Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.), P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum, a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency (E) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers

Gene expression profiles of antigenic proteins of third stage larvae of the zoonotic nematode Anisakis pegreffii in response to temperature conditions

Anisakis pegreffii, a recognised etiological agent of human anisakiasis, is a parasite of homeothermic hosts at the adult stage and of ectothermic hosts at the third larval stage. Among distinct factors, temperature appears to be crucial in affecting parasite hatching, moulting and to modulate parasite-host interaction. In the present study, we investigated the gene transcripts of proteins having an antigenic role among excretory secretory products (ESPs) (i.e., a Kunitz-type trypsin inhibitor, A.peg-1; a glycoprotein, A.peg-7; and the myoglobin, A.peg-13) after 24 h, in A. pegreffii larvae maintained in vitro, under controlled temperature conditions. Temperatures were 37 °C and 20 °C, resembling respectively homeothermic and ectothermic hosts conditions, and 7 °C, the cold stress condition post mortem of the fish host. Primers of genes coding for these ESPs to be used in quantitative real-time PCR were newly designed, and qRT-PCR conditions developed. Expression profiles of the genes A.peg-1 and A.peg-13 were significantly up-regulated at 20 °C and 37 °C, with respect to the control (larvae kept at 2 °C for 24 h). Conversely, transcript profiles of A.peg-7 did not significantly change among the chosen temperature conditions. In accordance with the observed transcript profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of the three target ESPs at 37 °C, while only A.peg-13 was observed at 7 °C. The results suggest that temperature conditions do regulate the gene expression profiles of A.peg-1 and A.peg-13 in A. pegreffii larvae. However, regulation of the glycoprotein A.peg-7 is likely to be related to other factors such as the host's immune response

Genetic relationships among species of Contracaecum railliet & Henry, 1912 and Phocascaris Höst, 1932 (Nematoda: Anisakidae) from pinnipeds inferred from mitochondrial cox2 sequences, and congruence with allozyme data

The genetic relationships among 11 taxa, belonging to the genus Contracaecum (C. osculatum A, C. osculatum B, C. osculatum (s.s.), C. osculatum D, C. osculatum E, C. osculatum baicalensis, C. mirounga, C. radiatum, C. ogmorhini (s.s.), C. margolisi) and Phocascaris IPhocascaris cystophorae), parasites as adults of seals, were inferred from sequence analysis (519 bp) of the mitochbndrial cytochrome c oxidase subunit II (mtDNA cox2) gene. Phylogenetic analyses obtained from Parsimony (MP) and Neighbour-Joining (NJ) K2P distance values generated similar topologies, each well supported at major nodes. All analyses delineated two main clades: the first encompassing the parasites of the phocid seals, i.e. the C. osculatum species complex, C. osculatum baicalensis, C. mirounga and C. radiatum, with the latter two species forming a separate subclade; the second including the parasites of otarids, i.e. C. ogmorhini (s.s.) and C. margolisi. An overall high congruence between mtDNA inferred tree topologies and those produced from nuclear data sets (20 allozyme loci) was observed. Comparison of the phylogenetic hypothesis here produced for Contracaecum spp. plus Phocascaris with those currently available for their definitive hosts (pinnipeds) suggests parallelism between hosts and parasite phylogenetic tree topologies.Fil: Mattiucci, Simonetta. Università di Roma; ItaliaFil: Paoletti, M.. Università di Roma; Italia. Università degli Studi della Tuscia; ItaliaFil: Webb, S.C.. Cawthron Institute; Nueva ZelandaFil: Sardella, Norma Haydee. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología. Laboratorio de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Timi, Juan Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; ArgentinaFil: Berland, B.. University of Bergen; NoruegaFil: Nascetti, G.. Università degli Studi della Tuscia; Itali

The Mediterranean European hake, Merluccius merluccius: Detecting drivers influencing the Anisakis spp. larvae distribution

