Histopathological investigation of four populations of abalone (Haliotis iris) exhibiting divergent growth performance.

The black-foot abalone (paua), Haliotis iris, is a unique and valuable species to New Zealand with cultural importance for Maori. Abalone are marine gastropods that can display a high level of phenotypic variation, including slow-growing or 'stunted' variants. This investigation focused on identifying factors that are associated with growth performance, with particular interest in the slow-growing variants. Tissue alterations in H. iris were examined using histopathological techniques, in relation to growth performance, contrasting populations classified by commercial harvesters as 'stunted' (i.e., slow-growing) and 'non-stunted' (i.e., fast-growing) from four sites around the Chatham Islands (New Zealand). Ten adults and 10 sub-adults were collected from each of the four sites and prepared for histological assessment of condition, tissue alterations, presence of food and presence of parasites. The gut epithelium connective tissue, digestive gland, gill lamellae and right kidney tissues all displayed signs of structural differences between the slow-growing and fast-growing populations. Overall, several factors appear to be correlated to growth performance. The individuals from slow-growing populations were observed to have more degraded macroalgal fragments in the midgut, increased numbers of ceroid granules in multiple tissues, as well as increased prevalence of birefringent mineral crystals and haplosporidian-like parasites in the right kidney. The histopathological approaches presented here complement anecdotal field observations of reduced seaweed availability and increased sand incursion at slow-growing sites, while providing an insight into the health of individual abalone and sub-populations. The approaches described here will ultimately help elucidate the drivers behind variable growth performance which, in turn, supports fisheries management decisions and future surveillance programs.

Histopathological changes in the greenshell mussel, Perna canaliculus, in response to chronic thermal stress.

Climate change associated temperature challenges pose a serious threat to the marine environment. Elevations in average sea surface temperatures are occurring and increasing frequency of marine heatwaves resulting in mortalities of organisms are being reported. In recent years, marine farmers have reported summer mass mortality events of the New Zealand Greenshell mussel, Perna canaliculus, during the summer months; however, the etiological agents have yet to be determined. To elucidate the role of thermal stress, adult P. canaliculus were exposed to three chronic temperature treatments: a benign control of 17 °C and stressful elevations of 21 °C and 24 °C. Eight mussels per treatment were collected each month throughout a 14-month challenge period to identify and investigate histopathological differences among P. canaliculus populations exposed to the three temperatures. Histopathology revealed several significant deleterious alterations to tissues associated with temperature and exposure time. Increasing temperature and progression of time resulted in 1) an increase in the number of focal lipofuscin-ceroid aggregations, 2) an increase in focal hemocytosis, 3) an increase in the thickness of the sub-epithelial layer of the intestinal tract and 4) a decreased energy reserve cell (glycogen) coverage in the mantle. Prolonged exposure, irrespective of temperature, impacted gametogenesis, which was effectively arrested. Furthermore, increased levels of the heat shock protein 70 kDa (HSP 70) were seen in gill and gonad from thermally challenged mussels. The occurrence of the parasite Perkinsus olseni at month 5 in the 24 °C treatment, and month 7 at 21 °C was unexpected and may have exacerbated the fore-mentioned tissue conditions. Prolonged exposure to stable thermal conditions therefore appears to impact P. canaliculus, tissues with implications for broodstock captivity. Mussels experiencing elevated, temperatures of 21 and 24 °C demonstrated more rapid pathological signs. This research provides further insight into the complex host-pathogen-environment interactions for P. canaliculus in response to prolonged elevated temperature.

New gregarine species (Apicomplexa) from tunicates show an evolutionary history of host switching and suggest a problem with the systematics of Lankesteria and Lecudina.

Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts.

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.

A novel nuclear marker and development of an ARMS-PCR assay targeting the metallopeptidase 10 (nas 10) locus to identify the species of the Anisakis simplex (s. l.) complex (Nematoda, Anisakidae).

