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

No more time to stay ‘single’ in the detection of Anisakis pegreffii, A. simplex (s. s.) and hybridization events between them: a multi-marker nuclear genotyping approach

A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α−1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of ‘hybrids’ both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR–RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α−1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species

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

Atlantic 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

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

Wave Packet Echoes in the Motion of Trapped Atoms

We experimentally demonstrate and systematically study the stimulated revival (echo) of motional wave packet oscillations. For this purpose, we prepare wave packets in an optical lattice by non-adiabatically shifting the potential and stimulate their reoccurence by a second shift after a variable time delay. This technique, analogous to spin echoes, enables one even in the presence of strong dephasing to determine the coherence time of the wave packets. We find that for strongly bound atoms it is comparable to the cooling time and much longer than the inverse of the photon scattering rate

Systematics and Glacial Population History of the Alternifolium Group of the Flowering Plant Genus Chrysosplenium (Saxifragaceae)

The flowering plant genus Chrysosplenium comprises approximately 57 species of herbaceous perennials. These species are mainly distributed in the Northern Hemisphere where they occur in moist habitats. Though the center of diversity, and presumed location of origin, for the genus is east temperate Asia, more recently radiating taxa have invaded the arctic of North America and Europe. There are six species of Chrysosplenium in North America and four of them (i.e., C. iowense, C. tetrandrum, C. wrightii, and C. rosendahlii) belong to the section Alternifolia. Termed the Alternifolium group, this collection of species presents an excellent opportunity to study the evolution of variation in arctic and alpine environments. Similar to many arctic taxa, these species display very little morphologic or genetic variation, but they exhibit diversity in chromosome number, breeding system, geographic distribution, and ecology. Though the Alternifolium group has been the subject of numerous taxonomic studies, no thorough investigation of its evolutionary history has been conducted. This study used a combination of genetic and phenotypic data (e.g., DNA sequence, Inter-Simple Sequence Repeat, morphology) to determine the patterns of variation present within the Alternifolium group and then used these patterns to infer historical processes that might have contributed to them. Through the course of the study, however, it also became necessary to investigate the applicability of genetic estimates derived from different molecular markers and statistical methods. Appropriate comparisons among genetic estimates are critical to accurately interpret results and generate new predictions

The influence of race on behavior and the neural correlates of expectancy during an economic decision game

Dr. Bruce Bartholow, Dissertation Supervisor.Includes vita.Field of study: Psychology."May 2018."I investigated how another person's race influences resource sharing during a social economic decision-making game that depends on trustworthiness. Seventy-eight university students played a behavioral economics Trust Game with players of different races while EEG was recorded. I predicted that negative stereotype-based expectancies of Black players would cause participants to be more trusting of White than Black players, and also that these expectancies would be evident in the event-related potential (ERP) component, RewP, which is sensitive to expectancy violations (or prediction errors). Results largely conformed to predictions, in that White participants shared more money with White players than with Black players; this effect was weaker (and not statistically significant) when data from the full sample, including 11 non-White participants, was used in the model. This preferential treatment of White players was more pronounced in individuals who held more biased racial attitudes. Additionally, RewP was greater for feedback from Black relative to White players on trials when offer amounts were higher than average, implying that when feedback was most salient (because more money was at stake), it was more unexpected when Black players reciprocated generosity, and less expected when they did not. Exploratory analyses of an ERP component associated with racial categorization of faces, the P2, was larger to Black versus White faces, as in previous studies. Of interest here, the race effect in P2 amplitude was moderated by racial attitudes, such that less-biased individuals showed larger race differences in the P2, suggesting that these participants allocated more attention to social categories in an attempt to respond in a less biased way. These novel results were not entirely consistent with my original hypotheses, and thus more research is necessary to replicate and help explain the present results.Includes bibliographical references (pages 52-63)

Resurrection of genus Phocanema Myers, 1959, as a genus independent from Pseudoterranova Mozgovoĭ, 1953, for nematode species (Anisakidae) parasitic in pinnipeds and cetaceans, respectively

Species of the genus Pseudoterranova, infect kogiid cetaceans and pinnipeds. However, there is mounting molecular evidence that those from cetaceans and pinnipeds are not congeneric. Here, we provide further evidence of the non-monophyly of members of Pseudoterranova from phylogenetic analyses of the conserved nuclear LSU rDNA gene, entire ITS rDNA region and mtDNA cox2 gene, and identify morphological characters that may be used to distinguish the members of the two clades. We propose the resurrection of the genus Phocanema, with Ph. decipiens (sensu stricto) as the type species, to encompass Ph. decipiens, Ph. azarasi, Ph. bulbosa, Ph. cattani and Ph. krabbei, all parasites of pinnipeds. We propose to restrict the conception of genus Pseudoterranova, which now harbours two species infecting kogiid whales; Ps. kogiae (type species) and Ps. ceticola. Members of the genera Phocanema and Pseudoterranova differ by the shape and orientation of the lips, relative tail lengths, adult size, type of final host (pinniped vs. cetacean) and phylogenetic placement based on nuclear rDNA and mtDNA cox2 sequences.publishedVersio