2000 Progress Report: Ecohab: Pfiesteria Or Fungus? Etiology Of Lesions In Menhaden

EPA Grant Number: R828225
Title: Ecohab: Pfiesteria Or Fungus? Etiology Of Lesions In Menhaden
Investigators: Shields, Jeffrey , Haas, Larry , Kator, Howard , Vogelbein, Wolfgang K.
Current Investigators: Shields, Jeffrey , Blazer, Vicki , Haas, Larry , Kator, Howard , Kiryu, Yasunari , Vogelbein, Wolfgang K.
Institution: Virginia Institute of Marine Science
Current Institution: Virginia Institute of Marine Science , College of William and Mary-VA
EPA Project Officer: Packard, Benjamin H
Project Period: June 16, 2000 through June 15, 2003
Project Period Covered by this Report: June 16, 2000 through June 15, 2001
Project Amount: $508,937
RFA: Ecology and Oceanography of Harmful Algal Blooms (1999) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Water Quality , Water , Ecosystems

Objective:

The overall objective of this research project is to identify the interrelationships between menhaden, Pfiesteria, and Aphanomyces, as well as environmental conditions that may modulate or contribute to the epizootics of ulcers on the fish. In addition, the objective is to determine whether Pfiesteria or a fungus, Aphanomyces invadans, is responsible for the ulcerous lesions in fish that dwell in the Chesapeake Bay and elsewhere. The specific objectives of this project are to conduct controlled laboratory exposure studies with menhaden to: (1) identify the causal agents responsible for the ulcerous lesions, and (2) identify contributory environmental and biological conditions required for the development and progression of the lesions.

Menhaden, Brevoortia tyrannus, develop ulcerous skin lesions that have been attributed to exposure to Pfiesteria piscicida toxins. The presence of these lesions in conjunction with counts of presumptive Pfiesteria-like cells in water samples are the primary criteria for river closures in Maryland and Virginia because of local, recent Pfiesteria activity. However, there is controversial evidence that the lesions are caused by an oomycete fungus, Aphanomyces sp., either as a primary or secondary invader. The fungus almost always is associated with the lesions, with hyphae penetrating the tissues and organs of the infected fish.

Progress Summary:

We further investigated the infectivity and pathogenicity of A. invadans in menhaden, B. tyrannus, in several laboratory challenge studies. In a dose-response study, secondary zoospores of WIC (an endemic isolate of A. invadans in menhaden from Maryland) were subcutaneously injected into menhaden at doses of 0, 1, 10, 50, 100, and 500 zoospores per fish. The fungus was highly pathogenic with an LD50 of approximately 10 zoospores, with a single zoospore sufficient to cause frank lesions (Kiryu, et al., in press). We demonstrated that fungal zoospores were highly infectious and highly pathogenic to menhaden. We speculate that the fungus may contribute to mortalities of menhaden by compromising the energetics and survivorship of infected fish.

We repeated exposing menhaden in bath challenges with zoospores. Treatments consisted of handling fish with a net (Net-handled, exposed for 5 hours to 100 zoospores/mL), and acclimating fish with little handling stress (acclimated, untraumatized, exposed for 5.5 hours to 100 zoospores/mL), and unexposed fish as controls. Mortality and prevalence of the lesions were high (70-100 percent) for net-handled fish, although they were low (24-32 percent) in the "untraumatized" fish. This verifies that the fungus was highly invasive, and that it could serve as a primary pathogen and a portal of entry for enhanced transmission.

We have completed single-factor exposure studies with Pfiesteria. In several laboratory challenge studies, we investigated the toxigenicity or pathogenicity of P. shumwayae in menhaden and in tilapia. In the separate dose-response studies, fish were exposed to dinospores of P. shumwayae (CCMP2089) (an endemic isolate from the Pamlico River, NC) at doses of 1,000, 3,000, and 5,000 dinospores per ml in 38 L aquaria. We have generated LD50s for various time periods for exposed fish. Currently, we are examining the microscopic pathology of menhaden exposed to P. shumwayae. Interestingly, we have documented that menhaden have cysts of P. shumwayae in their guts.

The Pfiesteria-Fungus dual challenge study and the dual factor studies with hypoxia and fungus, have been delayed by several months because of inconsistencies in sporulation of batch cultures of the fungus. These studies involve very complex experimental designs with a suite of different exposures at different salinities. We have undertaken numerous culture studies in an attempt to optimize batch cultures for zoospore production. These are vital to the success of the exposure experiments.

