West coast slugs: faunal composition, diagnostic characters and future threats

1. West coast slugs: faunal composition, diagnostic characters and future threats Rory Mc Donnell Department of Entomology, University of California, Riverside

2. Coming up! • Background information • What invasive slug families and species occur here? • How do we identify them? • DNA and identification • Conclusions

3. Background Information • Traditionally a repulsive organism • Slug phobias – American Journal of Clinical Hypnosis

4. Slugs as Pests • Direct pests of agriculture and horticulture • Human food = Gastropod food • Vectors of human pathogens • Aesthetic damage

5. External morphology Ocular tentacles Keel Mantle Pneumostome Caudal mucus pore Sensory tentacles Foot fringe

6. Genital morphology Body wall Oviduct Atrium Penis Bursa copulatrix duct Bursa copulatrix Vas deferens Flagellum Hermaphrodite duct

7. West coast slugs! • California – 34 slugs species (17 native) Ariolimax dolichophallus

8. The Invasive Fauna • 17 invasive species on the west coast Predominantly European

9. Family Testacellidae Testacella haliotidea

10. Family Veronicellidae Veronicella cubensis – Cuban Slug

11. Family Milacidae Milax gagates

12. Other Milacidae Tandonia budapestensis Boettgerilla pallens

13. Slug Identification • Breathing pore used to distinguish Arionidae from both Limacidae and Agriolimacidae Arionidae Agriolimacidae Limacidae

14. Agriolimacidae vs Limacidae Limacidae Agriolimacidae

15. Family Limacidae Lehmannia valentiana – Valentia Slug

16. Family Limacidae Limacus flavus – Cellar slug Limax maximus – Leopard slug

17. Family Agriolimacidae Deroceras reticulatum – Gray Garden Slug

18. Family Agriolimacidae Deroceras laeve Deroceras panormitanum Deroceras invadens

19. Deroceras panormitanum Deroceras laeve Deroceras invadens

20. Family Arionidae How to identify an Arion? • Family contains many species complexes • Arion circumscriptus, distinctus, hortensis, intermedius, silvaticus, subfuscus and rufus

21. Family Arionidae Arion intermedius

22. Family Arionidae Arion rufus

23. Arion rufus Arion ater Arion vulgaris (= lusitanicus)

24. Arion subfuscus Arion subfuscus – Dusky Slug

25. Arion fuscus Arion subfuscus

26. Arion hortensis complex • Three species – Arion hortensis, distinctus and owenii Arion hortensis Arion distinctus

27. Arion distinctus Arion hortensis 2mm 2mm • Epiphallus structure – structure associated with outlet of epiphallus in atrium • Backeljau and Van Beeck (1987)

28. Arion fasciatus complex • Arion fasciatus, silvaticus and circumscriptus Arion silvaticus

29. Arion silvaticus Arion circumsciptus Arion fasciatus

30. My e-mail: rorym@ucr.edu

31. Slug Identification • Taxonomically challenging e.g.Arion • External morphological features (e.g. color) are unreliable • Internal morphological structures (e.g. genitalia) can also be unreliable Could we use DNA to design a molecular identification key for slugs?

33. Methods and Materials • DNA extraction using Qiagen Extraction Kit • Amplification of a 655bp fragment of COI using PCR (Polymerase Chain Reaction) • Fragment cleaning and sequencing (GATC, Germany) • Restriction enzymes and gel electrophoresis

34. Restriction enzymes • Break sequences into differently sized fragments based on their composition. • Restriction enzyme X cleaves at GTCA Species A …GCTAGTCATAGATTGCCAAGC... Species B …GCTAGTCATAGAGTCACAAGC… • For larger sequences the post-digestion fragments are visible on a standard agarose gel

35. Molecular Identification Key 1. AccI does not fragment sequence ………………………….…………………………..….2 AccI fragments sequence into at least 2 parts.....…………...…….……………...…..….4 2. EarI cuts sequence into 2 or more parts; largest post-digestion band <600bp ……....3 EarI does not fragment sequence; largest post-digestion band at 706bp ……………………………………………………………..…………………...A. subfuscus 3. Largest EarI post-digestion band >500bp …………...………………...……A. distinctus Largest EarI post-digestion band <500bp ………………...………..….…A. intermedius 4. BstUI cuts sequence into 2 parts ………………..………………..………….A. silvaticus BstUI does not cut sequence; single post-digestion band at 706bp ……….…………..5 5. Largest BfaI post-digestion band <700bp …………..……………………..……. A. rufus Largest BfaI post-digestion band >700bp ……………..……….……….….A. hortensis

36. 1. Enzyme AccI does not fragment sequence……………….………………………...… 2 Enzyme AccI fragments sequence into at least 2 parts.......………………...…..…..4

37. 4. BstUI cuts sequence into 2 bands …………………..……………….......A. silvaticus BstUI does not cut sequence; single post-digestion band at 706bp …………….5

38. 5. Largest BfaI post-digestion band <700 bp………………………………….A. rufus Largest BfaI post-digestion band >700 bp ………………..…..….…. A. hortensis Species is Arion rufus

39. 1. Enzyme AccI does not fragment sequence……………….………………………..… 2 Enzyme AccI fragments sequence into at least 2 parts.......………………...…..…..4

40. 4. BstUI cuts sequence into 2 bands …………………………………….......A. silvaticus BstUI does not cut sequence; single post-digestion band at 706bp …………….5 500bp 100bp Species is Arion silvaticus

41. Conclusions • 17 invasive slug species on west coast • Knowledge of genital morphology is important • Species to be vigilant for: • Any leatherback slug • Arion vulgaris • Tandonia budapestensis • DNA can be used for accurate identifications

42. Acknowledgements • Roy Anderson, Kurt Jordaens and Michal Manas for permission to use images • Marie Curie Outgoing International Fellowship David Yoo Sarinah Simmons