Abstract
To better understand sublethal effects of harmful algal blooms (HABs) on fish, mummichog, Fundulus heteroclitus (L.), were exposed in the laboratory to varying, environmentally relevant densities of Pfiesteria shumwayae (Glasgow et Burkholder, CCMP 2089, dinoflagellate) and Chaetoceros concavicornis (Mangin, CCMP 169, diatom). Two experiments were conducted during the spring of 2003 and 2004 to quantitatively examine the effects of acute (2 h) P. shumwayae and C. concavicornis algal exposure on mummichog brain activity using c-Fos expression as a marker of altered neuronal activity. Brains from HAB-exposed fish were removed, sectioned, and stained using immunocytochemistry prior to quantifying neuronal c-Fos expression. Fish exposed to P. shumwayae and C. concavicornis showed increased c-Fos expression compared to unexposed control fish. A significant dose-response relationship was observed, with increased labeling in brains of fish exposed to higher cell densities for both HAB species tested (P ≤ 0.01). Increased labeling was found in the telencephalon, optic lobes, midbrain, and portions of the medulla. The greatest increases in expression were observed in the telencephalon of P. shumwayae-exposed fish, and in the telencephalon and optic lobes of C. concavicornis-exposed fish (P ≤ 0.01). These increases in c-Fos expression are consistent with other physical and chemical stress exposures observed in fish. Neuronal stress, evidenced by c-Fos expression, demonstrates a sublethal effect of exposure and changes in brain activity in fish exposed to HAB species.
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References
Baraban SC, Taylor MR, Castro PA, Baier H (2005) Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131:759–768
Bell GR (1961) Penetration of spines from a marine diatom into gill tissue of lingcod (Ophiodon elongatus). Nature 192:279–280
Bell GR, Griffioen W, Kennedy O (1974) Mortalities of pen-reared salmon associated with blooms of marine algae. Northwest Fish Cutlure Conference, Seattle, WA, pp 58–60
Berry JP, Reece KS, Rein KS, Baden DG, Haas LW, Ribeiro WL, Shields JD, Snyder RV, Vogelbein WK, Gawley RE (2002) Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae. Proc Natl Acad Sci USA 99:10970–10975
Blazer VS, Vogelbein WK, Densmore CL, May EB, Lilley JH, Zwerner DE (1999) Aphanomyces as a cause of ulcerative skin lesions of menhaden from Chesapeake Bay tributaries. J Aquat Anim Health 11:340–349
Bosch TJ, Maslam S, Roberts BL (1995) A polyclonal antibody against mammalian Fos can be used as a cytoplasmic neuronal-activity marker in a teleost fish. J Neurosci Methods 58:173–179
Bosch TJ, Maslam S, Roberts BL (2001) Fos-like immunohistochemical identification of neurons active during the startle response of the rainbow trout. J Comp Neurol 439:306–314
Burkholder JM, Glasgow HB (1997) Pfiesteria piscicida and other Pfiesteria-like dinoflagellates: behavior, impacts, and environmental controls. Limnol Oceanogr 42:1052–1075
Burkholder JM, Noga EJ, Hobbs CH, Glasgow HB (1992) New phantom dinoflagellate is the causative agent of major estuarine fish kills. Nature 360:768–768
Burkholder JM, Glasgow HB, Hobbs CW (1995) Fish kills linked to a toxic ambush-predator dinoflagellate––distribution and environmental-conditions. Mar Ecol Prog Ser 124:43–61
Burkholder JM, Glasgow HB, Deamer-Melia N (2001a) Overview and present status of the toxic Pfiesteria complex (Dinophyceae). Phycologia 40:186–214
Burkholder JM, Marshall HG, Glasgow HB, Seaborn DW, Deamer-Melia NJ (2001b) The standardized fish bioassay procedure for detecting and culturing actively toxic Pfiesteria, used by two reference laboratories for Atlantic and Gulf Coast states. Environ Health Perspect 109:745–756
Burkholder JM, Gordon AS, Moeller PD, Mac Law J, Coyne KJ, Lewitus AJ, Ramsdell JS, Marshall HG, Deamer NJ, Cary SC, Kempton JW, Morton SL, Rublee PA (2005) Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: comparison of assay methods and strains. Proc Natl Acad Sci USA 102:3471–3476