Single-factor exposure studies currently are underway to document the possible trauma to epidermal cells because of hypoxia. A dual-factor study of hypoxia and fungal exposure has been delayed because of problems in production of fungal zoospores. However, we have been developing methods to quantify skin damage in menhaden and tilapia exposed to hypoxia, Pfiesteria, or other stress-inducing agents. Preliminary results with various markers look quite promising.

Larval fish assays have been refined to give further insights into the nature of fish-killing activity by P. shumwayae. The results of these assays are reported in a recent manuscript (Vogelbein, et al., 2002).

Cultures of P. shumwayae were provided to collaborators at the Rosensteil School of Marine Science, Miami, FL. Collaborative efforts have resulted in a paper documenting the lack of polyketide synthase capability in P. shumwayae (Berry, et al., 2002). Polyketide synthases are enzymes that facilitate production of ichythotoxins in dinoflagellates.

Future Activities:

Future activities planned for Year 3 of the research project include completion of the dual-factor study with Pfiesteria and fungus exposures. We also are planning to complete single- and dual-factor studies with hypoxia and fungus exposures. These studies are logistically complex, involving different exposures at different salinities. The premise for the hypoxia exposures is that fish may suffer epidermal damage during hypoxic events. The resultant epidermal damage then provides a portal of entry for fungal infection.

We also are undertaking additional dose-response studies with P. shumwayae and tilapia. These studies will focus on collecting blood electrolyte levels for pathology and brain receptor data for other collaborators. Transfection studies with hogchoker, spot, and croaker currently are underway. These studies will facilitate our understanding of the host specificity of these fungus infections in our estuarine fishes.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 41 publications 9 publications in selected types All 9 journal articles
Type Citation Project Document Sources
Journal Article Berry JP, Reece KS, Rein KS, Baden DG, Haas LW, Ribeiro WL, Shields JD, Snyder RV, Vogelbein WK, Gawley RE. Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae. Proceedings of the National Academy of Sciences of the United States of America 2002;99(17):10970-10975. R828225 (2000)
R828225 (2001)
R828225 (Final)
R826655 (Final)
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  • Journal Article Kiryu Y, Shields JD, Vogelbein WK, Zwerner DE, Kator H, Blazer VS. Induction of skin ulcers in Atlantic menhaden by injection and aqueous exposure to the zoospores of Aphanomyces invadans. Journal of Aquatic Animal Health 2002;14(1):11-24. R828225 (2000)
    R828225 (2001)
    R828225 (Final)
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  • Abstract: Taylor&Francis-Abstract
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  • Journal Article Kiryu Y, Shields JD, Vogelbein WK, Kator H, Blazer VH. Infectivity and pathogenicity of the oomycete Aphanomyces invadans in Atlantic menhaden Brevoortia tyrannus. Diseases of Aquatic Organisms 2003;54(2):135-146. R828225 (2000)
    R828225 (2001)
    R828225 (Final)
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  • Journal Article Vogelbein WK, Lovko VJ, Shields JD, Reece KS, Mason PL, Haas LW, Walker CC. Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion. Nature 2002;418(6901):967-970. R828225 (2000)
    R828225 (2001)
    R828225 (Final)
    R826791 (Final)
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  • Journal Article Vogelbein WK, Shields JD, Haas LW, Reece KS, Zwerner DE. Skin ulcers in estuarine fishes:a comparative pathological evaluation of wild and laboratory-exposed fish. Environmental Health Perspectives 2001;109(Suppl 5):687-694. R828225 (2000)
    R828225 (2001)
    R828225 (Final)
    R826791 (Final)
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  • Supplemental Keywords:

    estuary, marine, animal, pathology, toxicity, harmful algal blooms, fish kills, fish lesions, ecological effects, Pfiesteria, Aphanomyces., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Epidemiology, Oceanography, Ecological Effects - Environmental Exposure & Risk, algal blooms, Ecological Effects - Human Health, Ecological Risk Assessment, Ecology and Ecosystems, Ecological Indicators, Aphanomyces, marine ecosystem, dermal exposure, ecological exposure, ecological effects, pathology, dinoflagellates, fish kills, etiology of lesions, fish lesions, harmful algal blooms, algal growth, pfiesteria, ecological impacts, ECOHAB, water quality, oomycete fungus, laboratory studies

    Relevant Websites:

    http://www.vims.edu/pfiesteria Exit
    http://www.vims.edu/~jeff Exit

    Progress and Final Reports:

    Original Abstract
  • 2001 Progress Report
  • Final Report