Advertisement

Marine Biology

, Volume 161, Issue 2, pp 261–273 | Cite as

Genotypic variation in the parasitic dinoflagellate Hematodinium perezi along the Delmarva Peninsula, Virginia

  • Katrina M. Pagenkopp Lohan
  • Jan R. McDowell
  • Jeffrey D. Shields
  • Kimberly S. ReeceEmail author
Original Paper

Abstract

Hematodinium perezi (genotype III) is a parasitic dinoflagellate that infects blue crabs along the eastern seaboard and Gulf of Mexico, USA. In order to examine the intra-specific genetic variation of this parasite, eleven microsatellite markers from H. perezi (III) were amplified from 227 infected blue crabs collected during 2008–2009 from six sites in Virginia. Simultaneous infections with multiple genetic types in a single-host individual were common and observed in 42 % of the samples. The remaining 58 % of samples had a single allele per locus at all eight polymorphic loci suggesting that the life history stages of the parasite in the host hemolymph are likely haploid. The composition and distribution of multi-locus genotypes (MLG) from samples with infections of a single genetic type indicated high genotypic variation along the Delmarva Peninsula, Virginia, with no evidence of population structure. The lack of linkage disequilibrium combined with the large number of unique MLGs (84 %) is strong evidence for recombination in the life cycle, but the sexual stages remain undetermined. This is the first evidence of ploidy level, infections by multiple genetic types in an individual host animal, high levels of genotypic variation, and sexual reproduction for any species of Hematodinium.

Keywords

Microsatellite Locus Dinoflagellate Sexual Reproduction Collection Site Blue Crab 
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.

Notes

Acknowledgments

We are grateful to those who helped with the sample collection including Dr. Caiwen Li, Dr. Tom Dolan, Kersten Wheeler, Anna Coffey, Peter Coffey, Christopher Magel, Kelly Delaney, Bhae-Jin Peemoeller, and David Gibbs. Drs. Hamish Small and Jessica Moss Small helped with sample collection and discussion. We thank Sean Fate, Alan Birch, and Edward Smith of the VIMS Eastern Shore Laboratory for their able boat handling and critical help in the field. Gail Scott, Alanna MacIntyre, and Annie Dershem assisted with the molecular laboratory work. Drs. David Gauthier, Allen Place, Emmett Duffy, and three anonymous reviewers offered comments on early versions of this manuscript. This project was funded by Evolution and Ecology of Infectious Diseases Program Grant, National Science Foundation (OCE BE-UF #0723662). This manuscript is contribution number 3315 of the Virginia Institute of Marine Science, College of William & Mary.

