parasite’s biology (presence of a narrow host spectrum), the final host’s behaviour (small home range), the natural dispersion of the host-parasite dyad occurred in past or the recent passive men-mediated migration. Finally, the presence of unconventional shortened amplicons revealed the presence of NUMTs (nuclear copy of mitochondrial genes) in the P. oryctolagi nuclear genome, suggesting caution when using DNA barcode as unique marker for the identification of species belonging to this genus. “In short, if all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable”. Nathan Augustus Cobb, from "Nematodes and Their Relationships", 1915

Twenty-five zebrafish presenting three different colour variants were collected and analysed by molecular markers in order to define their species and to verify if the unusual colouration presented by two morphotypes was due to genetic engineering or were natural colour variants obtained by selective breeding or spontaneous mutations. The three morphotypes collected were (a) an unusual reddish-pink (suspected red GloFish®), 15 specimens confiscated by the operative ecological core of police officers; (b) the black/blue striped colouration typical of wildtype Danio rerio (five specimens) and (c) an hybrid morphotype with an atypical flashing red pigmentation of skeletal muscle and a blue-black striped pigmentation of skin (five specimens obtained by crossing suspected GM danios with wildtype line) obtained by crossing wild type danios with fish confiscated by police. Despite the three different colourations presented, all analysed samples were unequivocally Danio rerio, excluding the possibility of belonging to different species or natural variants, which should carry different skin colours. Except the five wild-type danios, all other samples analysed should belong to an engineered variant and thus forbidden by EU GMO legislation. Here we designed a new highly reliable and affordable PCR-based assay able to identify the presence of red fluorescent proteins not only in the 15 parental GM samples but also in the five F1 hybrids created ad hoc. The cross-breeding experiment clearly demonstrated that the exogenous construct made by red fluorescent protein under a fast-muscle promoter was inherited by F1. For this reason in our opinion also cross-hybrids obtained by GM danios and wildtype zebrafish should be included in the list of organisms regulated by GMO legislation. Finally, the obtained results suggest that the PCR-based assay here implemented could represent a cost-effective test to rapidly identify suspected GM fish, which carry RFP/dsRED isoforms.

The rayed trough-shellMactra corallinaLinnaeus 1758 is a surf clam that inhabits the Atlantic Ocean, Black Sea and Mediterranean Sea and represents a commercially important bivalve. This species is present with two different and well-defined sympatric morphotypes, which differ mainly for the colour of the shell (white in thecorallinamorph, and brown-banded in thelignariamorph). The aim of this work is to resolve the confused and contradictory systematics of the bivalves belonging toM. corallinaputative species by analysing molecular and morphological features. Fifteen specimens ofM. corallina corallina(white variant) and 19 specimens ofM. corallina lignaria(brown variant) were collected in the North Adriatic Sea and analysed by four molecular markers (12S, 16S, 18S and COI genes, partial sequences). Genetic analyses clearly support the presence of two different species, which were previously ascribed toM. corallina.In addition, 35 specimens identified on a morphological basis asM. c. corallinaand 28 specimens identified asM. c. lignariacollected in the same area were used for a morphometric analysis. A positive correlation was found between the maximum width of shell (W), antero-posterior length and between W and the height of specimens from umbo to ventral margin, thus adding to molecular data.