Cheng SB, Kuchiiwa S, Nagatomo I, Akasaki Y, Uchida M, Tominaga M, Hashiguchi W, Kuchiiwa T, Nakagawa S (2002) 2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment induces c-Fos expression in the forebrain of the Long-Evans rat. Brain Res 931:176–180
Dykstra MJ, Kane AS (2000) Pfiesteria piscicida and ulcerative mycosis of Atlantic menhaden - Current status of understanding. J Aquat Anim Health 12:18–25
Espana RA, Valentino RJ, Berridge CW (2003) Fos immunoreactivity in hypocretin-synthesizing and hypocretin-1 receptor-expressing neurons: effects of diurnal and nocturnal spontaneous waking, stress and hypocretin-1 administration. Neuroscience 121:201–217
Fairey ER, Edmunds JSG, Deamer-Melia NJ, Glasgow H, Johnson FM, Moeller PR, Burkholder JM, Ramsdell JS (1999) Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida. Environ Health Perspect 107:711–714
Flewelling LJ, Naar JP, Abbott JP, Baden DG, Barros NB, Bossart GD, Bottein MYD, Hammond DG, Haubold EM, Heil CA, Henry MS, Jacocks HM, Leighfield TA, Pierce RH, Pitchford TD, Rommel SA, Scott PS, Steidinger KA, Truby EW, Van Dolah FM, Landsberg JH (2005) Red tides and marine mammal mortalities. Nature 435:755–756
Glasgow HB, Burkholder JM, Schmechel DE, Tester PA, Rublee PA (1995) Insidious effects of a toxic estuarine dinoflagellate on fish survival and human health. J Toxicol Environ Health 46:501–522
Glasgow HB, Burkholder JM, Mallin MA, Deamer-Melia NJ, Reed RE (2001) Field ecology of toxic Pfiesteria complex species and a conservative analysis of their role in estuarine fish kills. Environ Health Perspect 109:715–730
Gordon AS, Dyer B (2005) Relative contribution of exotoxin and micropredation to icthyotoxicity of two strains of Pfiesteria shumwayae (Dinophyceae). Harmful Algae 4:423–431
Gordon AS, Dyer BJ, Seaborn D, Marshall HG (2002) Comparative toxicity of Pfiesteria spp., prolonging toxicity of Pfiesteria piscicida in culture and evaluation of toxin(s) stability. Harmful Algae 1:85–94
Herdegen T, Leah JD (1998) Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Rev 28:370–490
Jakobsen KS, Tengs T, Vatne A, Bowers HA, Oldach DW, Burkholder JM, Glasgow HB, Rublee PA, Klaveness D (2002) Discovery of the toxic dinoflagellate Pfiesteria in northern European waters. Proc R Soc Lond Ser B-Biol Sci 269:211–214
Kiryu Y, Shields JD, Vogelbein WK, Zwerner DE, Kator H, Blazer VS (2002) Induction of skin ulcers in Atlantic menhaden by injection and aqueous exposure to the zoospores of Aphanomyces invadans. J Aquat Anim Health 14:11–24
Kiryu Y, Shields JD, Vogelbein WK, Kator H, Blazer VS (2003) Infectivity and pathogenicity of the oomycete Aphanomyces invadans in Atlantic menhaden Brevoortia tyrannus. Dis Aquat Org 54:135–146
Law M (2001) Differential diagnosis of ulcerative lesions in fish. Environ Health Perspect 109:681–686
Lewitus AJ, Jesien RV, Kana TM, Burkholder JM, Glasgow HB, May E (1995) Discovery of the phantom dinoflagellate in Chesapeake Bay. Estuaries 18:373–378
Lewitus AJ, Hayes KC, Willis BM, Burkholder JM, Glasgow HB, Holland AF, Maier PP, Rublee PA, Magnien R (2002) Low abundance of the dinoflagellates, Pfiesteria piscicida, P. shumwayae, and Cryptoperidiniopsis spp., in South Carolina tidal creeks and open estuaries. Estuaries 25:586–597
Litaker RW, Steidinger KA, Mason PL, Landsberg JH, Shields JD, Reece KS, Haas LW, Vogelbein WK, Vandersea MW, Kibler SR, Tester PA (2005) The reclassification of Pfiesteria shumwayae (Dinophyceae): Pseudopfiesteria, gen. nov. J Phycol 41:643–651
Lovko VJ, Vogelbein WK, Shields JD, Haas LW, Reece KS (2003) A new larval fish bioassay for testing the pathogenicity of Pfiesteria spp. (Dinophyceae). J Phycol 39:600–609
Magnien RE (2001) The dynamics of science, perception, and policy during the outbreak of Pfiesteria in the Chesapeake Bay. Bioscience 51:843–852
