Genetic divergence among toxic and non-toxic cyanobacteria ...
THE LIFE CYCLES OF TOXIC PFIESTERIA SPECIES AND...
Transcript of THE LIFE CYCLES OF TOXIC PFIESTERIA SPECIES AND...
THE LIFE CYCLES OF TOXIC PFIESTERIA SPECIES AND OTHER ESTUARINEDINOFLAGELLATES: TOWARD VERIFICATION OF PFIESTER’S HYPOTHESIS
J.M. Burkholder, Ph.D; H.B. Glasgow, Ph.D; J. Springer, M.S.; and M.W. Parrow, B.S.
Center for Applied Aquatic Ecology, North Carolina State University, Raleigh, NC 27606
ABSTRACT
After working many years to contribute among the most elegantresearch published on dinoflagellates, the late Dr. Lois Pfiesterhypothesized that “many if not all dinoflagellates will be foundto have amoeboid stages.” Research previously confirmed(1916- early 1970s) dinoflagellates with amoeboid stages inmarine waters. Given that dinoflagellates with amoeboidstages were known from freshwater and marine habitats, weexpected that species with amoeboid stages also would befound in estuaries. During the early 1990s we describedtranformations from isolated cells and isolated populations(clones) of P. piscicida from zoospores to distinct filose, lobose,and rhizopodial amoebae (each of which can be maintained inculture for weeks to months – thus, these are not merelytransitional forms; cross-confirmed by 3 other laboratories withSEM and molecular probes) in response to changing preyavailability, temperature, salinity, and other factors. Since thediscovery of P. piscicida as the first estuarine dinoflagellate witha complex life cycle and amoeboid stages (length 5-750 _m),two other Pfiesteria species have been described withamoeboid stages (Landsberg et al. 1995, Glasgow et al. 2000),at least 1 of which is toxic (P. shumwayae sp. nov.); and severalspecies of thus-far-benign Cryptoperidiniopsis gen. nov. havebeen documented to have amoeboid stages (Marshall et al.accepted, Steidinger and coworkers pers. comm.). Thus,increasing evidence over a relatively short time providessupport for Dr. Pfiester’s hypothesis, and support for thehypothesis that many other estuarine dinoflagellates will befound with complex life cycles and amoeboid stages.
DISCUSSION
There is a general paucity of information on thedistribution and ecology of amoebae in estuarineecosystems. Various authors have hypothesized thatamoebae, which are common among estuarinemicrofauna, play more important ecological roles in theseecosystems then previously conceived.
A number of freshwater and marine dinoflagellate specieshave been observed to have amoeboid stages.Dinoflagellates with amoeboid stages have beendescribed from such diverse habitats as peat bogs, cold-temperate and sub-arctic coastal waters, and alpinestreams. However, until the discovery of Pfiesteria, therewas no information on estuarine dinoflagellates withamoeboid stages. Pfiesteria spp. were also the first toxicdinoflagellates with documented amoeboid stages.
An intensive sampling effort has yielded dinoflagellateclones with amoeboid stages, including several‘cryptoperidiniopsoid’ species, scripsielloids, andgymnodinioids in addition to the two Pfiesteria species).These isolates were obtained from the Indian River InlandBay of Delaware; eight tributaries of Chesapeake Bay inMaryland and Virginia; the Neuse, Pamlico, and NewRiver Estuaries in North Carolina; and four majorestuaries of Florida (east and west coasts). Commonmorphotypes of amoe-bae observed in these clonalcultures have included filose, heliozoan, lobose, andrhizopodial forms. These amoeboid stages commonly co-occur in clonal cultures with motile, zoo-spore stages ofthe dinoflagellate species.
The development of primers specific to the 18s rDNA ofP. piscicida, P. shumwayae , and Cryptoperidinopsisbrodyii sp. nov. (Steidinger & coworkers) have enabledmolecular diagnosis of these amoeboid stages. The datafrom PCR and fluorescent in-situ hybridization testingsupport the observations from clonal cultures (e.g.,Burkholder & Glasgow 1995, Marshall et al. in press), andthe transformations described in single cells by Steidingeret al. (1996, J. Phycol.) that three estuarine dinofla-gellates each have amoeboid stages in their complex lifecycle.
ACKNOWLEDGMENTS
We thank the National Science Foundation, the U.S. EPA, the Z.Smith Reynolds Foundation, and an anonymous foundation forfunding support. We would also like to thank members of theCenter for Applied Aquatic Ecology for the provision of technicalassistance.
Predominantly marine(parasites of fish)
Gymnodinium austriacum
Popovsky andPfiester (1990)
Ci tat ionHabi tat
Figure 1. Representative amoeboid stages commonly observedunder light microscopy in c lonal cultures of estuarinedinof lagel lates. The amoeboid stages that we have reported havealso been separately maintained for at least 4 wk in clonal culture;that is, they are not merely transitional forms.
SELECTED REFERENCESBu ckla n d- Nicks, J.A., R eimch e n, T .E. & F .J.R . T ay lor (1 99 0) . A no ve la sso ciati o n b etw e en an en d emic sti ckle b ack a n d a p ar asi tic d in ofl a g el l ate . 2 .Mo r ph olo gy a n d l i fe cycle . Jo u r na l of Ph yco lo gy 2 6: 5 3 9- 54 8.
Bu rkh o ld e r, J.M. & H .B. G l asg o w ( 19 9 5) Inte rac tio ns of a to xi c e stu ar in ed i no fl ag e ll a te w ith micr ob ia l p re dato rs a n d p r ey. Arch ive s für Pr otis ten ku n de1 4 5: 1 77 -1 88 .