The genus Anisakis represents one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognized in the Anisakis simplex (s. l.) complex: A. pegreffii, A. simplex (s. s.) and A. berlandi. They are widely distributed in populations of their intermediate/paratenic hosts (fish and squids) and definitive hosts (cetaceans). A novel nuclear gene locus, metallopeptidase 10 (nas 10) (451 bp), was sequenced and validated on a total of 219 specimens of the three species of Anisakis, collected in fish and cetacean hosts from allopatric areas included in their ranges of distribution. The specimens of Anisakis were first identified by allozymes and sequence analysis of the mtDNA cox2 and EF1α-1 nDNA. The novel nuclear marker has shown fixed alternative nucleotide positions in the three species, i.e. diagnostic at 100%, permitting the species determination of a large number of specimens analyzed in the present study. In addition, primers to be used for amplification-refractory mutation system (ARMS) PCR of the same gene locus were designed at these nucleotide positions. Thus, direct genotyping determination, by double ARMS, was developed and validated on 219 specimens belonging to the three species. Complete concordance was observed between the tetra-primer ARMS-PCR assays and direct sequencing results obtained for the nas 10 gene locus. The novel nuclear diagnostic marker will be useful in future studies on a multi-locus genotyping approach and also to study possible hybridization and/or introgression events occurring between the three species in sympatric areas.

TITLE: Un nouveau marqueur nucléaire et développement d'un test ARMS-PCR ciblant le locus de la métallopeptidase 10 (nas 10) pour identifier les espèces du complexe Anisakis simplex (s. l.) (Nematoda, Anisakidae). ABSTRACT: Le genre Anisakis représente l'un des groupes de nématodes ascaridoïdes les plus répandus dans l'écosystème marin. Trois taxons étroitement apparentés sont reconnus dans le complexe Anisakis simplex (s. l.) : A. pegreffii, A. simplex (s. s.) et A. berlandi. Ils sont largement répartis dans les populations de leurs hôtes intermédiaires/paraténiques (poissons et calmars) et définitifs (cétacés). Un nouveau locus de gène nucléaire, la métallopeptidase 10 (nas 10) (451 pb), a été séquencé et validé sur un total de 219 spécimens des trois espèces d'Anisakis, collectés chez des hôtes poissons et cétacés de zones allopatriques incluses dans leur aire de répartition. Les échantillons d'Anisakis ont d'abord été identifiés par des allozymes et une analyse des séquences de l'ADNmt cox2 et de l'ADNn EF1α-1. Le nouveau marqueur nucléaire a montré des positions de nucléotides alternatives fixes dans les trois espèces, c'est-à-dire qu'il a permis un diagnostic à 100%, permettant la détermination de l'espèce d'un grand nombre d'échantillons analysés dans la présente étude. De plus, des amorces à utiliser pour la PCR par système de mutation réfractaire à l'amplification (ARMS) du même locus génique ont été conçues à ces positions nucléotidiques. Ainsi, la détermination directe du génotypage, par double ARMS, a été développée et validée sur 219 spécimens appartenant aux trois espèces. Une concordance complète a été observée entre les dosages ARMS PCR tétra-amorces et les résultats de séquençage direct obtenus pour le locus du gène nas 10. Le nouveau marqueur de diagnostic nucléaire sera utile dans les travaux futurs d'une approche de génotypage multi-locus et également pour étudier les éventuels événements d'hybridation et/ou d'introgression se produisant entre les trois espèces dans des zones sympatriques.

Cross-species utility of microsatellite loci for the genetic characterisation of Anisakis berlandi (Nematoda: Anisakidae).

