Marine Biology

, Volume 161, Issue 6, pp 1429–1440 | Cite as

Atypical plant–herbivore association of algal food and a kleptoplastic sea slug (Elysia clarki) revealed by DNA barcoding and field surveys

  • M. L. Middlebrooks
  • S. S. Bell
  • N. E. Curtis
  • S. K. Pierce
Original Paper


The identity of food sources and feeding preferences of specialist herbivores have been commonly inferred from spatial associations between consumer and food items. However, such basic information for well-known marine herbivores, sacoglossans (sea slugs), and their algal diets remains disappointingly lacking, especially from field studies. The sacoglossan, Elysia clarki (Pierce et al. in Molluscan Res 26:23–38, 2006), is kleptoplastic and sequesters chloroplasts from algal food to photosynthesize, so DNA identification of sequestered chloroplasts was employed to verify the algal species fed upon by the slug across its geographic range. The molecular information on the algae consumed by E. clarki was combined with field surveys of slugs and algae in slug habitats in the Florida Keys in July and August of 2008 in order to evaluate whether the diet of this herbivore could be predicted based on its spatial association with algae in the field. A considerable mismatch between food availability and kleptoplast identity was recorded. E. clarki commonly occupied areas devoid of potential food and often contained symbiotic plastids from algal species different from those most frequently found in the surveyed habitats. In three of the four study sites, algal species present were poor predictors of slug diet. These findings suggest that the photosynthetic capability of E. clarki may release the slug from the constraint of requiring proximity to its food sources and may allow for the potential lack of spatial coupling between this herbivore and its algal food. This combination of field surveys and DNA barcoding provided critical and previously unavailable information on herbivore feeding in this marine system.


Algal Species Spatial Association Halimeda Online Resource Table Specialist Herbivore 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Financial support for this research was provided by a Lerner Gray Fellowship and a Tharp Graduate Award, USF, to MLM, and a private donor, to SKP, who wishes to remain anonymous. Specimens were collected under permit SAL-11-0616-SR issued to SKP by the State of Florida Fish and Wildlife Conservation Commission. We thank Julie Schwartz for logistical assistance and Peter Stiling and Margaret Hall for comments on a previous version of the manuscript. We also thank the Keys Marine Lab for logistical assistance.

Supplementary material

227_2014_2431_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1096 kb)


