Background: Nematodes of the family Anisakidae are parasites of marine organisms, such as fish and sea mammals, pose a threat to humans and cause the disease anisakiasis. In Australia, little has been done to evaluate the risk of exposure for consumers of infected fish to these parasites. The aim of the present study is to partially address this gap by a small-scale survey of five local fish species from southern Australia destined for consumption. Methods: Fish were collected and examined for intestinal worms, including anisakids. The parasite larvae collected were fixed and identified both by morphological/morphometric analysis and by molecular investigation of the PCR-amplified internal transcribed spacer region of nuclear ribosomal DNA. Both datasets were combined and interpreted together with other biological data. Results: Infection rates of anisakid nematodes in the respective fish species ranged between 20 % and 100 %. Larval stages of seven anisakid species of three different genera, namely Anisakis, Contracaecum and Hysterothylacium, were identified. Some species discovered represent molecularly uncharacterized specimens and await unequivocal identification. Conclusion: The data show that Anisakis and related species are prevalent in southern Australia, in some fish hosts in large numbers. Further research will provide a better understanding of the parasite and other factors linked to anisakiasis.

Text Sample: Chapter 2.2, Parasite collection: Most samples were collected from fishes of local fish markets or provided by a fish-food processing plant from Melbourne. Apart from Seriola lalandi, which viscera had been handed over directly, the abdominal cavity of the fish were opened ventrally. Nematodes were collected from the internal organs, including digestive tract, gonads, liver, body cavity and kidney. They were washed extensively in PBS solution immediately after collection. A small section of the middle part (<1 mm) of the worm, which is dispensable for morphologic studies, was cut out with a clean scalpel, transferred into a tube and kept frozen at -80°C. The anterior and posterior ends were transferred to 70 % ethanol for preservation (see Figure 2.3. Dorsal view of Platycephalus bassensis Figure 2.4. Lateral view of Sardinops sagax Figure 2.5. Lateral view of Seriola lalandi). Morphological examination: Prior to watching the specimen under the light microscope, it was cleared in lactophenol to remove any stainings. Characters of systematic importance were measured by an eyepiece micrometer and sketch drawings made using a camera lucida. All measures are given as the arithmetic mean in millimeters unless stated otherwise, followed by the range in parentheses. These parameters include total body length, maximum body width, distance of the nerve ring to the anterior end, length of the esophagus, ventriculus, ventricular appendix, intestinal caecum, and distance of the anus to the posterior end. Special consideration was taken for the morphology of the lips, tail and the position of the excretion porus. Larval stages of anisakid nematodes were classified among different morphotypes”. Genomic DNA extraction: Genomic DNA from the frozen mid sections were isolated according to a standard sodium dodecyl sulphate (SDS)-Proteinase K method. Briefly, samples get transferred into individual 1.5 ml tubes, each containing 500 µl of extraction solution (containing 350 µg/ml Proteinase K in DNA extraction buffer). At 37°C, they were incubated for 18 h. Purification was performed using WizardTM DNA Clean-Up minicolumns (Promega) according to the manufacturer’s protocol. Control DNA samples from the host muscle tissue were also prepared. All purified samples were stored at -20°C until further use.

• Anisakis

• Anisakiasis

• ITS-2

• SSCP

• sequencing

• parasites

• fish species

Albert Eisenbarth, Diplom-Biologe, Biologie-Studium an der Eberhard Karls Universität in Tübingen, Abschluss 2008 als Diplom-Biologe. Derzeit tätig als Wissenschaftliche Hilfskraft im Bereich Parasitologie.