Eight microsatellite loci, recently developed in the species Anisakis pegreffii, were successfully amplified in Anisakis berlandi, sibling species of the A. simplex (s. l.) complex. They were validated on adult specimens (n = 46) of the parasite species, collected from two individuals of the definitive host, the long-finned pilot whale Globicephala melas from New Zealand waters. Among the eight loci scored, one, Anisl 07132, had null alleles in A. berlandi and was thus excluded from the subsequent genetic analysis. Two loci, Anisl 00314 and Anisl 10535, were monomorphic. In addition, as also previously detected in the other species of the A. simplex (s. l.) complex, the Anisl 7 locus was seen to be sex-linked, showing hemizygosity in male specimens. Differential allele frequency distributions of A. berlandi, with respect to those previously observed in A. pegreffii and A. simplex (s. s.), were found at some microsatellite loci. The Anisl 7 locus provided 100% diagnosis between A. berlandi and A. pegreffii, while others resulted in 99% diagnosis between A. berlandi and the other two species. Simple sequence repeat (SSR) loci also allowed us to estimate the genetic differentiation of A. berlandi from A. pegreffii (F st ≈ 0.45, Dc = 0.82) and A. simplex (s. s.) (F st ≈ 0.57, Dc = 0.73). The results suggest that SSRs provide a set of candidate markers for population genetics analysis of A. berlandi, as well as for the investigation, through a multi-locus genotyping approach, of possible patterns of hybridisation/introgression events between A. berlandi and the other two Anisakis species in sympatric conditions.

TITLE: Utilité des loci microsatellites pour la caractérisation génétique interspécifique d'Anisakis berlandi (Nematoda, Anisakidae). ABSTRACT: Huit loci microsatellites, récemment développés chez l'espèce Anisakis pegreffii, ont été amplifiés avec succès chez Anisakis berlandi, espèce sœur du complexe A. simplex (s. l.). Ils ont été validés sur des spécimens adultes (n = 46) de l'espèce, récoltés chez deux individus de l'hôte définitif, le globicéphale commun Globicephala melas, des eaux néo-zélandaises. Parmi les huit loci notés, l'un, Anisl 07132, avait des allèles nuls chez A. berlandi et a donc été exclu de l'analyse génétique ultérieure. Deux loci, Anisl 00314 et Anisl 10535, étaient monomorphes. De plus, comme cela a également été détecté précédemment dans les autres espèces du complexe A. simplex (s. l.), le locus Anisl 7 était lié au sexe, montrant une hémizygosité chez les spécimens mâles. Chez A. berlandi, des distributions de fréquences d'allèles, différentielles par rapport à celles précédemment observées chez A. pegreffii et A. simplex (s. s.), ont été trouvées pour certains loci microsatellites. Le locus Anisl 7 a fourni un diagnostic à 100 % entre A. berlandi et A. pegreffii, tandis que d'autres ont abouti à un diagnostic à 99 % entre A. berlandi et les deux autres espèces. Les loci des SSR ont également permis d'estimer la différenciation génétique d'A. berlandi par rapport à A. pegreffii (F st ≈ 0,45, Dc = 0,82) et A. simplex (s. s.) (F st ≈ 0,57, Dc = 0,73). Les résultats suggèrent que les répétitions de séquences simples (SSR) fournissent un ensemble de marqueurs candidats pour l'analyse génétique des populations d'A. berlandi, ainsi que pour l'investigation, dans une approche de génotypage multilocus, des modèles possibles d'hybridation/introgression entre A. berlandi et les deux autres espèces d'Anisakis dans des conditions sympatriques.

Morphological and molecular diversity of rissoellids (Mollusca, Gastropoda, Heterobranchia) from the Northwest Pacific island of Hokkaido, Japan.

