Fish feed composition by high-throughput sequencing analysis: Parasite risk assessment.

The use of wild small fish species as feed for aquaculture has clearly an economic incentive by speeding the growth of farmed species. Since feed ingredients are sourced from wild fisheries the farmed species could contain natural contaminants which may introduce food safety concerns. In this study, we used High-Throughput Sequencing (HTS) to explore the whole DNA profile of ten dry commercial feeds commonly used by Spanish fish farming companies. The feeds were mainly made of species within the genus Sprattus, Ammodytes and Clupea, and vegetables of the genus Triticum. In the feeds, DNA sequences of parasitic nematodes of fishes (˂1 % total OTUs) were also identified. A taxonomic assignment of query sequences, using a phylogeny-based approach, estimation of pairwise nucleotide identities within and between sequence groups and haplotype network analysis, allow assign short query sequences to the species Phocanema krabbei (Anisakidae) and Hysterothylacium aduncum (Rhaphidascarididae). Both species were identified as ingredient in two and six fish feeds, respectively. This result is of highly concern regarding dietetic recommendations to sensitized patients to anisakids, considering the growing evidence on the possible allergenic potential of both genera, and the recent data on the transfer of anisakid heat-resistant allergens from fishmeal to farm and aquaculture animals.

Population Genetic Structure of Anisakis simplex Infecting the European Hake from North East Atlantic Fishing Grounds.

The European hake, one of the most commercially valuable species in ICES fishing areas, is considered an important neglected source of zoonotic risk by nematode parasites belonging to the genus Anisakis. Merluccius merluccius is, by far, the most important host of Anisakis spp. at the European fishing grounds, in terms of demographic infection values, and carries the highest parasite burden. These high parasite population densities within an individual fish host offer a chance to explore new sources of variations for the genetic structure of Anisakis spp. populations. A total of 873 Anisakis spp. third-stage larvae, originally sampled from viscera and muscular sections of hake collected at ten fishing grounds, were primarily identified using ITS rDNA region as molecular marker. After that, we used mtDNA cox2 gene to reveal the high haplotype diversity and the lack of genetic structure for A. simplex. Dominant haplotypes were shared among the different fishing areas and fish sections analyzed. Results indicate a clear connection of A. simplex from European hake along the Northern North Sea to the Portuguese coast, constituting a single genetic population but revealing a certain level of genetic sub-structuring on the Northwest coast of Scotland. This study also provides useful information to advance the understanding of parasite speciation to different fish host tissues or microenvironments.

The hidden companion of non-native fishes in north-east Atlantic waters.

A tropicalization phenomenon of ichthyofauna has been described in the last decades in Galicia (north-eastern Atlantic), with increasing reports of tropical and subtropical fishes appearing northward this distribution range. A search for parasites was carried out in the digestive tract of two specimens first captured in Galician waters: the prickly puffer Ephippion guttifer (Tetraodontidae) and the African stripped grunt Parapristipoma octolineatum (Haemulidae). Examination of E. guttifer showed high intensity of nematodes, from three different genera: Cucullanus (Cucullanidae), Hysterothylacium (Raphidascaridae) and Anisakis (Anisakidae), with demonstrated pathogenicity to humans. Molecular identification allowed the identification of Anisakis pegreffii, already described in the area, and first reports for European waters of Cucullanus dodsworthi, Hysterothylacium reliquens and a new Hysterothylacium sp. P. octolineatum showed a far lower level of parasitization, with two Hysterothylacium larvae, genetically identified as Hysterothylacium deardorffoverstreetorum, also its first report in the eastern Atlantic. Thus, possible ecological impact of the occurrence of two non-native individual fishes in a new area could be remarkably higher if we see this issue through the lens of the parasitological perspective, as far as only two individual fish can harbour more of one hundred nematode parasites belonging to different species, most of them also new species for that area.

Occurrence of Anisakis and Hysterothylacium larvae in commercial fish from Balearic Sea (Western Mediterranean Sea).

