Persistence of SARS-CoV-2 infection and viral intra- and inter-host evolution in COVID-19 hospitalized patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion.

Phenotype transition from wild mouflon to domestic sheep.

The domestication of animals started around 12,000 years ago in the Near East region. This "endless process" is characterized by the gradual accumulation of changes that progressively marked the genetic, phenotypic and physiological differences between wild and domesticated species. The main distinctive phenotypic characteristics are not all directly attributable to the human-mediated selection of more productive traits. In the last decades, two main hypotheses have been proposed to clarify the emergence of such a set of phenotypic traits across a variety of domestic species. The first hypothesis relates the phenotype of the domesticated species to an altered thyroid hormone-based signaling, whereas the second one relates it to changes in the neural crest cells induced by selection of animals for tameness. These two hypotheses are not necessarily mutually exclusive since they may have contributed differently to the process over time and space. The adaptation model induced by domestication can be adopted to clarify some aspects (that are still controversial and debated) of the long-term evolutionary process leading from the wild Neolithic mouflon to the current domestic sheep. Indeed, sheep are among the earliest animals to have been domesticated by humans, around 12,000 years ago, and since then, they have represented a crucial resource in human history. The aim of this review is to shed light on the molecular mechanisms and the specific genomic variants that underlie the phenotypic variability between sheep and mouflon. In this regard, we carried out a critical review of the most recent studies on the molecular mechanisms that are most accredited to be responsible for coat color and phenotype, tail size and presence of horns. We also highlight that, in such a complicate context, sheep/mouflon hybrids represent a powerful and innovative model for studying the mechanism by which the phenotypic traits related to the phenotypic responses to domestication are inherited. Knowledge of these mechanisms could have a significant impact on the selection of more productive breeds. In fact, as in a journey back in time of animal domestication, the genetic traits of today's domestic species are being progressively and deliberately shaped according to human needs, in a direction opposite to that followed during domestication.

Changing and Evolution of Influenza Virus: Is It a Trivial Flu?

BACKGROUND: Influenza viruses are etiological agents which cause contagious respiratory, seasonal epidemics and, for influenza A subtypes, pandemics. The clinical picture of influenza has undergone continuous change over the years, due to intrinsic viral evolution as well as "reassortment" of its genomic segments. The history of influenza highlights its ability to adapt and to rapidly evolve, without specific circumstances. This reflects the complexity of this pathology and poses the fundamental question about its assumption as a "common illness" and its impact on public health. SUMMARY: The global influenza epidemics and pandemics claimed millions of deaths, leaving an indelible mark on public health and showing the need for a better comprehension of the influenza virus. The clear understanding of genetic variations during the influenza seasonal epidemics is a crucial point for developing effective strategies for prevention, treatment, and vaccine design. The recent advance in next-generation sequencing approaches, model systems to virus culture, and bioinformatics pipeline played a key role in the rapid characterization of circulating influenza strains. In particular, the increase in computational power allowed the performance of complex tasks in healthcare settings through machine learning algorithms, which analyze different variables, such as medical and laboratory outputs, to optimize medical research and improve public health systems. The early detection of emerging and reemerging pathogens is a matter of importance to prevent future pandemics. KEY MESSAGES: The perception of influenza as a "trivial flu" or a more serious public health concern is a subject of ongoing debate, reflecting the multifaceted nature of this infectious disease. The variability in the severity of influenza sheds light on the unpredictability of the viral characteristics, coupled with the challenges in accurately predicting circulating strains. This adds complexity to the public health burden of influenza and highlights the need for targeted interventions.

Genome-based survey of the SARS-CoV-2 BF.7 variant from Asia.

The SARS-CoV-2 BF.7 variant represents one of the most recent subvariant under monitoring. At the beginning of the 2023 it caused several concerns especially in Asia because of a resurge in COVID-19 cases. Here we perform a genome-based integrative approach on SARS-CoV-2 BF.7 to shed light on this emerging lineage and produce some consideration on its real dangerousness. Both genetic and structural data suggest that this new variant currently does not show evidence of an high expansion capability. It is very common in Asia, but it appears less virulent than other Omicron variants as proved by its relatively low evolutionary rate (5.62 × 10-4 subs/sites/years). The last plateau has been reached around December 14, 2022 and then the genetic variability, and thus the viral population size, no longer increased. As already seen for several previous variants, the features that may be theoretically related to advantages are due to genetic drift that allows to the virus a constant adaptability to the host, but is not strictly connected to a fitness advantage. These results have further pointed that the genome-based monitoring must continue uninterruptedly to be prepared and well documented on the real situation.