  1. Bell TM, Sotka EE (2012) Local adaptation in adult feeding preference and juvenile performance in the generalist herbivore Idotea balthica. Oecologia 170:383–393. doi: 10.1007/s00442-012-2302-3 CrossRefGoogle Scholar
  2. Bernays EA, Funk DJ (1999) Specialists make faster decisions than generalists: experiments with aphids. Proc R Soc B-Biol Sci 266:151–156CrossRefGoogle Scholar
  3. Blankenship LE, Yayanos AA (2005) Universal primers and PCR of gut contents to study marine invertebrate diets. Mol Ecol 14:891–899. doi: 10.1111/j.1365-294X.2005.02448.x CrossRefGoogle Scholar
  4. Bohmann K, Monadjem A, Noer CL, Rasmussen M, Zeale MRK, Clare E, Jones G, Willerslev E, Gilbert MTP (2011) Molecular diet analysis of two African free-tailed bats (Molossidae) using high throughput sequencing. PLoS One 6(6):e21441. doi: 10.1371/journal.pone.0021441 CrossRefGoogle Scholar
  5. Bourlat SJ, Nakano H, Akerman M, Telford MJ, Thorndyke MC, Obst M (2008) Feeding ecology of Xenoturbella bocki (Phylum Xenoturbellida) revealed by genetic barcoding. Mol Ecol Resour 8:18–22. doi: 10.1111/j.1471-8286.2007.01959.x CrossRefGoogle Scholar
  6. Bucklin A, Steinke D, Blanco-Bercial L (2011) DNA barcoding of marine metazoa. In: Carlson CA, Giovannoni SJ (eds) Annu Rev Mar Sci, Vol 3. Annual Reviews, Palo Alto, pp 471–508Google Scholar
  7. Christa G, Wescott L, Schaberle TF, Konig GM, Wagele H (2013) What remains after 2 months of starvation? Analysis of sequestered algae in a photosynthetic slug, Plakobranchus ocellatus (Sacoglossa, Opisthobranchia), by barcoding. Planta 237:559–572. doi: 10.1007/s00425-012-1788-6 CrossRefGoogle Scholar
  8. Clark KB (1994) Ascoglossan (=sacoglossa) mollusks in the Florida Keys: rare marine invertebrates at special risk. Bull Mar Sci 54:900–916Google Scholar
  9. Clark KB, Jensen KR, Stirts HM (1990) Survery for functional kleptoplasty among West Atlantic ascoglossa (=sacoglossa) (Mollusca Opisthobranchia). Veliger 33:339–345Google Scholar
  10. Curtis NE, Massey SE, Pierce SK (2006) The symbiotic chloroplasts in the sacoglossan Elysia clarki are from several algal species. Invert Biol 125:336–345. doi: 10.1111/j.1744-7410.2006.00065.x CrossRefGoogle Scholar
  11. Curtis NE, Pierce SK, Massey SE, Schwartz JA, Maugel TK (2007) Newly metamorphosed Elysia clarki juveniles feed on and sequester chloroplasts from algal species different from those utilized by adult slugs. Mar Biol 150:797–806. doi: 10.1007/s00227-006-0398-x CrossRefGoogle Scholar
  12. Curtis NE, Dawes CJ, Pierce SK (2008) Phylogenetic analysis of the large subunit Rubisco gene supports the exclusion of Avrainvillea and Cladocephalus from the Udoteaceae (Bryopsidales, Chlorophyta). J Phycol 44:761–767. doi: 10.1111/j.1529-8817.2008.00519.x CrossRefGoogle Scholar
  13. Curtis NE, Schwartz JA, Pierce SK (2010) Ultrastructure of sequestered chloroplasts in sacoglossan gastropods with differing abilities for plastid uptake and maintenance. Invert Biol 129:297–308. doi: 10.1111/j.1744-7410.2010.00206.x CrossRefGoogle Scholar
  14. Duffy JE, Hay ME (1994) Herbivore resistance to seaweed chemical defense—the roles of mobility and predation risk. Ecology 75:1304–1319. doi: 10.2307/1937456 CrossRefGoogle Scholar
  15. Egan SP, Funk DJ (2006) Individual advantages to ecological specialization: insights on cognitive constraints from three conspecific taxa. Proc R Soc B-Biol Sci 273:843–848. doi: 10.1098/rspb.2005.3382 CrossRefGoogle Scholar
  16. Evertsen J, Johnsen G (2009) In vivo and in vitro differences in chloroplast functionality in the two north Atlantic sacoglossans (Gastropoda, Opisthobranchia) Placida dendritica and Elysia viridis. Mar Biol 156:847–859. doi: 10.1007/s00227-009-1128-y CrossRefGoogle Scholar
  17. Gallop A, Bartrop J, Smith D (1980) The biology of chloroplasts acquisition by Elysia viridis. Proc R Soc Lond (B) 207:335–349CrossRefGoogle Scholar
  18. Garcia-Robledo C, Erickson DL, Staines CL, Erwin TL, Kress WJ (2013) Tropical plant-herbivore networks: reconstructing species interactions using DNA barcodes. PLoS One 8(1):e52967. doi: 10.1371/journal.pone.0052967 CrossRefGoogle Scholar
  19. Gimènez-Casalduero F, Muniain C (2008) The role of kleptoplasts in the survival rates of Elysia timida (Risso, 1818): (Sacoglossa : Opisthobranchia) during periods of food shortage. J Exp Mar Biol Ecol 357:181–187. doi: 10.1016/j.jembe.2008.01.020 CrossRefGoogle Scholar