The European hake Merluccius merluccius is one of the most commercially important and widely distributed fish species, occurring both in European and Mediterranean Sea fisheries. We analyzed the distribution and infection rates of different species of Anisakis in M. merluccius (N = 1130 hakes), by site of infection in the fish host (viscera, dorsal and ventral fillets) from 13 different fishing grounds of the Mediterranean Sea (FAO area 37). The fillets were examined using the UV-Press method. A large number of Anisakis specimens (N = 877) were identified by diagnostic allozymes, sequence analysis of the partial EF1 α-1 region of nDNA and mtDNA cox2 gene. Among these, 813 larvae corresponded to A. pegreffii, 62 to A. physeteris, 1 to A. simplex (s. s.), whereas one resulted as a F1 hybrid between A. pegreffii and A. simplex (s. s.). Remarkably high levels of infection with A. pegreffii were recorded in hakes from the Adriatic/Ionian Sea compared to the fish of similar length obtained from the western Mediterranean fishing grounds. A positive correlation between fish length and abundance of A. pegreffii was observed. Concerning the localization of A. pegreffii larvae in the fish, 28.3% were detected in the liver, 62.9% in the rest of the viscera, 6.6% in the ventral part of the flesh, whereas 2.1% in the dorsal flesh

Population genetic structure of the parasite Anisakis simplex (s. s.) collected in Clupea harengus L. from North East Atlantic fishing grounds

The Atlantic herring is a schooling, pelagic species that inhabits both sides of the North Atlantic Ocean. Herring stock identification is usually based on several approaches, including fish meristic characters, population genetic analysis and the use of parasite species composition. A total of 654 Anisakis spp. larvae collected from herring of four fishing grounds in the Norwegian Sea, Baltic Sea, North Sea, and the English Channel off the French coast, was identified to species level using diagnostic allozymes and sequence analysis of EF1 α−1 nDNA and the mtDNA cox2 genes. Population genetic differentiation of Anisakis simplex (s. s.) among the different fishing areas was estimated, at the intraspecific level, on the basis of mtDNA cox2 sequences analysis. Spatial comparison based on molecular variance analysis and Fst values was performed for the collected specimens (among regions). Haplotype network construction showed relevant differences in haplotype frequencies between samples of A. simplex (s. s.) from the different geographical areas. Results indicate a genetic sub-structuring of A. simplex (s. s.) obtained from herring in different areas, with the population from the Norwegian Sea being the most differentiated one, and with North Sea and Baltic Sea populations being most similar. The population genetic structure of A. simplex (s. s.) was in accordance with the herring population genetic structure throughout the host’s geographical range in the NE Atlantic. Results suggest that mtDNA cox2 is a suitable genetic marker for A. simplex (s. s.) population genetic structure analysis and a valuable tool to elucidate the herring stock structure in the NE Atlantic Ocean

Investigating the genetic structure of the parasites Anisakis pegreffii and A. berlandi (Nematoda: Anisakidae) in a sympatric area of the southern Pacific Ocean waters using a multilocus genotyping approach: first evidence of their interspecific hybridization

The southern Pacific Ocean, off the New Zealand coast, has been reported as one sympatric area of the two parasite species Anisakis pegreffii and A. berlandi. Here, a multilocus genotyping approach, based on a panel of eleven DNA microsatellite (SSR) loci plus the sequences analysis of the nuclear nas10 nDNA and the mitochondrial mtDNA cox2 gene loci, was applied to a total of N = 344 adults and larvae of Anisakis spp. from cetacean and fish species, respectively. Out of the newly scored SSR loci, Anisl 15 and Anisl 2 showed fixed alternative alleles between A. pegreffii and A. berlandi resulting as 100% diagnostic loci. Out of SSRs Anisl 00314 and Anisl 7 previously disclosed, two additional loci, i.e., Anisl 4 and Anisl 22, were found to be sex-linked. The Bayesian genotypes clustering approach (STRUCTURE) allowed identification, with a 100% of probability value, N = 208 specimens to the “pure parental” A. pegreffii, N = 133 to the “pure parental” A. berlandi, while one adult and two larval stages showed mixed ancestry between the two groups having, in all cases, a Q-value = 0.50. NEWHYBRIDS analysis assigned (100% of probability) those specimens to their F1 hybrid category. This represents the first evidence of contemporary hybridization between the two parasite species in a sympatric area. The pairwise FST values estimated at intraspecific and interspecific level, inferred from both SSR loci and mitochondrial mtDNA cox2 sequences, have also demonstrated the existence of two distinct panmictic units in this study area, corresponding respectively to A. pegreffii and A. berlandi. The results obtained support the useful application of a multilocus approach in the identification of sibling species and their hybrid categories in sympatric areas. The possible use of sex-linked SSR loci of the two species of the A. simplex (s. l.), for sex determination of their larval stages, is also suggested.publishedVersio

