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Prymnesiophytes of New Zealand's coastal

waters: taxonomy, physiology

and ecology.

A thesis presented in partial fulfilment of the requirements

for the degree

of Doctor of Philosophy

in Plant Biology and Biotechnology at

Massey University

lesley louise Rhodes

1 994

11\

Abstract

Prymnesiophytes are an important component of the marine phytop lankton of

New Zealand coastal waters , but there is l ittle knowledge of the taxonomy,

physiology or eco logy of local stra ins. The Class comprises four orders which

conta in putative fami l i es and genera of the microscopic organ isms, the

microa lgae . The Prymnesiophytes are known for the i r abi l ity to explode i n

population numbers into "blooms " ; one of New Zealand ' s most common

bloom-formers i s the coccol ithophore Emiliania huxleyi (Prymnesiophyceae) ,

which dominated the extensive phytop lankton blooms observed around New

Zealand ' s coast l i ne dur ing 1 9 93 - 94 . Aspects of the physio logy of and

factors contr ibut ing to b loom format ion i n E. huxleyi are i nvest igated .

The phenomenon of seasonal b looms, a common occurrence amongst the

microa lgae, i s reviewed . Studies of the blooms which occurred a long the

north-east coast l i ne of New Zeal and In 1 9 92-93 showed that these were

unusual events that were l i nked to the c l i matic condit ions at that t ime, i n

part icular co ld sea temperatures associated with a n "EI-N ino " phase o f the

Southern Osc i l l at ion I ndex in the southern Pacific Ocean . The dominant

microalgae were Geph yrocapsa oceanica (Prymnesiophyceae) and Fibrocapsa

japonica ( Ra phidophyceae) and it is probable that the condit ion ing of the

coastal w aters by these microalgae had a ro le in the succeed ing tox ic event, i n

which human i l l nesses were defi n it ively l i nked to shel l f ish toxins due to

microa lgae for the first t ime in New Zealand .

Al le lopath ic , or chemica l , i nteract ions between microa lgae were i nvestigated In

this study and Prymnesium parvum and P.patellifera caused inhi b it ion of the

growth of spec ies from several microalga l c lasses . Unfortunately P.patellifera

rap id ly lost its inhibitory activity in vitro , but P.parvum remai ned act ive for

IV

severa l years in culture and was therefore se lected as a posit ive contro l for the

bioassays for ichthyotoxic ity that were developed .

The c lass Prymnesiophyceae i ncludes toxi c species of three genera, n amely

Chrysochromulina, Prymnesium and Phaeoc ys tis. Ichthyotoxi n b ioassays based

on toxi n sensit ive microalgae ( Chattonella antiqua and Heterocapsa triquetra) ,

she l lf i sh l arvae (Haliotis iris) , br ine shr imps (Artemia salina) a n d sa lmon

erythrocytes ( Oncorhynchus tshawytscha) , were developed o r refined and

evaluated to enab le the rapid and i nexpensive detection of the h aemolyt ic and

cyto lytic prymnes iophyte tox in , prymnesi n .

The n ovel quadr if lagel late species C. quadrikonta, which b loomed i n nort h-east

N ew Zea land , May 1 994, exhibited low levels of haemolytic activity i n

stat ionary phase cultures grown i n standard nutrient medium (at 1 8°C , 1 00

)1mo l m-2s -1) as determined by the erythrocyte assay; no othe r

Chrysochromulina species tested was toxic .

S ix loca l l y occurr ing species were identifi ed b y electron m icroscop ic

exami nation of scales and l ight and e lectron microscop ic observat ion of

cultured iso lates i n this study. Seventeen of the nearly fifty named species of

Chrysochromulina have now been i dentif ied in New Zealand . The average ce l l

s izes and unmineral ised sp ine sca le lengths of the New Zea land isolates of

C. ericina, C. hirta and C. quadrikonta were s l ightly l arger than for the i r type

species and the calcareous scales of E. huxleyi and G. oceanica were more

heavi ly ca lcif ied than their northern hemisphere counterparts . N o g radation of

ca lc if icat ion with i ncreased l at itude was observed for the coccol ithophores, as

had been noted previously, and th is m ight reflect the consistently lower sea

temperatures p revai l ing , due to the unusual ly protracted flE I-N ino " c l imat ic

condit ions.