This study investigates the occurrence of anisakids and raphidascarids in commercial fish from Balearic Sea (Western Mediterranean). A total of 335 fish including 19 black anglerfish (Lophius budegassa), 33 white anglerfish (L. piscatorius), 129 European hake (Merluccius merluccius), 30 red mullet (Mullus barbatus), and 124 striped mullet (M. surmuletus) were examined using enzymatic digestion. A total of 948 nematode larvae were isolated (prevalence 52.53%) being the highest prevalence observed in striped mullet. Forty-six larvae were identified using molecular analyses which included PCR and sequencing of the 629-bp fragment of mitochondrial cox2 gene region. Anisakis pegreffii (80.43%), A. physeteris (8.69%), Hysterothylacium fabri (6.52%), and A. simplex (4.35%) were detected based on molecular analyses of larvae. Total nematode prevalence was positively correlated with weight, length, condition factor, and maturity stage of the host and also with fishing ground depth. Statistical differences between total nematode prevalence and geographical sector of capture were observed when fishing hauls were grouped according to the abundance of sperm whales or common bottlenose dolphins. The results also corroborate that fishing water depth may play an important role in anisakid and raphidascarid parasitization.

Parasites, pathological conditions and resistance to Marteilia cochillia in lagoon cockle Cerastoderma glaucum from Galicia (NW Spain).

A histopathological survey revealed parasites and pathological conditions affecting lagoon cockles Cerastoderma glaucum along the Galician coast; serious pathological threats were not detected because the potentially pathogenic conditions (infections with a Marteilia-like parasite and bucephalid sporocysts, disseminated neoplasia and a condition involving large foci of heavy haemocytic reaction) were rare, while more prevalent parasites had negligible or limited pathogeny. Considering that C. edule and C. glaucum are sympatric in some Galician rias, it is remarkable that C. glaucum was not seriously affected by Marteilia cochillia while C. edule suffered an intense outbreak of this parasite associated with massive mortality. Comparison of the digestive gland between cockle species showed co-occurrence of digestive tubules in different phases, with abundant disintegrated tubules, in the case of C. glaucum, while C. edule showed synchronicity and absence of fully disintegrated tubules; these differences could influence their susceptibility to M. cochillia because the main location of this parasite in common cockles is the epithelia of the digestive gland. Moreover, the observation of histological sections through the digestive gland easily allows differentiating the 2 cockle species.

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.

Perkinsus olseni and P. chesapeaki detected in a survey of perkinsosis of various clam species in Galicia (NW Spain) using PCR-DGGE as a screening tool.

A survey on perkinsosis was performed involving 15 locations scattered along the Galician coast (NW Spain) and four clam species with high market value (Ruditapes decussatus, Ruditapes philippinarum, Venerupis corrugata and Polititapes rhomboides). The prevalence of Perkinsus parasites was estimated by PCR using genus-specific primers. The highest percentage of PCR-positive cases for perkinsosis corresponded to clams R. decussatus and V. corrugata, while lower values were detected in R. philippinarum and no case was found in P. rhomboides. The discrimination of Perkinsus species was performed by PCR-RFLP and by a new PCR-DGGE method developed in this study. Perkinsus olseni was identified in every clam species, except in P. rhomboides, using both PCR-DGGE and PCR-RFLP. Additionally, Perkinsus chesapeaki was only detected by PCR-DGGE infecting two Manila clams R. philippinarum from the same location, reporting the first case in Galicia. P. chesapeaki identification was further confirmed by in situ hybridisation assay and phylogenetic analysis of ITS region and LSU rDNA.

Update of information on perkinsosis in NW Mediterranean coast: Identification of Perkinsus spp. (Protista) in new locations and hosts.

This study addressed perkinsosis in commercially important mollusc species in the western Mediterranean area. Perkinsus olseni was found in Santa Gilla Lagoon (Sardinia) infecting Ruditapes decussatus, Cerastoderma glaucum and Venerupis aurea, in Balearic Islands infecting Venus verrucosa and in Delta de l'Ebre (NE Spain) parasitising Ruditapes philippinarum and R. decussatus. Perkinsus mediterraneus was detected infecting Ostrea edulis from the Gulf of Manfredonia (SE Italy) and Alacant (E Spain), V. verrucosa and Arca noae from Balearic Islands and Chlamys varia from Balearic Islands, Alacant and Delta de l'Ebre.

