Shokoofeh Shamsi

Genetic and morphological evidences for the existence of sibling species within Contracaecum rudolphii (Hartwich, 1964) (Nematoda: Anisakidae) in Australia

The aims of this study were to examine specimens of Contracaecum rudolphii from Australian hosts, both morphologically and genetically, based on the ITS-1 and ITS-2 sequence data, to provide a re-description for the species and to investigate the genetic make-up of this species in Australia. Morphological examination of adult specimens of C. rudolphii sensu lato Hartwich 1964 from two species of cormorants Phalacrocorax carbo and P. varius in Australia, showed that they can be divided into two distinct morphotypes. Distinction of C. rudolphii sensu lato was supported by characterisation of the ITS-1 and ITS-2 regions of rDNA which showed that they belong to two different genotypes, different from those occuring in the northern hemisphere and studied previously. Base-pair nucleotide differences of 0.9–2.2% and 1.1–6.8% for ITS-1 and ITS-2, respectively, have been detected among different genotypes of C. rudolphii sensu lato. Herein, the two new species have been designated as Contracaecum rudolphii D and Contracaecum rudolphii E.

Redescription and genetic characterization of selected Contracaecum spp. (Nematoda: Anisakidae) from various hosts in Australia

In the present study, five species of Contracaecum Railliet & Henry 1912 (Nematoda: Ascaridida), including Contracaecum bancrofti, Contracaecum microcephalum, Contracaecum variegatum, Contracaecum eudyptulae, and Contracaecum ogmorhini, were redescribed using light and scanning electron microscopy. In addition, in order to elucidate their taxonomic status, first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA of each morphospecies were characterized. Analyses of sequence and morphological data sets suggested that C. bancrofti, infecting the Australian pelican Pelecanus conspicillatus, is a valid species and is distinct from C. micropapillatum reported from pelicans in the northern hemisphere. C. microcephalum from cormorants Phalacrocorax melanoleucos and C. variegatum from the darter Anhinga melanogaster and from P. conspicillatus as well as C. eudyptulae from the little penguin Eudyptula minor were also considered as distinct species, which can be differentiated from one another morphologically based on the lengths of spicules and genetically based on the sequences of ITS-1 and ITS-2. Comparison of sequence data of ITS-1 and ITS-2 for the members of C. ogmorhini sensu lato from pinnipeds with those of previous studies suggested that only ITS-2 can be used for differentiation between C. ogmorhini sensu stricto and Contracaecum margolisi, occurring in the southern and northern hemispheres, respectively. Analyses of the ITS-1 and ITS-2 sequence data of Contracaecum spp. in the present study supported the distinction among species of Contracaecum based on morphological data and were useful in confirming the taxonomic status of individual species in Australia.

Description and genetic characterisation of Hysterothylacium (Nematoda: Raphidascarididae) larvae parasitic in Australian marine fishes.

Nematodes belonging to the genus Hysterothylacium (family Raphidascarididae) infect various species of marine fish in both the larval and adult stages. Humans can be accidentally infected upon eating infected seafood. In spite of their importance, relatively little is known of their occurrence and systematics in Australia. An examination of various species of marine teleosts in Australian waters revealed a high prevalence of Hysterothylacium larval types. In the present study, seven previously undescribed Hysterothylacium larval morphotypes (V to VII and IX to XII) were discovered. In total we found 10 different morphotypes and we genetically characterised nine morphotypes identified. A morphological dichotomous identification key has been established to differentiate these morphotypes. Since some larvae of Hysterothylacium from marine fishes cannot be differentiated morphologically from other nematode larvae, such as Paraheterotyphlum, Heterotyphlum, Iheringascaris and Lapetascaris, the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of these larvae were characterised to confirm their taxonomic status. This genetic characterisation implied that some distinct morphotypes belong to different developmental stages of the same species. In addition, it revealed that some morphotypes can comprise distinct genotypes. No match was found between ITS-1 and ITS-2 sequences obtained from larvae in the present study and those from adults available in the GenBank, highlighting the lack of knowledge on occurrence of adult nematodes infecting Australian fish.