This study deals with four species of marine microgastropods of the family Rissoellidae. Rissoella elatior (Golikov, Gulbin Sirenko, 1987), R. golikovi (Gulbin, 1979), R. japonica n. sp., and Rissoella sp. 1 were collected in different locations around the island of Hokkaido, Japan. Light and scanning electron microscopy (SEM) were used to study the general morphology of the shell and radula, and a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified for 26 specimens. Rissoella elatior is morphologically characterized by a highly asymmetrical radula with a deep notch encircled by 10-13 minute secondary cusps on the left dorsal margin of the central tooth. Rissoella golikovi is characterized by a skeneiform shell and possession of three teeth per row on the radula. Rissoella japonica n. sp. shows five teeth per row on the radula; central tooth higher than wide; lateral and marginal teeth narrow with an outer lateral projection at the base; all teeth presenting numerous small cusps on the cutting edge. Rissoella sp. 1 is distinguished from R. japonica n. sp. in having i) very short oral lobes, ii) a mantle with a large, black patch and whitish blotches inside, and iii) different color patterns associated with the visceral mass. Although Rissoella sp. 1 probably represents an undescribed species, additional specimens are needed to complete its description. This study represents a first molecular approach to the family Rissoellidae. Studies of traditional morphological characters indicated four species, the addition of COI data raised the count to eight potential species, suggesting the occurrence of cryptic species among rissoellids.

PCR test to specifically detect the apicomplexan 'X' (APX) parasite found in flat oysters Ostrea chilensis in New Zealand.

Described here is a polymerase chain reaction (PCR) test to detect the apicomplexan-X (APX) parasite of a flat oyster species, Ostrea chilensis, endemic to New Zealand. The test primers target sequences in the in situ hybridisation probes identified to bind specifically to APX 18S rRNA and amplify a 723 bp DNA product. The test did not amplify 18S rRNA gene sequences of other apicomplexan species, including Toxoplasma gondii, Neospora caninum, Selenidium spp., Cephaloidophorida spp., Lecudina spp. and Thiriotia sp. Of 73 flat oysters identified by histology to be infected with APX at different severities, 69 (95%) tested PCR-positive. Failure to amplify an internal control indicated the presence of PCR inhibitors in the 4 PCR-negative samples. The high analytical sensitivity, specificity and speed of the PCR test should make it a useful tool for detecting APX.

Distribution of Tetrodotoxin in the New Zealand Clam, Paphies australis, Established Using Immunohistochemistry and Liquid Chromatography-Tandem Quadrupole Mass Spectrometry.

Tetrodotoxin (TTX) is one of the most potent neurotoxins known. It was originally thought to only occur in puffer fish but has now been identified in twelve different classes of freshwater and marine organisms, including bivalves. Despite being one of the world’s most studied biotoxins, its origin remains uncertain. There is contradictory evidence regarding the source of TTX and its pathway through food webs. To date, the distribution of TTX has not been examined in bivalves. In the present study, 48 Paphies australis, a TTX-containing clam species endemic to New Zealand, were collected. Thirty clams were dissected, and organs and tissues pooled into five categories (siphons, digestive gland, adductor muscles, and the ‘rest’) and analyzed for TTX using liquid chromatography-mass spectrometry (LC-MS). The micro-distribution of TTX was visualized in the remaining 18 individuals using an immunohistological technique incorporating a TTX-specific monoclonal antibody. The LC-MS analysis revealed that siphons contained the highest concentrations of TTX (mean 403.8 µg/kg). Immunohistochemistry analysis showed TTX in the outer cells of the siphons, but also in the digestive system, foot, and gill tissue. Observing TTX in organs involved in feeding provides initial evidence to support the hypothesis of an exogenous source in P. australis.

Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

Genetic and morphological approaches distinguish the three sibling species of the Anisakis simplex species complex, with a species designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae).

Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacépède, from the northeastern Atlantic waters. No F1 hybrids were detected among the 3 species using the nuclear markers. The phylogenetic inference, obtained by maximum parsimony (MP) analysis of separate nuclear (ITS rDNA region), combined mitochondrial (mtDNA cox2 and rrnS) sequences datasets, and by concatenated analysis obtained at both MP and Bayesian inference (BI) of the sequences datasets at the 3 studied genes, resulted in a similar topology. They were congruent in depicting the existence of the 3 species as distinct phylogenetic lineages, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n. sp. (= A. simplex sp. C) represent a monophyletic group. The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species. Morphological analysis using principal component analysis, and Procrustes analysis, combining morphological and genetic datasets, showed the specimens clustering into 3 well-defined groups. Nomenclatural designation and formal description are given for A. simplex species C: the name Anisakis berlandi n. sp. is proposed. Key morphological diagnostic traits are as follows between A. berlandi n. sp. and A. simplex (s.s.): ventriculus length, tail shape, tail length/total body length ratio, and left spicule length/total body length ratio; between A. berlandi n. sp. and A. pegreffii: ventriculus length and plectane 1 width/plectane 3 width ratio; and between A. simplex (s.s.) and A. pegreffii: ventriculus length, left and right spicule length/total body length ratios, and tail length/total body length ratio. Ecological data pertaining to the geographical ranges and host distribution of the 3 species are updated.

Anisakis nascettii n. sp. (Nematoda: Anisakidae) from beaked whales of the southern hemisphere: morphological description, genetic relationships between congeners and ecological data.

A new anisakid nematode, Anisakis nascettii n. sp., is described from beaked whales Mesoplodon spp. off the coast of New Zealand and South Africa. Morphological and molecular (allozymes and mtDNA cox2 sequence) data were used for diagnostic and identification purposes. Among the 19 allozymes studied, 10 were found to be unique and characteristic for A. nascettii n. sp. Analysis of allozymes demonstrated reproductive isolation from A. ziphidarum Paggi, Nascetti, Webb, Mattiucci, Cianchi & Bullini, 1998 and mtDNA cox2 sequences depict this Anisakis species as a distinct and unique entity. Key morphological diagnostic traits for A. nascettii with respect to the genetically closely related species A. ziphidarum include: spicule length, the spicule/body length ratio, the arrangement of the caudal papillae and the shape of the plectanes of the adult males. Genetic data confirmed that Anisakis sp. A of Pontes et al. (2005), which was partly described by Iglesias et al. (2008), and Anisakis sp. of Valentini et al. (2006) are conspecific with A. nascettii. Both molecular and morphological data indicate that the new species belongs to the 'ziphidarum-group'; however, it is genetically very distinct from A. ziphidarum (D ( Nei ) = 0.69, K2P = 0.09), as well as from all of the previously genetically characterised Anisakis spp. All tree topologies inferred by different methods (MP, NJ and Bayesian) support the finding that A. nascettii n. sp. and A. ziphidarum are sister-species. It is also confirmed that A. nascettii n. sp. is, at the adult stage, a parasite of beaked whales of the genus Mesoplodon, whereas, as a larva, it has been identified from the squid Moroteuthis ingens Smith. Furthermore, Mesoplodon bowdoini Andrews represents a new host record for A. ziphidarum. The parallelism between the clade formed by these two anisakine taxa, i.e. A. ziphidarum and A. nascettii, and that formed by their definitive hosts further supports the hypothesis of host-parasite co-evolutionary relationships, as previously suggested for Anisakis spp. and their cetacean hosts.

Towards cryopreservation of Greenshell mussel (Perna canaliculus) oocytes.

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshelltrade mark mussel (Perna canaliculus), New Zealand's main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2-0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to -10 degrees C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to -10 degrees C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

Genetic relationships among Anisakis species (Nematoda: Anisakidae) inferred from mitochondrial cox2 sequences, and comparison with allozyme data.

