1. Life cycle studies on Renicola spp. (Digenea: Renicolidae) infecting sea birds: DNA tools provide an update N. Campbell 1, C. Collins 2, M. A. Cross 3, J. C. Chubb 3, C. O. Cunningham 2, K. MacKenzie 1, E. Hatfield 2 1.School of Biological Sciences (Zoology) University of Aberdeen, Aberdeen AB24 2TZ. 2. FRS Marine Laboratory, 375 Victoria Road, Aberdeen, AB11 9DB. 3. School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB. Introduction As part of a multidisciplinary EU funded research project (WESTHER), which aims to identify stocks of Herring (Clupea harengus L.) to the west of the British Isles, the parasite fauna of herring is being studied with the aim of identifying potentially useful biological tags. Results of earlier studies have shown metacercariae of Renicola spp (Fig. 1) may be informative tags. The complete life cycles of many renicolid digeneans are still unknown due to the difficulty of identifying morphologically similar adult stages in kidneys of seabird hosts to species level. One way of solving this problem is to compare DNA from adult and larval parasite stages. DNA sequences obtained from different life stages will be the same irrespective of morphology, and can be used to associate the relatively easily identifiable metacercarial stage with its adult form. Fig 1. Renicolid Cercariae doricha left and Cercariae pythionike metacercarial cysts. Fig. 2. Life Cycle of Renicola spp. which infect herring. Sea Birds Herring Turitella communis Results and Discussion ITS PCR products of 950 bases were obtained from metacercarial and adult Renicola. ITS PCR products of 950 bases were obtained from metacercarial and adult Renicola. 85% similarity was found between the ITS sequences of C. doricha and C. pythionike. 85% similarity was found between the ITS sequences of C. doricha and C. pythionike. Identical ITS sequences were obtained from C. pythionike and adult forms of Renicola from all of the seabird species examined, indicating that C. pythionike is the larval form of Renicola which infect fulmars, puffins and guillemots. Identical ITS sequences were obtained from C. pythionike and adult forms of Renicola from all of the seabird species examined, indicating that C. pythionike is the larval form of Renicola which infect fulmars, puffins and guillemots. Methods Renicola metacercariae were extracted from the abdominal cavity of herring collected in sea-lochs on the west coast of Scotland. Adult Renicola specimens were isolated from kidneys of puffin (Fratercula arctica), fulmar (Fulmarus glacialis) and common guillemot (Uria aalge) (Fig. 3 a, 3 b & 3 c ) collected from an RSPB site on Skye, on the west coast of Scotland. Crude DNA lysates were obtained from Renicola metacercariae and adults. These lysates were used in PCR reactions to amplify the Internal Transcribed Spacer (ITS) region of the parasites’ ribosomal DNA using primers and conditions as described in Cunningham (1997). Fig. 3 a.(left) Puffin, Fratercula arctica. Fig. 3 b (centre) Fulmar, Fulmarus glacialis. Fig. 3 c (right) Guillemot, Uria aalge. The ITS region of the ribosomal DNA is widely used for species identification, as it is robust and displays little geographical variation. The ITS PCR products from larval and adult Renicola were purified and sequenced, and the sequences compared with each other, and to sequence databases. Sequences were obtained from three individuals of C. pythionike and C. doricha from herring, and from three adult Renicola from each seabird species. Three renicolid species, R. glacialis, R. wrighti and R. pseudosloanei have been described from the fulmar, puffin and common guillemot respectively. The identical DNA sequences obtained for adult worms from these seabirds questions the validity of these as different species. R. glacialis and R. wrighti may now have to be considered as junior synonyms of R. pseudosloanei (Wright, 1954), first described from the common guillemot. The identity of the final host for C. doricha remains unknown. Combining DNA and morphological analysis is a useful approach to investigating parasite life-cycles especially where morphologically indistinguishable stages occur. Further analysis of Renicola specimens from fish and bird hosts will help clarify the taxonomy of the genus and improve the efficiency of their use as biological tags for herring. References Cunningham CO. (1997) Species variation within the internal transcribed spacer (ITS) region of Gyrodactylus (Monogenea:Gyrodactylidae) ribosomal RNA genes. J Parasitol. 83(2):215-9. C. pythionikeC. doricha PuffinFulmarGuillemot R. pseudosloanei Renicola spp. This project is funded by the European Union under the 5 th Framework, Contract no. QLRT-2001-01056 Fig. 4 Analysis of ITS sequences reveals puffins, guillemots and fulmars are infected by the same species of Renicola, while the final host of Renicola cercaria doricha remains unknown. R. glacialis R. wrighti R. pseudosloanei