Occurrence and abundance of anisakid nematode larvae in five species of fish from southern Australian waters

The aim of the present study was to conduct, in southern Australian waters, a preliminary epidemiological survey of five commercially significant species of fish (yellow-eye mullet, tiger flathead, sand flathead, pilchard and king fish) for infections with anisakid nematodes larvae using a combined morphological–molecular approach. With the exception of king fish, which was farmed and fed commercial pellets, all other species were infected with at least one species of anisakid nematode, with each individual tiger flathead examined being infected. Five morphotypes, including Anisakis, Contracaecum type I and II and Hysterothylacium type IV and VIII, were defined genetically using mutation scanning and targeted sequencing of the second internal transcribed spacer of nuclear ribosomal DNA. The findings of the present study provide a basis for future investigations of the genetic composition of anisakid populations in a wide range of fish hosts in Australia and for assessing their public health significance.

Mutation scanning-coupled sequencing of nuclear ribosomal DNA spacers as a tool for the specific identification of different Contracaecum (Nematoda Anisakidae)larval types.

In the present study, various morphotypes of Contracaecum (Nematoda) larvae in different developmental stages were described and then genetically characterised using sequence data obtained from the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The alignment of ITS-1 and ITS-2 sequence data for larvae investigated with those from well characterised adults in previous studies showed that fourth-stage Contracaecum larvae from the Australian pelican, Pelecanus conspicillatus, had the same sequence as Contracaecum microcephalum reported from the little pied cormorant, Phalacrocorax melanoleucos. In addition, four different morphotypes of third-stage larvae, including types I to IV, were found in various species of fish. Contracaecum type I larvae were composed of two distict genotypes; those collected from mullet, Mugil cephalus, had the same sequence as C. multipapillatum, and those from an unknown species of hardyhead (family Atherinidae) were C.xa0pyripapillatum. Contracaecum type II larvae from mackerel, Scomber australasicus, had the same sequence as adult C. ogmorhini sensu stricto, found in the Australian and New Zealand fur seals (Arctocephalus spp). Contracaecum type III larvae from flathead, Platycephalus laevigatus, had the same sequence as C. rudolphii D found in the black cormorant, Phalacrocorax carbo. The approach used herein was an effective means of matching incompletely identifiable larval nematodes with reference sequences and provides a basis for exploring the composition of populations of anisakid larvae in fish as well as their ecology, particularly their life cycles and transmission patterns.

Contracaecum pyripapillatum n. sp. (Nematoda: Anisakidae) and a description of C. multipapillatum (von Drasche, 1882) from the Australian pelican, Pelecanus conspicillatus

A new species of nematode, Contracaecum pyripapillatum, is reported from the Australian pelican, Pelecanus conspicillatus. This species resembles Contracaecum multipapillatum which was found in the same host. These two species can be differentiated from one another based on the shape of the preanal papillae, being pyriform in C. pyripapillatum and circular in C. multipapillatum. Genetically, the two species differ in the sequences of first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of ribosomal DNA (rDNA). C. pyripapillatum and C. multipapillatum differed in the ITS-1 (443bp in both species) and ITS-2 (between 231 and 233bp) sequences by 3.4–3.8% and 6.0%, respectively. Based on previous allozyme and mtDNA datasets, genotypes of C. multipapillatum A, B, and C have been reported from Europe and USA. Therefore, C. multipapillatum from Australia has been designated as C. multipapillatum D. A morphological examination of these genotypes is necessary to determine whether they represent distinct species.

Recent advances in our knowledge of Australian anisakid nematodes

Graphical abstract Larval stages of anisakid nematodes (arrows) encysted on the surface of the internal organs in snapper.