Infection of Manila clams Ruditapes philippinarum from Galicia (NW Spain) with a Mikrocytos-like parasite.

The name 'microcells' is frequently used to refer to small-sized unicellular stages of molluscan parasites of the genera Bonamia (Rhizaria, Haplosporidia) and Mikrocytos (Rhizaria). Histological examination of Manila clams Ruditapes philippinarum revealed microcells in the connective tissue of adductor muscle, foot, mantle, gills, siphon and visceral mass. The clams had been collected from 4 beds on the coast of Galicia, Spain. The prevalence of these microcells ranged from 73 to 93% in surface clams and from 3 to 33% in buried clams. However, the detection of brown ring disease signs in clams from every bed prevented us from making the assumption that the microcells alone were responsible for clam mortality. PCR assays using primer pairs designed to detect Bonamia spp. and haplosporidians gave negative results, whereas positive results were obtained with primers for the genus Mikrocytos. A consensus sequence of 1670 bp of the ribosomal gene complex of the microcells was obtained. It contained a section of the 18S region, the whole first internal transcribed spacer, the 5.8S region, the second internal transcribed spacer and a section of the 28S region. Comparison of this sequence with those of M. mackini infecting Crassostrea gigas and Mikrocytos sp. infecting Ostrea edulis showed that the microcells of Galician clams were the most divergent among the compared parasites. This is the first report of a Mikrocytos-like parasite infecting Manila clams. Care must be taken to avoid the spread of this parasite through Manila clam transfers.

Species-specific oligonucleotide probe for detection of Bonamia exitiosa (Haplosporidia) using in situ hybridisation assay.

Bonamiosis is a disease affecting various oyster species and causing oyster mass mortalities worldwide. The protozoans Bonamia exitiosa and B. ostreae (Haplosporidia) are included in the list of notifiable diseases of the World Organisation for Animal Health as the causative agents of this disease. Although the geographic range of both species was considered different for years, both species are now known to co-occur in some European areas affecting the same host, Ostrea edulis, which strengthens the need of species-specific methods to unequivocally identify the species of Bonamia. An oligonucleotide probe for specific detection of B. exitiosa (BEX_ITS) was designed to be used in in situ hybridisation (ISH) assays. ISH assay with BEX_ITS probe showed species-specificity and more sensitivity than traditional histology to visualise the parasite inside host tissue. ISH assay showed that the oyster gonad was the area where the parasite was most frequently located, and was the exclusive organ of infection in some oysters. A recommendation arising from the study is that more than 1 organ (including gonad and gills) should be used for PCR-based diagnosis of B. exitiosa, to maximise the sensitivity.

Interlaboratory variability in screening for Bonamia ostreae, a protistan parasite of the European flat oyster Ostrea edulis.

The spread of the protozoan parasite Bonamia ostreae is of major concern to the European flat oyster Ostrea edulis industry. Many studies have looked at the sensitivity of individual methods available to screen for B. ostreae, but in this study, 3 separate laboratories examined 4 methods of diagnosis currently used routinely in laboratories: heart imprints, histology, polymerase chain reaction (PCR) and in situ hybridisation (ISH). The results were compared to estimate interlaboratory variability. Heart imprints and histology had the highest reproducibility amongst the 3 laboratories, with greatest agreement between detection of infected and uninfected individuals. PCR had the highest detection level in every laboratory. These positives were related to the presence of confirmed infections but also in unconfirmed infections, possibly due to the presence of traces of B. ostreae DNA in oysters where clinical infections were not observed. PCR, in combination with histology or ISH, provided the most reliable detection levels in every laboratory. Variation in results for PCR and ISH observed between laboratories may be due to the different protocols used by each laboratory for both methods. Overall, the findings from the 3 laboratories indicated that at least 2 methods, with fixed protocols, should be used for the accurate detection and determination of infection prevalence within a sample. This combination of methods would allow for a clearer and more precise diagnosis of B. ostreae, preventing further spread of the disease and providing more accurate detection levels and epidemiological information.

Oyster parasites Bonamia ostreae and B. exitiosa co-occur in Galicia (NW Spain): spatial distribution and infection dynamics.