  20. Händeler K, Wägele H (2007) Preliminary study on molecular phylogeny of Sacoglossa and a compilation of their food organisms. Bonn Zool Beitr 55:231–254Google Scholar
  21. Händeler K, Wägele H, Wahrmund U, Rudinger M, Knoop V (2010) Slugs’ last meals: molecular identification of sequestered chloroplasts from different algal origins in Sacoglossa (Opisthobranchia, Gastropoda). Mol Ecol Resour 10:968–978. doi: 10.1111/j.1755-0998.2010.02853.x CrossRefGoogle Scholar
  22. Hay ME, Duffy JE, Paul VJ, Renaud PE, Fenical W (1990) Specialist herbivores reduce their susceptibility to predation by feeding on the chemically defended seaweed Avrainvillea longicaulis. Limnol Oceanogr 35:1734–1743CrossRefGoogle Scholar
  23. Jensen KR (1980) A review of sacoglossan diets with comparative notes on radular and buccal anatomy. Malacol Rev 13:55–78Google Scholar
  24. Jensen KR (1994) Behavioral adaptations and diet specificity of sacoglossan opisthobranchs. Ethol Ecol Evol 6:87–101CrossRefGoogle Scholar
  25. Jurado-Rivera JA, Vogler AP, Reid CAM, Petitpierre E, Gomez-Zurita J (2009) DNA barcoding insect-host plant associations. Proc R Soc B 276:639–648. doi: 10.1098/rspb.2008.1264 CrossRefGoogle Scholar
  26. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefGoogle Scholar
  27. Maeda T, Hirose E, Chikaraishi Y, Kawato M, Takishita K, Yoshida T, Verbruggen H, Tanaka J, Shimamura S, Takaki Y, Tsuchiya M, Iwai K, Maruyama T (2012) Algivore or Phototroph? Plakobranchus ocellatus (Gastropoda) continuously acquires kleptoplasts and nutrition from multiple algal species in nature. PLoS One 7(7):e42024. doi: 10.1371/journal.pone.0042024 CrossRefGoogle Scholar
  28. Marques LV, Villaca R, Pereira RC (2006) Susceptibility of macroalgae to herbivorous fishes at Rocas Atoll, Brazil. Bot Mar 49:379–385. doi: 10.1515/bot.2006.049 Google Scholar
  29. Middlebrooks ML, Pierce SK, Bell SS (2011) Foraging behavior under starvation conditions is altered via photosynthesis by the marine gastropod, Elysia clarki. PLoS One 6(7):e22162. doi: 10.1371/journal.pone.0022162 CrossRefGoogle Scholar
  30. Middlebrooks ML, Bell SS, Pierce SK (2012) The kleptoplastic sea slug Elysia clarki prolongs photosynthesis by synthesizing chlorophyll a and b. Symbiosis 57:127–132CrossRefGoogle Scholar
  31. Miller MA, Muller GC, Kravchenko VD, Junnila A, Vernon KK, Matheson CD, Hausmann A (2006) DNA-based identification of Lepidoptera larvae and plant meals from their gut contents. Russ Entomol J 15:427–432Google Scholar
  32. Pearre SJ (1982) Estimating prey preference by predators: uses of various indices, and proposal of another based on χ2. Can J Fish Aqua Sci 39:914–923CrossRefGoogle Scholar
  33. Pennings SC, Paul VJ (1993) Secondary chemistry does not limit dietary range of the specialist sea hare Stylocheilus-longicauda (Quoy-et-Gaimard 1824). J Exp Mar Biol Ecol 174:97–113. doi: 10.1016/0022-0981(93)90253-k CrossRefGoogle Scholar
  34. Pennings SC, Nadeau MT, Paul VJ (1993) Selectivity and growth of the generalist herbivore Dolabella auricularia feeding upon complementary resources. Ecology 74:879–890. doi: 10.2307/1940813 CrossRefGoogle Scholar
  35. Pierce SK, Curtis NE (2012) Cell biology of the chloroplast symbiosis in sacoglossan sea slugs. Int Rev Cell Mol Biol 293:123–148CrossRefGoogle Scholar
  36. Pierce SK, Curtis NE, Massey SE, Bass AL, Karl SA, Finney CM (2006) A morphological and molecular comparison between Elysia crispata and a new species of kleptoplastic sacoglossan sea slug (Gastropoda: Opisthobranchia) from the Florida Keys, USA. Molluscan Res 26:23–38Google Scholar
  37. Pierce SK, Curtis NE, Schwartz JA (2009) Chlorophyll a synthesis by an animal using transferred algal nuclear genes. Symbiosis 49:121–131. doi: 10.1007/s13199-009-0044-8 CrossRefGoogle Scholar
  38. Poore AGB, Hill NA, Sotka EE (2008) Phylogenetic and geographic variation in host breadth and composition by herbivorous amphipods in the family ampithoidae. Evolution 62:21–38. doi: 10.1111/j.1558-5646.2007.00261.x Google Scholar
  39. Raye G, Miquel C, Coissac E, Redjadj C, Loison A, Taberlet P (2011) New insights on diet variability revealed by DNA barcoding and high-throughput pyrosequencing: chamois diet in autumn as a case study. Ecol Res 26:265–276. doi: 10.1007/s11284-010-0780-5 CrossRefGoogle Scholar
  40. Rogers CN, De Nys R, Steinberg PD (2000) Predation on juvenile Aplysia parvula and other small anaspidean, ascoglossan, and nudibranch gastropods by pycnogonids. Veliger 43:330–337Google Scholar