Genetic characterisation and taxonomy of species of Anisakis (Nematoda : Anisakidae) parasitic in Australian marine mammals

Abstract. Three species of Anisakis from Australian marine mammals, including Anisakis brevispiculata, A. simplex C and A. pegreffii, are described and characterised genetically on the basis of sequence data for the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear rDNA. Parasite specimens were collected from Delphinus delphis, Tursiops truntatus and Kogia sima in Australia. A. brevispiculata is reported for the first time in Australia. However, analyses of sequence data suggests that A. brevispiculata in Australia is genetically distinct from specimens considered to represent the same species from other parts of the world. Fourth larval and adult stages of A. pegreffii were found in dolphins. Assigning larvae to A. pegreffii was based on the ITS-1 and ITS-2 sequences. A description of these larvae also is provided. Furthermore, fourth-stage larvae of A. simplex C were found in Kogia sima. Alignments of ITS-1 and ITS-2 sequences for members of A. simplex sensu lato revealed that nucleotide differences in ITS-1 can be used to differentiate among members of A. simplex sensu lato. This study reinforces the use of a combined molecular and morphological approach for the specific identification of anisakid nematodes.

Larval anisakid nematodes in teleost fishes from Lizard Island, northern Great Barrier Reef, Australia

A survey was undertaken to characterise larval anisakid nematodes present in teleosts at Lizard Island on the northern Great Barrier Reef. In total, 464 fish were examined from 32 families, 62 genera and 107 species. Anisakid nematodes were found in 46 (9.9%) of the fish examined. Infections in Atherinidae, Lethrinidae, Lutjanidae and Serranidae were moderately prevalent, with the intensities of infection ranging from 1 to 80; whereas in the Sphyraenidae and Scombridae, the prevalence of infection was very high, with intensities ranging from 1 to >375 anisakids. A combined morphological and molecular-phylogenetic approach was employed to identify larval anisakid nematodes to species and/or genotypes. The nematodes examined were identified as Anisakis typica (three genotypes based on molecular characterisation), Terranova Types I (five genotypes) and II (five genotypes) and Hysterothylacium Types IV, V (four genotypes), VI and X. The findings of the present study provide some insights into the distribution of larval anisakid nematodes in coral-reef fishes and a basis for future investigations of anisakid populations in marine fishes.

Genetic variation in the mitochondrial cytochrome c oxidase subunit 1 within Progamotaenia festiva (Cestoda: Anoplocephalidae) from macropodid marsupials.

Genetic variation was examined in the anoplocephalid cestode Progamotaenia festiva, from Australian marsupials, in order to test the hypothesis that P. festiva, is a complex of sibling species and to assess the extent of host switching reported previously based on multilocus enzyme electrophoresis (MEE). Polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) was used for the analysis of sequence variation in the cytochrome c oxidase subunit 1 (cox1) gene among 179 specimens of P. festiva (identified based on morphology and predilection site in the host) from 13 different host species, followed by selective DNA sequencing. Fifty-three distinct sequence types (haplotypes) representing all specimens were defined. Phylogenetic analyses of these sequence data (utilizing maximum parsimony and neighbour-joining methods) revealed 12 distinct clades. Other heterologous species, P. ewersi and P. macropodis, were used as outgroups and the remaining bile-duct inhabiting species, P. diaphana and P. effigia, were included in the analysis for comparative purposes. The latter 2 species were nested within the clades representing P. festiva. Most clades of P. festiva identified were restricted to a single host species; one clade primarily in Macropus robustus was also found in the related host species M. antilopinus in an area of host sympatry; another clade occurring primarily in M. robustus occurred also in additional kangaroo species, M. rufus and M. dorsalis. High levels of genetic divergence, the existence of distinct clades and their occurrence in sympatry provide support for the hypothesis that P. festiva represents a complex of numerous species, most of which, but not all, are host specific. Three distinct clades of cestodes were found within a single host, M. robustus, but there was no evidence of within-host speciation.