Genome-based comparison between the recombinant SARS-CoV-2 XBB and its parental lineages.

Recombination is the main contributor to RNA virus evolution, and SARS-CoV-2 during the pandemic produced several recombinants. The most recent SARS-CoV-2 recombinant is the lineage labeled XBB, also known as Gryphon, which arose from BJ.1 and BM.1.1.1. Here we performed a genome-based survey aimed to compare the new recombinant with its parental lineages that never became dominant. Genetic analyses indicated that the recombinant XBB and its first descendant XBB.1 show an evolutionary condition typical of an evolutionary blind background with no further epidemiologically relevant descendant. Genetic variability and expansion capabilities are slightly higher than parental lineages. Bayesian Skyline Plot indicates that XBB reached its plateau around October 6, 2022 and after an initial rapid growth the viral population size did not further expand, and around November 10, 2022 its levels of genetic variability decreased. Simultaneously with the reduction of the XBB population size, an increase of the genetic variability of its first sub-lineage XBB.1 occurred, that in turn reached the plateau around November 9, 2022 showing a kind of vicariance with its direct progenitors. Structure analysis indicates that the affinity for ACE2 surface in XBB/XBB.1 RBDs is weaker than for BA.2 RBD. In conclusion, at present XBB and XBB.1 do not show evidence about a particular danger or high expansion capability. Genome-based monitoring must continue uninterrupted to individuate if further mutations can make XBB more dangerous or generate new subvariants with different expansion capability.

Genetic and structural analyses reveal the low potential of the SARS-CoV-2 EG.5 variant.

The severe acute respiratory syndrome coronavirus 2 EG.5 lineage is the latest variant under monitoring, and it is generating significant concern due to its recent upward trend in prevalence. Our aim was to gain insights into this emerging lineage and offer insights into its actual level of threat. Both genetic and structural data indicate that this novel variant presently lacks substantial evidence of having a high capacity for widespread transmission. Their viral population sizes expanded following a very mild curve and peaked several months after the earliest detected sample. Currently, neither the viral population size of EG.5 nor that of its first descendant is increasing. The genetic variability appear to be flattened, as evidenced by its relatively modest evolutionary rate (9.05 × 10-4 subs/site/year). As has been observed with numerous prior variants, attributes that might theoretically provide advantages seem to stem from genetic drift, enabling the virus to continually adjust to its host, albeit without a clear association with enhanced dangerousness. These findings further underscore the necessity for ongoing genome-based monitoring, ensuring preparedness and a well-documented understanding of the unfolding situation.

Epidemic history and evolution of an emerging threat of international concern, the severe acute respiratory syndrome coronavirus 2.

This comprehensive review focuses on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact as the cause of the COVID-19 pandemic. Its objective is to provide a cohesive overview of the epidemic history and evolutionary aspects of the virus, with a particular emphasis on its emergence, global spread, and implications for public health. The review delves into the timelines and key milestones of SARS-CoV-2's epidemiological progression, shedding light on the challenges encountered during early containment efforts and subsequent waves of transmission. Understanding the evolutionary dynamics of the virus is crucial in monitoring its potential for adaptation and future outbreaks. Genetic characterization of SARS-CoV-2 is discussed, with a focus on the emergence of new variants and their implications for transmissibility, severity, and immune evasion. The review highlights the important role of genomic surveillance in tracking viral mutations linked to establishing public health interventions. By analyzing the origins, global spread, and genetic evolution of SARS-CoV-2, valuable insights can be gained for the development of effective control measures, improvement of pandemic preparedness, and addressing future emerging infectious diseases of international concern.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Integrative Genome-Based Survey of the SARS-CoV-2 Omicron XBB.1.16 Variant.