Bu rsa , A .S. ( 19 7 0a ). D in amo e bid iu m c o lo r ad en se s p. n ov. a n d Kato d i niu ma u ratu m sp . n o v. i n C o mo cre ek, Bo uld er C ou nty, C olo ra do . Ar cti c a n d Alp in eR ese a rch 2: 1 45 - 15 1.
Bu rsa , A.S. ( 19 70 b). D in amo e bid iu m h yp e rb or eu m sp. n o v. i n c oa sta lp l an kto n o ff El l e sme re Isla nd , N.W .T ., C a na d a. Arcti c a n d Alp in e R ese a rch 2 :1 5 2- 15 4.
C ach o n, J.M. ( 19 68 ). F i l od in iu m h ova sse i g en . n ov. sp . n ov. Pr oti sto lo g ica T .IV, facs.
G lasg ow, H .B., Bu rkh old er, J.M., Mo rton , S.L. a nd J. Sp r in g er ( 20 00 ) Ase co nd sp e cies o f i chth yo to xi c Pfi este r ia ( Din amo e ba le s, Py r rho ph yta ).Ph yco lo gia.
Po p ovsk y_, J . & L .A. Pfi este r ( 19 9 0) Sü ßw asse rfl o ra vo n Mi tte le u ropa . Ed s., H.Ettl , J. G e r loff, H. H eyn ig , and D . Mo ll e n ha ue r. G usta v F i sch e r V er la g ,Stu g ga rt.
Ste i din ge r, K.A., J.M. Bu rkh o ld e r, H .B. G l asg o w, C.W . H ob bs, E. T r ub y, J .G a r rett, E.J. N o ga & S.A. Smi th ( 19 9 6) Pfi este r ia p isci cid a g en. et sp . n ov .(P fi e ste ri ace a e, fa m. n o v.), a n e w to xi c d in ofl a ge ll a te g e n us a n d sp e cies w itha co mp lex li fe c ycle a n d b e ha vi or. J. Phyco l. 3 2: 1 57 - 16 4.
T impano, P. & L.A. Pfiester (1986) Observations on “Vampyrella penula-Stylodinium sphaera” and the ultrastructure of the reproductive cyst. A m.. J.Bot . 7 3: 1341-1350.
Table 1. Examples of dinoflagellate species with multiphasiclife cycles including amoeboid stage(s).
SEM of Amoeba
Figure 2. Scanning electron micrograph of a P .piscicida lobose
amoeba adjacent to P . piscicida zoospores(Neuse clone).
10 µmZoospore
10 µm 40µm
F i lose Lobose
MATERIALS AND METHODS
Clona l c ul tures of amoebae and zoospores (routinelyisolated and cleaned using flow cytometric procedures)originated from estuarine locations in mid-Atlantic andsoutheastern U.S. estuaries, mostly the Albemarle-PamlicoEstuarine System in NC and Chesapeake Bay in MD. Cultureswere maintained on a diet of Cryptomonas sp. in anenvironmental incubator (15 ppt, 21 oC, 14:10 L/D). Lightmicro-scopic observations were made using an Olympus AX-70 research microscope equipped with water immersion opticsunder Nomarski DIC. SEM was completed with a JEOL 505Tat 15kV, following the protocols of Burkholder & Glasgow (1995)and Glasgow et al. (2000).
An 18s rDNA-based PCR protocol was used to identifywhether cul-tured isolates were P. piscicida, P. shumwayae, orCryptoperidiniopsis brodyii Alexa Fluor labelled in-situhybridization (FISH) probes based on the 18s rDNA PCRprimer pairs were applied to enable fluorescent identification ofthe above species. Epifluorescence (light microscopeequipped with the appropriate band-specific filters -- 350, 488,532 nm) was used to image labeled cells.
Figure 3. Application of fluorescent in-situ hybridization(FISH) probes toward the molecular identification of free-living amoe- boid stages in c lonal cultures ofestuar ine dinoflagellates.
Figure 4. Gel electrophoresis of PCR products amplifiedusing a 18s rDNA primer developed for the detection of P.piscicida . Assayed cultures included c lona l isolates ofamoebae collected from estuarine sites in Maryland andNorth Carolina.
1,00075050030015050
Ladd
er- C
ontro
l47
1AN
euse
472A
113-
2P
am
lico
1011
59
Poc
omok
eS
eagu
l+
Con
trol
574A
480A
598A
612A
617A
666A
-Con
trol
Ladd
er
Amoebae testing positive for P. piscicida
Zoospore
Amoebae
Zoospo re s
P. shumwayae P. piscicida Cryptoperidiniopsis brodyii Alexa Fluor 350 Alexa Fluor 488Alexa Fluor 532
Spec ie s
Cystodiniumbataviense
(Dinococcusoedogoni i)Dinamoebidin ium
coloradense
D. hyperboreum
Bursa(1970a)
Bursa(1970b)
Fi lodin iumhovassei
Marine (parasite of Appendicular ians)
Cachon (1968)
Oodinium spp
Pfiesteriapiscicida
P. shuumwayae
Burkholder e t al .(1992)
Glasgow et al.(2000)Buckland-Nicks et al.
(1990)
Popovsky & Pfiester (1990)
Freshwater(ol igotrophic lakes in
Europe and N. America)Popovsky & Pfiester
(1990)
Timpano & Pfiester (1986)
Piscinodinium-l ikesp.
Freshwater (parasite ofstickle- back,
Gasterosteus sp.)
Estuarine (U.S. Atlantic,Gul f )
Estuarine (similardistr ibution)
Stylodiniumsphaera
(= Vampryel lapenula)
Freshwater (parasit ic onOedogonium spp.)
Freshwater (alpine;amoeboid stage
resembles Amoebaverrucosa)
Marine (Arctic, coastal)
Peat Bogs, Clay Pools(Central Europe, Java,USA)