1662 PRIMER NOTES © 2000 Blackwell Science Ltd, Molecular Ecology , 9, 1661–1686 The number of alleles, product size and heterozygosity at each of the eight microsatellite loci are shown in Table 1. All of the examined loci showed a distinct allelic variation ranging from 2 – 8 alleles in the bears examined. Alleles at each locus differed by multiples of two in size. All the polymorphic loci conformed to Hardy–Weinberg expectations except for MSUT-3, which may have null alleles (Table 1). The eight loci showed relatively low allelic variations and low heterozygosities. This may be caused by a small sampling area for this species or by the population being isolated. To elucidate the cause, the genetic diversity of the present population has to be compared with that of other larger populations. To the best of our knowledge, no original microsatellite DNA loci for this species have yet been described, although several sets of microsatellite primers have been developed for other bear species (Paetkau & Strobeck 1994; Paetkau et al . 1995; Taberlet et al . 1997), some of which may be applicable to this species. The present microsatellite loci will become a potent DNA marker to investigate genetic variations in the Asiatic black bear. Acknowledgements We wish to thank T. Kawahara and N. Ohnishi for their kind assist- ance for genetic analyses. We are also indebted to T. Shimada, Y. Segawa, and M. Yabuta for specimen management. References Avise JC (1994) Molecular Markers, Natural History and Evolution . Chapman & Hall. New York. Burke T, Rainey WE, White TJ (1992) Molecular variation and ecological problems. In: Genes in Ecology (eds Berry RJ, Crawford TJ, Hewitt GM), pp. 229 – 254. Blackwell Scientific Publications, Oxford. Hashimoto Y, Yasutake A (1999) Seasonal changes in body weight of female Asiatic black bears under captivity. Mammal Study , 24 , 1– 6. Horino S, Miura S (2000) Population viability analysis of a Japanese black bear population. Population Ecology , 42 , 37–44. Isagi T, Kanazashi T, Suzuki W, Tanaka H, Abe T (1999) Poly- morphic microsatellite DNA markers for Magnolia obovata Thunb. and their utility in related species. Molecular Ecology , 8 , 698–700. Paetkau D, Calvert W, Stirling I, Strobeck C (1995) Microsatellite analysis of population structure in Canadian polar bears. Molecular Ecology , 4 , 347 – 354. Paetkau D, Strobeck C (1994) Microsatellite analysis of genetic variation in black bear populations. Molecular Ecology , 3 , 489–495. Raymond M, Rousset F (1995) genepop (Version 1.2): population genetics software for exact tests and ecumenicism. Journal of Heredity , 86 , 248 – 249. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a Laboratory Manual . 2nd edn. Cold Spring Harbor Laboratory Press, New York. Servheen C (1990) The Status and Conservation of the Bears of the World. International Conference on Bear Research and Management Monograph Series , 2 , 32pp. Taberlet P, Camarra J-J, Grifin S et al. (1997) Noninvasive genetic tracking of the endangered Pyrenean brown bear population. Molecular Ecology , 6 , 869 – 876. The Mammalogical Society of Japan, ed. (1997) Red List of Japanese Mammals . (in Japanese with a list in English) Bun-ichi Sogo Shuppan, Tokyo. 92000 1040 PRIMER NOTES PRIMER NOTES PRIMER NOTES 1000 Graphicraft Limited, Hong Kong Microsatellites in the hermaphroditic snail, Lymnaea truncatula , intermediate host of the liver fluke, Fasciola hepatica S. TROUVÉ,*† L. DEGEN,* C. MEUNIER,‡ C. TIRARD,§ S. HURTREZ-BOUSSÈS,‡ P. DURAND,‡ J. F. GUÉGAN,‡ J. GOUDET* and F. RENAUD‡ * Institut Zoologie et Ecologie Animale, Université, 1015 Lausanne, Switzerland, † Laboratoire Ecologie-Evolution, UMR CNRS 5561 Biogéosciences, 6 Bd Gabriel, Université Bourgogne, 21000 Dijon, France, ‡ Centre d’Etude sur le Polymorphisme des Micro-Organismes, Centre IRD Montpellier, 911 av. Agropolis, 34032 Montpellier Cedex 1, France, § Laboratoire Fonctionnement et Evolution des Systèmes Ecologiques, Université Pierre et Marie Curie, 7 quai St. Bernard, 75252 Paris Cedex 05, France Keywords : DNA markers, genetic variability, heterozygosity, Lymnaeidae, mating system, mollusc Received 20 February 2000; revision accepted 11 May 2000 Correspondence: S. Trouvé. Laboratoire Ecologie-Evolution, UMR CNRS 5561 Biogéosciences, 6 Bd Gabriel, Université Bourgogne, 21000 Dijon, France. Fax: 33 3 80 39 62 31; E-mail: sandrine.trouve@u-bourgogne.fr Host-parasite interactions are strongly affected by differential gene flow in host and parasite populations. In this context, genetic markers are particularly useful to estimate population structure and heterozygosity level associated with infection. The freshwater snail, Lymnaea truncatula (Gastropod, Mollusc), is the main species acting as intermediate host in the life cycle of the liver fluke, Fasciola hepatica (Trematoda, Platyhelminth), which is responsible for important human health and veterinary problems worldwide. The mollusc is hermaphroditic and usually inhabits small temporary ponds and streams. While isoenzymatic markers have already been developed in L. truncatula , a total monomorphism was encountered at 18 enzymatic loci in each of 19 populations originating from France, Portugal, Morocco and Bolivia ( Jabbour-Zahab et al. 1997). Extinction colonization events due to the temporality of the habitat as well as a reproduction through self-fertilization could explain the low level of variability observed. To investigate the role of mating systems and population dynamics in the genetic variability of L. truncatula as well as to analyse population genetic of host–parasite interactions, we developed polymorphic microsatellite markers. A genomic library of 2174 clones was constructed and screened for (CA) 10 , and (GA) 10 repeats using standard hybridization techniques (Estoup et al. 1993). A total of 18 positive clones were sequenced. We selected clones for which appropriate flanking sequence could be defined (i.e. nine loci: Table 1). For amplification of microsatellite loci, primers were designed using Primer 0.5 program (Lincoln & Daly 1991). Each polymerase chain reaction (PCR) consisted of a 10.5- μ L mixture containing 0.076 m m each of dCTP, dTTP,