The genetic relationships among 9 taxa of Anisakis Dujardin, 1845 (A. simplex (sensu stricto), A. pegreffii, A. simplex C., A. typica, A. ziphidarum, A. physeteris, A. brevispiculata, A. paggiae, and Anisakis sp.) were inferred from sequence analysis (629 bp) of the mitochondrial cox2 gene. Genetic divergence among the considered taxa, estimated by p-distance, ranged from p = 0.055, between sibling species of the A. simplex complex, to p = 0.12, between morphologically differentiated species, i.e., A. ziphidarum and A. typica. The highest level was detected when comparing A. physeteris, A. brevispiculata, and A. paggiae versus A. simplex complex (on average p = 0.13) or versus A. typica (on average p = 0.14). Sequence data from the newly identified Anisakis sp. poorly aligned with other Anisakis species but was most similar to A. ziphidarum (p = 0.08). Phylogenetic analyses based upon Parsimony and Bayesian Inference, as well as phenetic analysis based upon Neighbor-Joining p-distance values, generated similar tree topologies, each well supported at major nodes. All analyses delineated two main claides, the first encompassing A. physeteris, A. brevispiculata, and A. paggiae as a sister group to all the remaining species, and the second comprising the species of the A. simplex complex (A. simplex (s.s.), A. pegreffii and A. simplex C), A. typica, A. ziphidarum, and Anisakis sp. In general, mtDNA-based tree topologies showed high congruence with those generated from nuclear data sets (19 enzyme-loci) and with morphological data delineating adult and larval stages of the Anisakis spp.; however, precise positioning of A. typica and A. ziphidarum remain poorly resolved, though they consistently clustered in the same clade as Anisakis sp. and the A. simplex complex. Comparison of anisakid data with those currently available for their cetacean-definitive hosts suggests parallelism between host and parasite phylogenetic tree topologies.

Evidence for a new species of Anisakis Dujardin, 1845: morphological description and genetic relationships between congeners (Nematoda: Anisakidae).

In the present study, a new biological species of Anisakis Dujardin, 1845, was detected in Kogia breviceps and K. sima from West Atlantic waters (coast of Florida) on the basis of 19 (nuclear) structural genes studied by multilocus allozyme electrophoresis. Fixed allele differences at 11 enzyme loci were found between specimens of both adults and larvae of the new species and the other Anisakis spp. tested. Reproductive isolation from A. brevispiculata Dollfus, 1968 was demonstrated by the lack of hybrid or recombinant genotypes in mixed infections in K. breviceps. Genetic distance of the new species from its closest relative, A. brevispiculata, was D(Nei)=0.79. The new species is morphologically different from the other species which have been genetically characterised and from the other Anisakis retained by Davey (1971) as valid or as species inquirendae: the name of Anisakis paggiae n. sp. is proposed for the new taxon. Anisakis Type II larvae (sensu Berland, 1961) from the European hake Merluccius merluccius in the northeastern Atlantic Ocean (Galician coast) and from the scabbard fish Aphanopus carbo in Central Atlantic waters (off Madeira), were identified as A. paggiae n. sp. Its genetic relationships with respect to the seven species previously characterised (A. simplex (Rudolphi, 1809) sensu stricto), A. pegreffii Campana-Rouget & Biocca, 1955, A. simplex, (A. typica (Diesing, 1860), A. ziphidarum Paggi et al., 1998, A. physeteris Baylis, 1923 and A. brevispiculata) were also inferred. Overall, a low genetic identity was detected at allozyme level between the eight Anisakis species. Interspecific genetic identity ranged from I(Nei)=0.68, between the sibling species of the A. simplex complex, to I(Nei)=0.00 (no alleles shared at the considered loci) when A. physeteris, A. brevispiculata and the new species were compared with the other species of the genus. Concordant topologies were obtained using both UPGMA and NJ tree analyses for the considered species. In both analyses, A. paggiae n. sp. clustered with A. brevispiculata. They also indicated two main clades, the first including A. physeteris, A. brevispiculata and A. paggiae n. sp., the second containing all of the remaining species (i.e. A. simplex (s.s.), A. pegreffii, A. simplex, A. typica and A. ziphidarum). A deep separation between these two main Anisakis clades, also supported by high bootstrap values at the major nodes, was apparent. This is also supported by differences in adult and larval morphology, as well as with respect to their main definitive hosts. A morphological key for distinguishing adult A. paggiae n. sp., A. physeteris and A. brevispiculata is presented. Allozyme markers for the identification of any life-history stage of the Anisakis spp. so far studied, as well as ecological data on their definitive host preferences and geographical distribution, are updated.