Fluorescent probes proved to be useful tools for the d ifferent iat ion of some

morpholog ica l ly- l i ke species under the l ight microscope : Calcofluor wh ite

he lped d isti nguish between cel l s of C. ericina and C. quadrikonta . The

d ifferentiat ion of the genera Prymnesium and Chrysochromulina was e nab led

through the spec ific b ind ing of fluorescently-tagged wheat germ lect in to

Chrysochromulina species.

v

The g rowth characterist ics and cul tura l id iosyncrasies of severa l southern

hemisphere Chrysochromulina i so lates have been described and compared with

the toxic northern hemisphere relative, C.po/y/epis. C.po/y/epis, C. ericina a nd

C. hirta fel l i nto a temperate g roup on the basis of opt imum growth rates

(doubl ings d-1) , whi l e C. acantha, C. aphe/es, C. came/la and C. quadrikonta fe l l

i nto a sub-tropica l g roup . A l l but C. acantha grew equal ly wel l with potassium

n itrate , urea or a mmonium chloride; C. acantha grew s ignif icantly s lower with

urea as n itrogen source . O nly C. quadrikonta had a sel en ium requ i rement for

growth. Maximum growth rates (doub l ings d-1 ) recorded in vitro were

C. acantha, 1 .2 ; G. aphe/es , 0 . 9 ; C. came//a, 1 . 1 ; G. ericina, 1 . 5 ; C. hirta, 2 . 4 and

C. quadrikonta, 1 . 4. The Chrysochromu/ina species and E. hux/eyi and

G. oceanica grew at low l i ght i ntensit ies ( 2 5 tlmo l m-2s-\ which could g ive

these prymnes iophytes a competitive advantage in b loom s ituat ions , where

shad i ng due to the phytop lankton biomass can occur.

The New Zeal and i solate of G. oceanica g rew optima l ly at sa l i n i t ies of 1 7 -

2 9 0/00, pH of 8 . 4 - 8 . 9 and temperatures of 20 - 2 50C; E. hux/eyi grew

opt ima l ly at 2 9 0/00 , pH 7 . 5 - 8 . 9 and 1 5 - 2 50C. G. oceanica grew equal ly wel l

with ammon ium chloride , urea or n i trate a s n itrogen source; E.hux/eyi grew

opt ima l ly w ith ammonium chlor ide. Maximum growth rates recorded were 1 . 9

doub l i ngs d-1 for E. huxleyi and 1 . 4 doub l ings d-1 for G. oceanica .

Acknowledgments

Specia l tha n ks are due to David Founta in , Char ley 0 I Kel ly, Ju l ie H a l l and D o n

G rant . I have a pp reciated their r igorous supe rv is ion and positive crit ic i s m ,

which have a lways been carr ied out i n a thoughtfu l a n d constructive way.

Thanks to my co l l eagues at Cawthron I n stitute, Ne lso n , for the i r

contribut ions t o my work: Hen ry Kaspar a n d Doug Mountfort have g iven

exce l lent feed-back on pub l ication and thes is drafts and the s u pport o f

Graeme Robertson , CEO at Cawthron I n stitute, has been vital and a l ways

forthcom i n g . Lincoln Mac Kenzie co l lected the f irst iso l ate of

Chrysochromufina quadrikonta and has cons istently shared i n formation w ith

me and B rendon B urke provided the first iso late of G. ericina. I have e nj oyed

co l laborat ing with A l l ison H aywood on the studies of Northland and H a u raki

G ulf blooms and va lue her enthusiasm. D avid White carr ied out the acryl ic

acid ana lyses , Doug Mountfort rendered Heterocapsa triquetra axenic for the

b ioassay exper iments and Brendon Burke, Rod Asher , Maggie Atk inson and

Aaron Quarterm a n have contributed at var ious t imes and i n var ious ways to

the thesis with exce l lent technical s upport . I n particu lar Brendon has put

t ime and effort into sett ing up a bank of maps, which I have i ncorporated

i nto thi s thesis. I have a lso appreciated the support of John Stark and John

H ayes with computing and stat istical p rob lems.

The consistent and excel lent assistance of Doug Hopcroft and R aymond

Bennett, H o rt R esearch, Pa lmerston North, New Zea land with e lectron

m icroscopy is acknowledged and a lso the assistance of Rob Thomson and

Teeba Lundy at Victoria U niversity's EM Un i t .

I w ish to acknowledge Isao I nouye and M asanobu Kawachi , U niversity of

Tsukuba, I barak i , Japan, for m aking avai l ab le the i r unpub l ished results on the

Japanese stra in of C. quadrikonta and Tony Edwards, Strat igraphic S o l ut ions,

Wel l i ngto n , New Zealand for sharin g his coccol ithophorid expert ise .

VI

Assistance with obtain ing f ish erythrocytes was wi l l ing ly given by Regal

Sa lmon Ltd . , B lenheim; Southern Ocean Seafoods , Ra i Va l ley; Co l in

Anderson , Wa l lacevi l le An ima l Research Centre, U pper Hutt and Jan de

Zwart , MAF Regu l atory Authority, B lenhe i m . Than ks a lso to Jan H o l land ,

Crop and Food Research, Nelso n .