Air-dried stockfish of Northeast Arctic cod do not carry viable anisakid nematodes

A total of 80 stockfish fillets of Northeast Arctic cod (Gadus morhua), traditionally open-air-dried in northern Norway, was examined for the presence and viability of larval parasitic nematodes of the family Anisakidae. Anisakids (particularly those belonging to genera Anisakis and Pseudoterranova) are of public health and economic concern globally, since they are responsible for an underestimated fish-borne zoonotic disease called anisakidosis (anisakiasis when caused by members of the Anisakis genus). Stockfish fillets were inspected for anisakids by candling and artificial (pepsin) digestion methodologies. The recovered nematodes (n = 342) were morphologically identified to genus level and their viability assessed. Subsamples of anisakid larvae (n = 31) were identified by molecular/genetic markers inferred from sequences analyses and real time polymerase chain reaction (RT-PCR) of the mtDNA cox2 gene, as Anisakis simplex sensu stricto (s.s.) (n = 29) and as Pseudoterranova decipiens (s.s.) (n = 2). This is the first time a RT-PCR primer/probe system was used to identify anisakids in a processed fishery product. Anisakis simplex (s.s.) larvae were found in 81% of the fillets, with average (range) 4 (0–35). In total, 338 A. simplex (s.s.) and 4 P. decipiens (s.s.) larvae, all dead, were recovered from the fillets. Anisakids were devitalised by the air-dried stockfish production process in 7.5 months (common stockfish production time from sea to plate). The results suggest that there is a negligible risk of acquiring anisakidosis from consumption of air-dried stockfish. Further research is recommended to evaluate if anisakids can be devitalised in five months (i.e. minimum stockfish production time). The health risk for sensitized consumers posed by the potential presence of anisakid allergens in stockfish needs to be assessed. This is the first report on the viability of anisakid larvae in an unsalted, naturally dried fishery product. Drying could represent an alternative and efficient treatment for the inactivation of anisakids in fishery products. Trimming of the belly flaps of highly parasitized cod may reduce the number of anisakids in stockfish by 74%.publishedVersio

Nucleation, reorganization and disassembly of an active network from lactose-modified chitosan mimicking biological matrices

Developing synthetic materials able to mimic micro- and macrorheological properties of natural networks opens up to novel applications and concepts in materials science. The present contribution describes an active network based on a semi-synthetic polymer, a lactitol-bearing chitosan derivative (Chitlac), and a transient inorganic cross-linker, boric acid. Due to the many and diverse anchoring points for boric acid on the flanking groups of Chitlac, the cross-links constantly break and reform in a highly dynamic fashion. The consequence is a network with unusual non-equilibrium and mechanical properties closely resembling the rheological behavior of natural three-dimensional arrangements and of cytoskeleton. Concepts like network nucleation, reorganization and disassembly are declined in terms of amount of the cross-linker, which acts as a putative motor for remodeling of the network upon application of energy. The out-of-equilibrium and non-linear behavior render the semi-synthetic system of great interest for tissue engineering and for developing in-vitro mimics of natural active matrices

Impacts of air pollution on human and ecosystem health, and implications for the National Emission Ceilings Directive. Insights from Italy