Bonamiosis constrains the flat oyster industry worldwide. The protistan species Bonamia ostreae had been considered solely responsible for this disease in Europe, but the report of B. exitiosa infecting Ostrea edulis 5 yr ago in Galicia (NW Spain), and subsequently in other European countries, raised the question of the relevance of each species in bonamiosis. The spatial distribution of B. exitiosa and B. ostreae in Galicia was addressed by sampling 7 natural O. edulis beds and 3 culture raft areas, up to 3 times in the period 2009 to 2010. B. ostreae infected flat oysters in every natural bed and every raft culture area. True B. exitiosa infections (histological diagnosis) were detected in every raft culture area but only in 2 natural beds, i.e. in 4 rías. PCR-positive results for B. exitiosa were recorded in 4 out of 5 beds where true infections were not found, thus the occurrence of B. exitiosa in those 4 beds cannot be ruled out. Additionally, 4 cohorts of hatchery-produced oyster spat were transferred to a raft to analyse Bonamia spp. infection dynamics through oyster on-growing. The highest percentages of oysters PCR-positive for both Bonamia spp. were recorded in the first months of on-growing; other peaks of PCR-positive diagnosis were successively lower. Differences in the percentage of PCR-positive cases and in the prevalence of true infection between B. exitiosa and B. ostreae through on-growing were not significant. Our results support that B. exitiosa is adapted to infect O. edulis in the Galician marine ecosystem.

Cockle Cerastoderma edule fishery collapse in the Ría de Arousa (Galicia, NW Spain) associated with the protistan parasite Marteilia cochillia.

The highest shellfishery catch in Galicia (NW Spain) has traditionally been cockle Cerastoderma edule. The shellfish bed located in Lombos do Ulla (Ría de Arousa) used to be among those with the highest cockle production; however, cockle mortality rate increased sharply in this bed in April 2012, reaching 100% in May 2012. Salinity and temperature were discounted as potential causes of the mortality. Marteiliosis, which was first detected in February 2012 and reached 100% prevalence in April 2012, was identified as the most probable cause. Marteiliosis had never been detected in Galician cockles, but extensive surveillance of the Galician coast in May to July 2012 detected marteiliosis in most cockle beds of the Ría de Arousa, whereas it was not found in other rías; 2 mo later, the cockle catch in the Ría de Arousa became negligible. Examination of the aetiological agent of marteiliosis with light and transmission electron microscopy supported its assignation to the genus Marteilia; morphological features showed similarity, but not complete identity, with the recently described species M. cochillia Carrasco et al., 2013. Regarding its molecular characterisation, a consensus sequence of 4433 bp containing a partial sequence of the intergenic spacer region, the complete 18S rRNA gene and a partial sequence of the first internal transcribed spacer region was obtained. The obtained sequences were compared with those available for Marteilia spp. and other Paramyxida. Molecular data support that this parasite corresponds to the species M. cochillia, and a PCR assay was designed for its specific diagnosis. The association of huge cockle mortality with M. cochillia infection urges extreme caution to avoid spreading this disease.

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR.

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

First report of the protozoan parasite Perkinsus marinus in South America, infecting mangrove oysters Crassostrea rhizophorae from the Paraíba River (NE, Brazil).

The present work aimed to study the infection by Perkinsus sp. in the mangrove oysters Crassostrea rhizophorae from the estuary of the Paraíba River (Paraíba State, Brazil). Perkinsosis was detected by incubation of oyster gill pieces in Ray's fluid thioglycollate medium. The monthly prevalence values were all above 70%, thus infection was not likely to be a transient event. Perkinsus sp. parasites isolated from eight oysters were propagated in vitro. PCR-RFLP analysis of in vitro cultured cells as well as the sequences of the rDNA ITS region allowed the identification of the in vitro propagated parasites as Perkinsus marinus. Phylogenetic analyses using rDNA ITS region sequences strongly supported the Perkinsus sp. from Paraíba in a monophyletic group with P. marinus. Thus, the results confirmed the species affiliation of Paraíba Perkinsus sp. as P. marinus. This is the first report of P. marinus in Brazil and South America and the first report of P. marinus naturally infecting C. rhizophorae.