The XBB.1.16 SARS-CoV-2 variant, also known as Arcturus, is a recent descendant lineage of the recombinant XBB (nicknamed Gryphon). Compared to its direct progenitor, XBB.1, XBB.1.16 carries additional spike mutations in key antigenic sites, potentially conferring an ability to evade the immune response compared to other circulating lineages. In this context, we conducted a comprehensive genome-based survey to gain a detailed understanding of the evolution and potential dangers of the XBB.1.16 variant, which became dominant in late June. Genetic data indicates that the XBB.1.16 variant exhibits an evolutionary background with limited diversification, unlike dangerous lineages known for rapid changes. The evolutionary rate of XBB.1.16, which amounts to 3.95 × 10-4 subs/site/year, is slightly slower than that of its direct progenitors, XBB and XBB.1.5, which have been circulating for several months. A Bayesian Skyline Plot reconstruction suggests that the peak of genetic variability was reached in early May 2023, and currently, it is in a plateau phase with a viral population size similar to the levels observed in early March. Structural analyses indicate that, overall, the XBB.1.16 variant does not possess structural characteristics markedly different from those of the parent lineages, and the theoretical affinity for ACE2 does not seem to change among the compared variants. In conclusion, the genetic and structural analyses of SARS-CoV-2 XBB.1.16 do not provide evidence of its exceptional danger or high expansion capability. Detected differences with previous lineages are probably due to genetic drift, which allows the virus constant adaptability to the host, but they are not necessarily connected to a greater danger. Nevertheless, continuous genome-based monitoring is essential for a better understanding of its descendants and other lineages.

A Multigene-Panel Study Identifies Single Nucleotide Polymorphisms Associated with Prostate Cancer Risk.

The immune system plays a critical role in modulating cancer development and progression. Polymorphisms in key genes involved in immune responses are known to affect susceptibility to cancer. Here, we analyzed 35 genes to evaluate the association between variants of genes involved in immune responses and prostate cancer risk. Thirty-five genes were analyzed in 47 patients with prostate cancer and 43 healthy controls using next-generation sequencing. Allelic and genotype frequencies were calculated in both cohorts, and a generalized linear mixed model was applied to test the relationship between prostate cancer risk and nucleotide substitution. Odds ratios were calculated to describe the association between each single nucleotide polymorphism (SNP) and prostate cancer risk. Significant changes in allelic and genotypic distributions were observed for IL4R, IL12RB1, IL12RB2, IL6, TMPRSS2, and ACE2. Furthermore, a generalized linear mixed model identified statistically significant associations between prostate cancer risk and SNPs in IL12RB2, IL13, IL17A, IL4R, MAPT, and TFNRS1B. Finally, a statistically significant association was observed between IL2RA and TNFRSF1B and Gleason scores, and between SLC11A1, TNFRSF1B and PSA values. We identified SNPs in inflammation and two prostate cancer-associated genes. Our results provide new insights into the immunogenetic landscape of prostate cancer and the impact that SNPs on immune genes may have on affecting the susceptibility to prostate cancer.

Genetic and Structural Data on the SARS-CoV-2 Omicron BQ.1 Variant Reveal Its Low Potential for Epidemiological Expansion.

The BQ.1 SARS-CoV-2 variant, also known as Cerberus, is one of the most recent Omicron descendant lineages. Compared to its direct progenitor BA.5, BQ.1 has some additional spike mutations in some key antigenic sites, which confer further immune escape ability over other circulating lineages. In such a context, here, we perform a genome-based survey aimed at obtaining a complete-as-possible nuance of this rapidly evolving Omicron subvariant. Genetic data suggest that BQ.1 represents an evolutionary blind background, lacking the rapid diversification that is typical of a dangerous lineage. Indeed, the evolutionary rate of BQ.1 is very similar to that of BA.5 (7.6 × 10-4 and 7 × 10-4 subs/site/year, respectively), which has been circulating for several months. The Bayesian Skyline Plot reconstruction indicates a low level of genetic variability, suggesting that the peak was reached around 3 September 2022. Concerning the affinity for ACE2, structure analyses (also performed by comparing the properties of BQ.1 and BA.5 RBD) indicate that the impact of the BQ.1 mutations may be modest. Likewise, immunoinformatic analyses showed moderate differences between the BQ.1 and BA5 potential B-cell epitopes. In conclusion, genetic and structural analyses on SARS-CoV-2 BQ.1 suggest no evidence of a particularly dangerous or high expansion capability. Genome-based monitoring must continue uninterrupted for a better understanding of its descendants and all other lineages.

Seroprevalence and risk factors associated with Leishmania infantum in dogs in Sardinia (Italy), an endemic island for leishmaniasis.