References

  1. Ajzenberg D, Banuls AL, Tibayrenc M, Darde ML (2002) Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. Int J Parasitol 32:27–38CrossRefGoogle Scholar
  2. Alpermann TJ, Beszteri B, John U, Tillmann U, Cembella AD (2009) Implications of life-history transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense. Mol Ecol 18:2122–2133CrossRefGoogle Scholar
  3. Alpermann TJ, Tillmann U, Beszteri B, Cembella AD, John U (2010) Phenotypic variation and genotypic diversity in a planktonic population of the toxigenic marine dinoflagellate Alexandrium tamarense (Dinophyceae). J Phycol 46:18–32CrossRefGoogle Scholar
  4. Anderson TJC, Haubold B, Williams JT, Estrada-Franco JG, Richardson L, Mollinedo R, Bockarie M, Mokili J, Mharakurwa S, French N, Whitworth J, Velez ID, Brockman AH, Nosten F, Ferreira MU, Day KP (2000) Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol 17:1467–1482CrossRefGoogle Scholar
  5. Audemard C, Reece KS, Burreson EM (2004) Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters. Appl Environ Microbiol 70:6611–6618CrossRefGoogle Scholar
  6. Babiker HA, Walliker D (1997) Current views on the population structure of Plasmodium falciparum: implications for control. Parasitol Today 13:262–267CrossRefGoogle Scholar
  7. Balmer O, Stearns SC, Schotzau A, Brun R (2009) Intraspecific competition between co-infecting parasite strains enhances host survival in African trypanosomes. Ecology 90:3367–3378CrossRefGoogle Scholar
  8. Belkhir K, Borsa P, Chikhi L, Raufaste N, Catch F (1996–2002) GENETIX 4.04, software under Windows TM for the genetics of the populations. Laboratory Genome, Populations, Interactions, CNRS UMR 5000, University of Montpellier II, Montpellier, France. http://kimura.univ-montp2.fr/genetix/. Accessed 6 May 2009
  9. Brown SP, Hochberg ME, Grenfell BT (2002) Does multiple infection select for raised virulence? Trends Microbiol 10:401–405CrossRefGoogle Scholar
  10. Colwell RK (2011) EstimateS, Version 8.2: statistical estimation of species richness and shared species from samples. http://viceroy.eeb.uconn.edu/EstimateS/
  11. Doerder FP, Arslanyolu M, Saad Y, Kaczmarek M, Mendoza M, Mita B (1996) Ecological genetics of Tetrahymena thermophila: mating types, i-antigens, multiple alleles, and epistasis. J Eukaryot Microbiol 43:95–100CrossRefGoogle Scholar
  12. Eaton WD, Love DC, Botelho C, Meyers TR, Imamura K, Koeneman T (1991) Preliminary results on the seasonality and life cycle of the parasitic dinoflagellate causing Bitter Crab disease in Alaskan Tanner crabs (Chionoecetes bairdi). J Invertebr Pathol 57:426–434CrossRefGoogle Scholar
  13. Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5:435–445CrossRefGoogle Scholar
  14. Excoffier LGL, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50Google Scholar
  15. Frischer ME, Lee RF, Sheppard MA, Mauer A, Rambow F, Neumann M, Brofft JE, Wizenmann T, Danforth JM (2006) Evidence for a free-living stage of the blue crab parasitic dinoflagellate, Hematodinium sp. Harmful Algae 5:548–557CrossRefGoogle Scholar
  16. Giraud T, Enjalbert J, Fournier E, Delmotte F, Dutech C (2008a) Population genetics of fungal diseases of plants. Parasite 15:449–454CrossRefGoogle Scholar
  17. Giraud T, Yockteng R, López-Villavicencio M, Refrégier G, Hood ME (2008b) Mating system of the Anther Smut Fungus Microbotryum violaceum: selfing under heterothallism. Eukaryot Cell 7:765–775CrossRefGoogle Scholar
  18. Gruebl T, Frischer ME, Sheppard M, Neumann M, Maurer AN, Lee RF (2002) Development of an 18 s rRNA gene-targeted PCR-based diagnostic for the blue crab parasite Hematodinium sp. Dis Aquat Org 49:61–70CrossRefGoogle Scholar
  19. Hanif A, Bowers HA, Mcintosh D, Dyson WE, Pitula JS, Jagus R, Schott E (2011) Using PCR methodology to search for an environmental presence of Hematodinium sp., a lethal parasite of the blue crab Callinectes sapidus. Abstract. National Shellfisheries Association, BaltimoreGoogle Scholar
  20. Howells EJ, van Oppen MJH, Willis BL (2009) High genetic differentiation and cross-shelf patterns of genetic diversity among Great Barrier Reef populations of Symbiodinium. Coral Reefs 28:215–225CrossRefGoogle Scholar
  21. Kirk NL, Andras JP, Harvell CD, Santos SR, Coffroth MA (2009) Population structure of Symbiodinium sp. associated with the common sea fan, Gorgonia ventalina, in the Florida Keys across distance, depth, and time. Mar Biol 156:1609–1623CrossRefGoogle Scholar
  22. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefGoogle Scholar
  23. Li YC, Korol AB, Fahima T, Beiles A, Nevo E (2002) Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Mol Ecol 11:2453–2454CrossRefGoogle Scholar