Across the 28 EU member states there were nearly half a million premature deaths in 2015 as a result of exposure to PM2.5, O3 and NO2. To set the target for air quality levels and avoid negative impacts for human and ecosystems health, the National Emission Ceilings Directive (NECD, 2016/2284/EU) sets objectives for emission reduction for SO2, NOx, NMVOCs, NH3 and PM2.5 for each Member State as percentages of reduction to be reached in 2020 and 2030 compared to the emission levels into 2005. One of the innovations of NECD is Article 9, that mentions the issue of “monitoring air pollution impacts” on ecosystems. We provide a clear picture of what is available in term of monitoring network for air pollution impacts on Italian ecosystems, summarizing what has been done to control air pollution and its effects on different ecosystems in Italy. We provide an overview of the impacts of air pollution on health of the Italian population and evaluate opportunities and implementation of Article 9 in the Italian context, as a case study beneficial for all Member States. The results showed that SO42− deposition strongly decreased in all monitoring sites in Italy over the period 1999–2017, while NO3− and NH4+ decreased more slightly. As a consequence, most of the acid-sensitive sites which underwent acidification in the 1980s partially recovered. The O3 concentration at forest sites showed a decreasing trend. Consequently, AOT40 (the metric identified to protect vegetation from ozone pollution) showed a decrease, even if values were still above the limit for forest protection (5000 ppb h−1), while PODy (flux-based metric under discussion as new European legislative standard for forest protection) showed an increase. National scale studies pointed out that PM10 and NO2 induced about 58,000 premature deaths (year 2005), due to cardiovascular and respiratory diseases. The network identified for Italy contains a good number of monitoring sites (6 for terrestrial ecosystem monitoring, 4 for water bodies monitoring and 11 for ozone impact monitoring) distributed over the territory and will produce a high number of monitored parameters for the implementation of the NECD

Infection levels and species diversity of ascaridoid nematodes in Atlantic cod, Gadus morhua, are correlated with geographic area and fish size

13 pages, 6 figures, 4 tablesAtlantic cod (Gadus morhua) is among the most important commercial fish species on the world market. Its infection by ascaridoid nematodes has long been known, Pseudoterranova even being named cod worm. In the present study, 755 individuals were sampled in the Barents, Baltic and North Seas during 2012–2014. Prevalences for Anisakis in whole fish and in fillets in the different fishing areas varied from 16 to 100% and from 12 to 90% respectively. Abundance was also greatly influenced by the sampling area. Generalized additive model results indicate higher numbers of Anisakis in the North Sea, even after the larger body size was accounted for. Numbers and prevalence of Anisakis were positively related to fish length or weight. The prevalence of parasites in whole fish and in fillets was also influenced by the season, with the spring displaying a peak for the prevalence in whole fish and, at the same time, a drop for the prevalence in fillets. Whereas 46% of cod had Anisakis larvae in their fillets, the majority (39%) had parasites mainly in the ventral part of the fillet and only 12% had parasites in their dorsal part. This observation is of importance for the processing of the fish. Indeed, the trimming of the ventral part of the cod fillet would allow the almost total elimination of ascaridoids except for cod from the Baltic Sea where there was no difference between the dorsal and the ventral part. The presence of other ascaridoid genera was also noticeable in some areas. For Pseudoterranova, the highest prevalence (45%) in whole fish was observed in the Northern North Sea, whereas the other areas had prevalences between 3 and 16%. Contracaecum was present in every commercial size cod sampled in the Baltic Sea with an intensity of up to 96 worms but no Contracaecum was isolated from the Central North Sea. Non-zoonotic Hysterothylacium was absent from the Baltic Sea but with a prevalence of 83% in the Barents and the Northern North Sea. A subsample of worms was identified with genetic-molecular tools and assigned to the species A. simplex (s.s.), A. pegreffii, P. decipiens (s.s.), P. krabbei, C. osculatum and H. aduncum. In addition to high prevalence and abundance values, the cod sampled in this study presented a diversity of ascaridoid nematodes with a majority of fish displaying a co-infection. Out of 295 whole infected fish, 269 were co-infected by at least 2 genera.The present survey was financially supported by the European Union's 7th Framework Program for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant agreement (GA) no. 312068.Peer reviewe