Leishmaniasis is a widespread, vector-borne parasitosis causing clinical manifestations in animals and in humans. In dogs, Canine Leishmaniasis has been reported in as much as 50 countries and the Mediterranean basin is known to be one of the most affected zones. Within these areas, the Island of Sardinia (Italy) has long been considered endemic for leishmaniasis and the presence of two arthropod vectors has recently been reported there. Nevertheless, to date, no epidemiological surveys regarding CanL have been carried out on the island. Hence, for the first time, the seroprevalence and the risk factors were investigated. Blood samples, as well as clinical and general information from 1.147 dogs, were collected and analyzed. Dogs consisted of two distinct populations, namely "owned dogs" and "kennel dogs." Anti-Leishmania IgG antibodies were detected using IFAT and samples were scored as positive at a cut-off dilution of 1:80. Data was analyzed using a Chi-squared test and bivariate and multivariate analyses were performed. Overall, 15.4% of dogs were found to be infected with CanL while only 44.1% of these animals exhibited clinical signs. Owned dogs (27.2%) were found to be infected more often than kennel dogs (10.6%); male dogs were found to be more frequently infected than female dogs and the number of infected animals increases with age. The present survey confirmed the endemic nature of leishmaniasis in Sardinia with a similar seroprevalence as mainland Italy. The results obtained serve as validation for the hypothesis that, in endemic areas, clinical CanL representations constitute only a fraction of the leishmaniasis cases.

MTHFR, XRCC1 and OGG1 genetic polymorphisms in breast cancer: a case-control study in a population from North Sardinia.

BACKGROUND: Despite conflicting results, considerable evidence suggests the association between single nucleotide polymorphisms in MTHFR, XRCC1 and OGG1 genes and, risk of developing breast cancer. Here a case-control study is reported, including 135 breat cancer patients and 112 healthy women, all representative of Northern Sardinian population. METHODS: Polymerase chain reaction/restriction fragment length polymorphism method was used to determine the genotypes of five polymorphisms: MTHFR C677T (rs1801133) and A1298C (rs1801131), XRCC1 Arg194Trp (rs1799782) and Arg399Gln (rs25487) and OGG1 Ser326Cys (rs1052133). Allelic, genotypic and haplotype association analyses with disease risk and clinicopathological parameters were performed. RESULTS: A nominally significant association with breast cancer risk was observed for MTHFR C677T polymorphism heterozygous genotype in the codominant model (OR: 0.57, 95% CI: 0.32-1.00, p = 0.049) and for Cys/Cys genotype of the OGG1 Ser326Cys polymorphism in the recessive model (OR: 0.23, 95% CI: 0.05-1.11, p = 0.0465). No significant differences were found at genotype-level for A1298C polymorphism of the MTHFR gene and Arg194Trp and Arg399Gln of the XRCC1 gene. Furthermore, the OGG1 and XRCC1 rs25487 polymorphisms were nominally associated with PgR, Her2 status and with sporadic breast cancer, respectively. CONCLUSIONS: Based on genetic characteristics of individuals included in this study, results suggest that MTHFR CT and OGG1 Cys/Cys genotypes have a protective effect that may have an influence on breast cancer risk in a representative Northern Sardinian population.

Influence of genetic drift on patterns of genetic variation: The footprint of aquaculture practices in Sparus aurata (Teleostei: Sparidae).

Aquaculture finfish production based on floating cage technology has raised increasing concerns regarding the genetic integrity of natural populations. Accidental mass escapes can induce the loss of genetic diversity in wild populations by increasing genetic drift and inbreeding. Farm escapes probably represent an important issue in the gilthead sea bream (Sparus aurata), which accounted for 76.4% of total escapees recorded in Europe during a 3-year survey. Here, we investigated patterns of genetic variation in farmed and wild populations of gilthead sea bream from the Western Mediterranean, a region of long gilthead sea bream farming. We focused on the role that genetic drift may play in shaping these patterns. Results based on microsatellite markers matched those observed in previous studies. Farmed populations showed lower levels of genetic diversity than wild populations and were genetically divergent from their wild counterparts. Overall, farmed populations showed the smallest effective population size and increased levels of relatedness compared to wild populations. The small broodstock size coupled with breeding practices that may favour the variance in individual reproductive success probably boosted genetic drift. This factor appeared to be a major driver of the genetic patterns observed in the gilthead sea bream populations analysed in the present study. These results further stress the importance of recommendations aimed at maintaining broodstock sizes as large as possible and equal sex-ratios among breeders, as well as avoiding unequal contributions among parents.

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.

Y-chromosome and Surname Analyses for Reconstructing Past Population Structures: The Sardinian Population as a Test Case.

