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

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

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

Differences in Gene Expression Profiles of Seven Target Proteins in Third-Stage Larvae of Anisakis simplex (Sensu Stricto) by Sites of Infection in Blue Whiting (Micromesistius poutassou)

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CORYNOSOMA AUSTRALE JOHNSTON, 1937 AND C. CETACEUM JOHNSTON & BEST, 1942 (ACANTHOCEPHALA: POLYMORPHIDAE) FROM MARINE MAMMALS AND FISH IN ARGENTINIAN WATERS: ALLOZYME MARKERS AND TAXONOMIC STATUS

Genetic and morphological studies were carried out on acanthocephalans belonging to Corynosoma Luhe, 1904 and referable to the species C. cetaceum Johnston & Best, 1942 and C. australe Johnston, 1937, which were recovered from both definitive and intermediate hosts in Argentinian waters. The aims were to estimate the level of genetic differentiation between the two taxa at any stage of their life-cycle, to provide genetic ( allozyme) markers for their recognition and to analyse the systematic status of both taxa. Acanthocephalans were collected from the stomach and intestine of Arctocephalus australis (Zimmerman), the intestine of Mirounga leonina (Linnaeus) and the stomach of Pontoporia blainvillei Gervais & D'Orbigny (definitive hosts) in Argentinian waters. Alternative alleles at all the 13 enzymatic loci studied were observed for C. australe and C. cetaceum. The specimens from the stomach of both P. blainvillei and A. australis were identified, on the basis of the great number of diagnostic loci found, as C. cetaceum; those from intestine of both A. australis and M. leonina as C. australe. A high level of genetic differentiation (D-Nei= infinity: I-Nei= 0.00) between the two taxa was found, suggesting a generic distinction between the two species. Cystacanths of the two species from the body-cavity of the fish Cynoscion guatucupa (Cuvier) collected from the same geographical area were identified genetically. Morphological patterns, such as the number of hooks and hook rows on the proboscis, the distribution of somatic and genital armature, and other morphometric and meristic differences, in addition to ecological data, enabled the identification of these two species at cystacanth, juvenile and adult stages. However, a number of morphological and morphometric features of the Argentinian material were different to those of C. australe and C. cetaceum described from other regions of the world

Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) hidden in the mantle of European flying squid Todarodes sagittatus (Cephalopoda: Ommastrephidae) in NE Atlantic Ocean: Food safety implications

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A Comparison of Marketing Techniques Among Military Recruiters

The U.S. Department of Defense spent $11 billion in enlistment and retention bonuses from 2006 to 2010, which had only a marginally positive effect on the enlistment rate for the Army. The case study addressed this business problem of recruiting by exploring marketing strategies successful recruiting professionals used to motivate individuals to join the military. The purpose of this study was to determine effective recruiting strategies. Therefore, it incorporated the conceptual framework of emergent strategy theory, which postulated the best strategies are neither completely planned nor completely random, but are rather an adaptation to changing dynamics and circumstances. The population consisted of 2 former recruiters, 1 from the Army and another from the Marine Corps, as well as 38 college students located in Wilkes-Barre, Pennsylvania. Data for the study morphed from face-to-face interviews and 3 focus groups comprised of 10 to 15 students each, for the purpose of addressing the research question. Data analysis occurred through a process of coding and theming. The 9 themes identified included tell the story, advertising strategies, and fit for duty. A lesson learned from these themes was that the key for successful recruiting strategies lies in aligning with the wants and needs of individuals in the target demographic. If senior leaders in the Department of Defense followed the recommendations provided, each of the branches of the military service could potentially achieve higher recruiting rates at a lower cost. The study could result in social change whereby eligible recruits could view the Army and Marine Corps as professions of arms in which individuals can live out their ideals of patriotism but also have a good quality of life due to the benefits of military service

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

Assessing the risk of an emerging zoonosis of worldwide concern : anisakiasis

The authors sincerely thank the Biobanking platform at the PARASITE project (EU FP7 PARASITE project (GA no. 312068)) for providing host-parasite data. We thank Rosa Fernández and Cristina Martínez from CETMAR for their help during creation and divulgation of the questionnaires. We also thank Arturo del Rey Moreno (“Antequera” hospital) for his helpful comments. We are also grateful to “Subdirección General de Economía Pesquera” of “Ministerio de Agricultura, Alimentación y Medio Ambiente” (MAGRAMA) of the Spanish government for providing anchovy trade statistics for 2013. M. Bao is supported by a PhD grant from the University of Aberdeen and also by financial support of the contract from the EU Project PARASITE (grant number 312068).Peer reviewedPublisher PD