  41. Root RB (1973) Organization of a plant-arthropod association in simple and diverse habitats—fauna of collards (Brassica oleracea). Ecol Monogr 43:95–120. doi: 10.2307/1942161 CrossRefGoogle Scholar
  42. Sheppard SK, Harwood JD (2005) Advances in molecular ecology: tracking trophic links through predator-prey food-webs. Funct Ecol 19:751–762. doi: 10.1111/j.1365-2435.2005.01041.x CrossRefGoogle Scholar
  43. Sørensen T (1948) A method of establishing groups of equal amplitude in plant sociology based on similarity of species and its application to analyses of the vegetation on Danish commons. Biol Skr 5:1–34Google Scholar
  44. Sotka EE (2005) Local adaptation in host use among marine invertebrates. Ecol Lett 8:448–459. doi: 10.1111/j.1461-0248.2004.00719.x CrossRefGoogle Scholar
  45. Sotka EE (2007) Restricted host use by the herbivorous amphipod Peramphithoe tea is motivated by food quality and abiotic refuge. Mar Biol 151:1831–1838. doi: 10.1007/s00227-007-0612-5 CrossRefGoogle Scholar
  46. Sotka EE, Hay ME, Thomas JD (1999) Host-plant specialization by a non-herbivorous amphipod: advantages for the amphipod and costs for the seaweed. Oecologia 118:471–482. doi: 10.1007/s004420050750 CrossRefGoogle Scholar
  47. Steneck RS, Hacker SD, Dethier MN (1991) Mechanisms of competitive dominance between crustose coralline algae—an herbivore-mediated competitive reversal. Ecology 72:938–950. doi: 10.2307/1940595 CrossRefGoogle Scholar
  48. Symondson WOC (2002) Molecular identification of prey in predator diets. Mol Ecol 11:627–641. doi: 10.1046/j.1365-294X.2002.01471.x CrossRefGoogle Scholar
  49. Tahvanai JO, Root RB (1972) Influence of vegetational diversity on population ecology of a specialized herbivore, Phyllotreta cruciferae (Coleoptera: chrysomelidae). Oecologia 10:321–346. doi: 10.1007/bf00345736 CrossRefGoogle Scholar
  50. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  51. Tollit DJ, Schulze AD, Trites AW, Olesiuk PF, Crockford SJ, Gelatt TS, Ream RR, Miller KM (2009) Development and application of DNA techniques for validating and improving pinniped diet estimates. Ecol Appl 19:889–905. doi: 10.1890/07-1701.1 CrossRefGoogle Scholar
  52. Tosh CR, Krause J, Ruxton GD (2009) Theoretical predictions strongly support decision accuracy as a major driver of ecological specialization. Proc Natl Acad Sci USA 106:5698–5702. doi: 10.1073/pnas.0807247106 CrossRefGoogle Scholar
  53. Trench RK, Ohlhorst S (1976) Stability of chloroplasts from siphonaceous algae in symbiosis with sacoglossan mollusks. New Phytol 76:99–109CrossRefGoogle Scholar
  54. Trench RK, Greene RW, Bystrom BG (1969) Chloroplasts as functional organelles in animal tissues. J Cell Biol 42:404–417CrossRefGoogle Scholar
  55. Trowbridge CD (1991) Diet specialization limits herbivorous sea slugs capacity to switch among food species. Ecology 72:1880–1888CrossRefGoogle Scholar
  56. Trowbridge CD (1992) Mesoherbivory: the ascoglossan sea slug Placida dendritica may contribute to the restricted distribution of its algal host. Mar Ecol Prog Ser 83:207–220CrossRefGoogle Scholar
  57. Trowbridge CD (1998) Stenophagous, herbivorous sea slugs attack desiccation-prone, green algal hosts (Codium spp.): indirect evidence of prey-stress models (PSMs)? J Exp Mar Biol Ecol 230:31–53CrossRefGoogle Scholar
  58. Trowbridge CD (2002) Local elimination of Codium fragile ssp. tomentosoides: indirect evidence of sacoglossan herbivory? J Mar Biol Assoc UK 82:1029–1030CrossRefGoogle Scholar
  59. Trowbridge CD (2004) Emerging associations on marine rocky shores: specialist herbivores on introduced macroalgae. J Anim Ecol 73:294–308CrossRefGoogle Scholar
  60. Trowbridge CD, Todd CD (2001) Host-plant change in marine specialist herbivores: ascoglossan sea slugs on introduced macroalgae. Ecol Monogr 71:219–243CrossRefGoogle Scholar
  61. Warner RR (1997) Evolutionary ecology: how to reconcile pelagic dispersal with local adaptation. Coral Reef 16:S115–S120. doi: 10.1007/s003380050247 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • M. L. Middlebrooks
    • 1
    • 2
  • S. S. Bell
    • 1
  • N. E. Curtis
    • 1
    • 3
  • S. K. Pierce
    • 1
  1. 1.Department of Integrative BiologyUniversity of South FloridaTampaUSA
  2. 2.Department of BiologyUniversity of TampaTampaUSA
  3. 3.Department of BiologyRollins CollegeWinter ParkUSA

Personalised recommendations