Many anthropological, linguistic, genetic and genomic analyses have been carried out to evaluate the potential impact that evolutionary forces had in shaping the present-day Sardinian gene pool, the main outlier in the genetic landscape of Europe. However, due to the homogenizing effect of internal movements, which have intensified over the past fifty years, only partial information has been obtained about the main demographic events. To overcome this limitation, we analyzed the male-specific region of the Y chromosome in three population samples obtained by reallocating a large number of Sardinian subjects to the place of origin of their monophyletic surnames, which are paternally transmitted through generations in most of the populations, much like the Y chromosome. Three Y-chromosome founding lineages, G2-L91, I2-M26 and R1b-V88, were identified as strongly contributing to the definition of the outlying position of Sardinians in the European genetic context and marking a significant differentiation within the island. The present distribution of these lineages does not always mirror that detected in ancient DNAs. Our results show that the analysis of the Y-chromosome gene pool coupled with a sampling method based on the origin of the family name, is an efficient approach to unravelling past heterogeneity, often hidden by recent movements, in the gene pool of modern populations. Furthermore, the reconstruction and comparison of past genetic isolates represent a starting point to better assess the genetic information deriving from the increasing number of available ancient DNA samples.

Xenoligophoroides cobitis (Ergens, 1963) n. g., n. comb. (Monogenea: Ancyrocephalidae), a parasite of Gobius cobitis Pallas (Perciformes: Gobiidae) from the Mediterranean and Black seas.

Based on an integrative taxonomic approach, combining morphological characters and partial sequences of the nuclear 28S rRNA gene, a new genus and combination for the species Xenoligophoroides cobitis (Ergens, 1963) is proposed, to accommodate ancyrocephalid monogeneans, parasites on the gills of Gobius cobitis Pallas (Gobiidae) from the western Mediterranean Sea and the northern Black Sea. A morphological comparison of newly collected material with the descriptions of Ancyrocephalus cobitis Ergens, 1963 and Haliotrema cupensis Sasal, Pages & Euzet, 1998, recently synonymised and named as Haliotrema cobitis (Ergens, 1963), confirms their similarity and belonging to the same species. However, characters of this species, as the vas deferens not looping the caecal branch, the bilobed base of the male copulatory organ and the marginal hooks with an upright thumb, do not correspond to the diagnosis of Haliotrema Johnston & Tiegs, 1922. Morphologically, this species is close to members of Ligophorus Euzet & Suriano, 1977 and Kriboetrema Sarabeev, Rubtsova, Yang & Balbuena, 2013, but differs from the former in the accessory piece articulated with MCO and two prostatic reservoirs, and from the latter in the uncoiled MCO with bilobed base and the dextral vaginal pore. Moreover, all species of Ligophorus and Kriboetrema are parasites of grey mullets. A 28S rDNA-based phylogenetic analysis of sequences derived from specimens of X. cobitis from the Mediterranean and Black seas, along with sequences from several closely related genera of the Ancyrocephalidae, suggested the occurrence of a new taxonomic unit, which corresponded to the X. cobitis specimens. This finding supports the establishment of the new genus for the ancyrocephalid parasites on the gills of G. cobitis from the western Mediterranean Sea and the northern Black Sea.

Epidemiological and molecular data on heterophyid trematode metacercariae found in the muscle of grey mullets (Osteichthyes: Mugilidae) from Sardinia (western Mediterranean Sea).

This study is a contribution to the molecular taxonomy and epidemiology of heterophyid (Trematoda: Heterophyidae) metacercariae found in the muscle of Mugilidae (Osteichthyes) from Sardinia (western Mediterranean Sea). Sixty specimens of mugilids (13 Chelon labrosus, 18 Liza aurata, 6 Liza ramada, 8 Liza saliens, 15 Mugil cephalus) were examined and 17,899 metacercariae isolated in 95 % of the hosts. Four types of metacercariae were identified: Heterophyes sp. (n = 14,113), Heterophyes sp. -small (1225), Stictodora sp. (1606), and Ascocotyle (Phagicola) sp. (955). The experimental infection of a hamster with Heterophyes sp. metacercariae produced six adults identified as Heterophyes heterophyes and two as Heterophyes cf. nocens. The morphology of Heterophyes sp. -small metacercariae matched with that of Heterophyes dispar. The sequence analysis of the ITS2 and 28S portions of rDNA confirmed the morphological identification of metacercariae, showing four clusters. All adults grouped together with the Heterophyes sp. metacercariae, whereas adults of Heterophyes nocens from Korea clustered separately, showing that this species is distinguished from H. heterophyes, and suggesting caution in the exclusive use of the number of rodlets of the genital sucker to separate the two species. The presence of metacercariae was high in all hosts; the highest prevalence is of Heterophyes sp. (prevalence ≥78 %; mean intensity ≥135 metacercariae/100 g muscle), and the most heavily infected host is M. cephalus (prevalence = 100 %; mean intensity = 841 metacercariae/100 g muscle).