Marshall HG, Hargraves PE, Burkholder JM, Parrow MW, Elbrachter M, Allen EH, Knowlton VM, Rublee PA, Hynes WL, Egerton TA, Remington DL, Wyatt KB, Lewitus AJ, Henrich VC (2006) Taxonomy of Pfiesteria (Dinophyceae). Harmful Algae 5:481–496
Miller TR, Belas R (2003) Pfiesteria piscicida, P. shumwayae, and other Pfiesteria-like dinoflagellates. Res. Microbiol. 154:85–90
Moeller PDR, Beauchesne KR, Huncik KM, Davis WC, Christopher SJ, Riggs-Gelasco P, Gelasco AK (2007) Metal complexes and free radical toxins produced by Pfiesteria piscicida. Environ Sci Technol 41:1166–1172
Noga EJ, Khoo L, Stevens JB, Fan Z, Burkholder JM (1996) Novel toxic dinoflagellate causes epidemic disease in estuarine fish. Mar Pollut Bull 32:219–224
Profet EB, Mills B, Arrington JB, Sobin LH (1992) Laboratory methods in histotechnology. Armed Forces Institute of Pathology, Washington
Rhodes LL, Burkholder JM, Glasgow HB, Rublee PA, Allen C, Adamson JE (2002) Pfiesteria shumwayae (Pfiesteriaceae) in New Zealand. N.Z. J Mar Freshwat Res 36:621–630
Rublee PA, Kempton JW, Schaefer EF, Burkholder JM, Glasgow H, Oldach DW (1999) PCR and FISH detection extends the range of Pfiesteria piscicida in estuarine waters. Virginia J Sci 50:325–336
Sadananda M, Bischof HJ (2002) Enhanced fos expression in the zebra finch (Taeniopygia guttata) brain following first courtship. J Comp Neurol 448:150–164
Salierno JD, Snyder NS, Murphy AZ, Poli M, Hall S, Baden DG, Kane AS (2006) Harmful algal bloom toxins alter c-Fos protein expression in the brain of killifish, Fundulus heteroclitus. Aquat Toxicol 78:350–357
Shimomura-Umemura S, Ijiri K (2006) Effect of hypergravity on expression of the immediate early gene, c-fos, in central nervous system of medaka (Oryzias latipes). Adv Space Res 38:1082–1088
Vogelbein WK, Shields JD, Haas LW, Reece KS, Zwerner DE (2001) Skin ulcers in estuarine fishes: a comparative pathological evaluation of wild and laboratory-exposed fish. Environ. Health Perspect 109:687–693
Vogelbein WK, Lovko VJ, Shields JD, Reece KS, Mason PL, Haas LW, Walker CC (2002) Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion. Nature 418:967–970
Watson RE, Wiegand SJ, Clough RW, Hoffman GE (1986) Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 7:155–159
Wulliman MF, Rupp B, Reichert H (1996) Neuroanatomy of the zebrafish brain: A topological atlas. Birkhauser Verlag, Basel
Yang CZ, Albright LJ (1992) Effects of the harmful diatom Chaetoceros concavicornis on respiration of rainbow-trout Oncorhynchus mykiss. Dis Aquat Org 14:105–114
Yang CZ, Albright LJ (1994) The harmful phytoplankter Chaetoceros concavicornis causes high mortalities and leukopenia in chinook salmon (Oncorhynchus tshawytscha) and coho salmon (Oncorhyncus kisutch). Can J Fish Aquat Sci 51:2493–2500
Acknowledgments
Portions of this study were supported by the U.S. Environmental Protection Agency, Science to Achieve Results (STAR) program (#R82-8224), the Maryland Department of Health and Mental Hygiene, and the Centers for Disease Control and Prevention. The authors thank N. Snyder, A. Miller, and C. Squyars, for their assistance with the brain processing and data collection. We also thank R. Andersen, Bigelow Marine Laboratory, for providing the C. concavicornis cultures. Reported experiments complied with regulations set by the Institutional Animal Care and Use Committee of the University of Maryland (Protocol R-00-36B) and VIMS (IACUC protocol 0129, IBC protocols 9906 & 0206).
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Communicated by J.P. Grassle.
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Salierno, J.D., Shields, J.D., Murphy, A.Z. et al. Altered c-Fos expression demonstrates neuronal stress in mummichog, Fundulus heteroclitus, exposed to Pfiesteria shumwayae and Chaetoceros concavicornis . Mar Biol 152, 835–843 (2007). https://doi.org/10.1007/s00227-007-0734-9
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DOI: https://doi.org/10.1007/s00227-007-0734-9