  24. Li C, Shields JD, Miller TL, Small HJ, Pagenkopp KM, Reece KS (2010) Detection and quantification of the free-living stage of the parasitic dinoflagellate Hematodinium sp. in laboratory and environmental samples. Harmful Algae 9:515–521CrossRefGoogle Scholar
  25. Li C, Miller TL, Small HJ, Shields JD (2011) In vitro culture and developmental cycle of the parasitic dinoflagellate Hematodinium sp. from the blue crab Callinectes sapdius. Parasitol 138:1924–1934CrossRefGoogle Scholar
  26. Ligges U, Mächler M (2003) Scatterplot3d- an R package for visualizing multivariate data. J Stat Softw 8:1–20Google Scholar
  27. Lowe CD, Montagnes DJS, Martin LE, Watts PC (2010) High genetic diversity and fine-scale spatial structure in the marine flagellate Oxyrrhis marina (Dinophyceae) uncovered by microsatellite loci. PLoS ONE. doi: 10.1371/journal.pone.0015557 Google Scholar
  28. Magalon H, Baudry E, Huste A, Adjeroud M, Veuille M (2006) High genetic diversity of the symbiotic dinoflagellates in the coral Pocillopora meandrina from the South Pacific. Mar Biol 148:913–922CrossRefGoogle Scholar
  29. Masseret E, Grzebyk D, Nagai S, Genovesi B, Lasserre B, Laabir M, Collos Y, Vaquer A, Berrebi P (2009) Unexpected genetic diversity among and within populations of the toxic dinoflagellate Alexandrium catenella as revealed by nuclear microsatellite markers. Appl Environ Microbiol 75:2037–2045CrossRefGoogle Scholar
  30. Maynard Smith J, Smith NH, O’Rourke M, Spratt BG (1993) How clonal are bacteria? Proc Natl Acad Sci 90:4384–4388CrossRefGoogle Scholar
  31. McCauley LAR, Erdner DL, Nagai S, Richlen ML, Anderson DM (2009) Biogeographic analysis of the globally distributed harmful algal bloom species Alexandrium minutum (Dinophyceae) based on rRNA gene sequences and microsatellite markers. J Phycol 45:454–463CrossRefGoogle Scholar
  32. Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA- coding regions. Gene 71:491–499CrossRefGoogle Scholar
  33. Meirmans PG, Van Tienderen PH (2004) GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol Ecol Notes 4:792–794CrossRefGoogle Scholar
  34. Messick GA (1994) Hematodinium perezi infections in adult and juvenile blue crabs Callinectes sapidus from coastal bays of Maryland and Virginia, USA. Dis Aquat Org 19:77–82CrossRefGoogle Scholar
  35. Messick GA, Shields JD (2000) Epizootiology of the parasitic dinoflagellate Hematodinium sp. in the American blue crab Callinectes sapidus. Dis Aquat Org 43:139–152CrossRefGoogle Scholar
  36. Mosquera J, Adler FR (1998) Evolution of virulence: a unified framework for coinfection and superinfection. J Theor Biol 195:293–313CrossRefGoogle Scholar
  37. Moss JA, Xiao J, Dungan CF, Reece KS (2008) Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. parasite in oysters of southern China. J Eukaryot Microbiol 55:117–130CrossRefGoogle Scholar
  38. Nagai S, Lian C, Yamaguchi S, Hamaguchi M, Matsuyama Y, Itakura S, Shimada H, Kaga S, Yamauchi H, Sonda Y, Nishikawa T, Kim CH, Hogetsu T (2007) Microsatellite markers reveal population genetic structure of the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) in Japanese coastal waters. J Phycol 43:43–54CrossRefGoogle Scholar
  39. Nagai S, Nishitani G, Sakamoto S, Sugaya T, Lee CK, Kim CH, Itakura S, Yamaguchi M (2009) Genetic structuring and transfer of marine dinoflagellate Cochlodinium polykrikoides in Japanese and Korean coastal waters revealed by microsatellites. Mol Ecol 18:2337–2352CrossRefGoogle Scholar
  40. Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genet 89:583–590Google Scholar
  41. Newman MW, Johnson CA (1975) A disease of blue crabs (Callinectes sapidus) caused by a parasitic dinoflagellate, Hematodinium sp. J Parasitol 63:554–557CrossRefGoogle Scholar
  42. Oliveira RP, Broude NE, Macedo AM, Cantor CR, Smith CL, Pena SDJ (1998) Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci 95:3776–3780CrossRefGoogle Scholar
  43. Pagenkopp Lohan KM, Reece KS, Miller TL, Wheeler KN, Small HJ, Shields JD (2012a) The role of alternate hosts in the ecology and life history of Hematodinium sp., a parasitic dinoflagellate of the blue crab (Callinectes sapidus). J Parasitol 98:73–84CrossRefGoogle Scholar
  44. Pagenkopp Lohan KM, McDowell JR, Shields JD, Xiao J, Miller TL, Reece KS (2012b) Isolation and characterization of microsatellite loci for the parasitic dinoflagellate, Hematodinium perezi genotype III, a parasite of Callinectes sapidus. Mol Ecol Resour 12:570–572CrossRefGoogle Scholar
  45. Pagenkopp Lohan KM, Shields JD, Place AR, Small HJ, Reece KS (2013) Conservation in the first internal transcribed spacer (ITS1) region of Hematodinium perezi genotype III from Callinectes sapidus. Dis Aquat Org 13:65–76CrossRefGoogle Scholar
  46. Pitula JS, Dyson WD, Bakht HB, Njoku I, Chen F (2012) Temporal distribution of genetically homogenous ‘free-living’ Hematodinium sp. in a Delmarva coastal ecosystem. Aquat Biosyst 8:16CrossRefGoogle Scholar
  47. R Core Team (2012) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. ISBN: 3-900051-07-0. http://www.R-project.org/
  48. Raymond M, Rousset F (1995) GENEPOP (version 2.1): population genetics software for exact test and ecumenicism. J Hered 86:248–249Google Scholar
  49. Razakandrainibe FG, Durand P, Koella JC, Meeus TD, Rousset F, Ayala FJ, Renaud F (2002) “Clonal” population structure of the malaria agent Plasmodium falciparum in high-infection regions. Proc Natl Acad Sci 102:17388–17393CrossRefGoogle Scholar
  50. Reece KS, Bushek D, Hudson KL, Graves JE (2001) Geographic distribution of Perkinsus marinus genetic strains along the Atlantic and Gulf coasts of the USA. Mar Biol 139:1047–1055CrossRefGoogle Scholar
  51. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225CrossRefGoogle Scholar
  52. Santos SR, Coffroth MA (2003) Molecular genetic evidence that dinoflagellates belonging to the genus Symbiodinium Freudenthal are haploid. Biol Bull 204:10–20CrossRefGoogle Scholar
  53. Santos SR, Gutierrez-Rodriguez C, Lasker HR, Coffroth MA (2003) Symbiodinium sp. associations in the gorgonian Pseudopterogorgia elisabethae in the Bahamas: high levels of genetic variability and population structure in symbiotic dinoflagellates. Mar Biol 143:111–120CrossRefGoogle Scholar
  54. Shields JD, Squyars CM (2000) Mortality and hematology of blue crabs, Callinectes sapidus, experimentally infected with the parasitic dinoflagellate Hematodinium perezi. Fish Bull 98:139–152Google Scholar
  55. Small HJ, Shields JD, Hudson KL, Reece KS (2007) Molecular detection of Hematodinium sp. infecting the blue crab, Callinectes sapidus. J Shellfish Res 26:131–139CrossRefGoogle Scholar
  56. Small HJ, Shields J, Reece K, Bateman K, Stentiford G (2012) Morphological and molecular characterization of Hematodinium perezi (Dinophyceae: Syndiniales), a dinoflagellate parasite of the harbour crab, Liocarcinus depurator. J Eukaryot Microbiol 59:54–66CrossRefGoogle Scholar
  57. Stentiford GD, Shields JD (2005) A review of the parasitic dinoflagellates Hematodinium species and Hematodinium-like infections in marine crustaceans. Dis Aquat Org 66:47–70CrossRefGoogle Scholar
  58. Taylor F (1990) Phylum Dinoflagellata. In: Margulis L, Corliss J, Melkonian M, Chapman D (eds) Handbook of protoctista. Jones and Bartlett Publishers, Sudbury, pp 419–437Google Scholar
  59. Thompson PC, Rosenthal BM, Hare MP (2011) An evolutionary legacy of sex and clonal reproduction in the protistan oyster parasite Perkinsus marinus. Infect Genet Evol 11:598–609CrossRefGoogle Scholar
  60. Thornhill DJ, Xiang Y, Fitt WK, Santos SR (2009) Reef endemism, host specificity, and temporal stability in populations of symbiotic dinoflagellates from two ecologically dominant Caribbean corals. PLoS ONE. doi: 10.1371/journal.pone.0006262 Google Scholar
  61. Tibayrenc M, Ayala FJ (2002) The clonal theory of parasitic protozoa: 12 years on. Trends Parasitol 18:405–410CrossRefGoogle Scholar
  62. Tibayrenc M, Kjellberg F, Ayala FJ (1990) A clonal theor of parasitic protozoa: the population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas, and Trypanosoma and their medical and taxonomical consequences. Proc Natl Acad Sci 87:2414–2418CrossRefGoogle Scholar
  63. Van Baalen M, Sabelis MW (1995) The dynamics of multiple infection and the evolution of virulence. Am Nat 146:881–910CrossRefGoogle Scholar
  64. Vardo-Zalik AM, Schall JJ (2008) Clonal diversity within infections and the virulence of a malaria parasite, Plasmodium mexicanum. Parasitology 135:1363–1372CrossRefGoogle Scholar
  65. Vardo-Zalik AM, Ford AF, Schall JJ (2009) Detecting number of clones, and their relative abundance, of a malaria parasite (Plasmodium mexicanum) infecting its vertebrate host. Parasitol Res 105:209–215CrossRefGoogle Scholar
  66. Vilas R, Cao A, Pardo BG, Fernández S, Villalba A, Martínez P (2011) Very low microsatellite polymorphism and large heterozygote deficits suggest founder effects and cryptic structure in the parasite Perkinsus olseni. Infect Genet Evol 11:904–911CrossRefGoogle Scholar
  67. Wheeler K, Shields JD, Taylor DM (2007) Pathology of Hematodinium infections in snow crabs (Chionoecetes opilio) from Newfoundland, Canada. J Invertebr Pathol 95:93–100CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Katrina M. Pagenkopp Lohan
    • 1
    • 2
  • Jan R. McDowell
    • 1
  • Jeffrey D. Shields
    • 1
  • Kimberly S. Reece
    • 1
    Email author
  1. 1.The Virginia Institute of Marine ScienceThe College of William and MaryGloucester PointUSA
  2. 2.Smithsonian Conservation Biology InstituteCenter for Conservation and Evolutionary GeneticsWashingtonUSA

Personalised recommendations