Several m ic roa lga l species were provided b y Len Tong , MAF F isheries,

Mahanga Bay hatchery, Wel l i ngton and Prymnesium species by Hoe Chang ,

Oceanographic I nstitute, N IWA, Wel l i ngton . J ul ie Ha l l , Ecosystems , N IWA,

H a m i lton, p rovided the f luorescently l abe l l ed beads for the phagotrophy

exper iments .

Co l l aboration with Paul Lup i , New Zea land Mar ine Farmers Associ at ion ,

Blenhe im , Peter Smith and Brian Jones, MAF Fisheries, Wel l i ngto n , G reg

S loane and B i l l Trusewich, MAF Regu latory Authority, Wel l i ngto n , B i l l

Ba l l anti ne , U niversity of Auck land , Barbara Hickey, A uckland Reg iona l

Counci l , S imon M arwick, B ig G lory Seafoods , Stewart I s l and , Barry Peake,

U niversity of Otago, Duned in , Tony Beauchamp, North land Area H ea lth

Services and M a u rice Mi les , Waitemata Health Services, has been va luab le

and enjoyab le .

A New Zea land Universities Postgraduate Scholarship, a C awthron

Scholarship , M assey Un iversity Grants Committee fund ing a nd contracts

with the Found ation for Resea rch, Science and Technology have been

appreciated . The New Zealand Salmon Farmers Associat ion and the New

Zealand Marine Sciences Society a lso p rovided funds for t ravel to overseas

conferences .

V!1

viii

Preface

The fol lowing p apers were submitted for pub l ication dur ing the cours e of this

study . Copies of the publ ished papers a re found ins ide the back cover of the

thes is :

o Rhodes , L . L. , H aywood, A .J . , Ba l iantine ,W.J . , MacKenzie ,A . L . ( 1 9 9 3 )

A lga l b looms a n d c l imate anomal ies i n north-east New Zealand , August to

December, 1 9 9 2 . New Zealand journal of marine and freshwa ter research

27, 4 1 9-430.

@ Rhodes ,L . L . , O'Ke l ly ,C . J . , H a l l ,J .A . ( 1 9 94) Comparison of growth

characteristics of New Zealand isolates of the prymnesiophyte s

Chrysochromulina quadrikonta and C . cam ella with those of the

ichthyotoxic species C.poly/epis. Journal of plankton research 1 6, 6 9-82.

@) Jones,J . B . and Rhodes, L .L . ( 1 994) Suffocation of p i lchards .

(Sardinops sagax) by a green microa lga l b loom, Wel l ington H arbour , New

Zea land , December 1 99 3 . New Zealand journal of marine and

freshwater research, 28, 3 79-384.

o Rhodes , L . L . , Edwards ,A . R . , Peake , B . M . , M acKenzie,A . L. , M arwi c k , S .

( 1 9 94) B l o o m a n d growth characteristics of the coccol ithophores

Gephyrocapsa oceanica and Emiliania huxleyi ( Prymnesiophyceae ) i n New

Zealand ' s coastal waters. New Zealand journal of marine and freshwater

research ( submitted November 1 994) .

C ontents page

Abstract i i i

Acknowledgments vi

Preface viii

Contents ix

Figures x

Tables xiii

Abbreviations xv

GEN ERAL INTRODUCTION 1

Chapter one:

Chapter two :

Chapter thre e :

C hapter fou r :

C hapter five :

FLO RISTICS: New Zealand's Prymnesiophytes 1 1

FLUO RESCENT PRO BES AS TAXONOMIC

TOOLS

GROWTH CHARACTERISTICS OF

PRYMN ESIOPHYTES

PRYMN ESIOPHYTE BLOOMS IN NEW

ZEALAND

MICROALGAL TOXICITY INTERACTIONS

40

55

80

Section one : al lelopathy 1 02

Section two : m icroalgal toxicity bioassays 1 1 7

CON CLUSIO N 1 33

REFERENCES 1 36

IX

x

Figures page

1 .1 M ap s of New Zealand and M ar lborough Sounds, showing sites from which prymnesio phytes were isolated. 1 5

1 .2 U nstained transmission e lectron m icrographs of sp ine and p late sca les of Chrysochromulina acantha. 1 9

1 .3 Transmiss ion micrographs of scales of Chrysochromulina

apheles. 1 9

1 .4 Transmiss ion electron micrographs of scales of Chrysochromulina camella. 2 1

1 .5 Light m icrograph of Chrysochromulina ericina with phagocytosed f luorescently l abe l led bead . 2 1

1 .6 Tran s miss ion electron micrographs of p late and sp ine sca les of Chrysochromulina ericina. 23

1 .7 Chrysochromulina hirta scales (transmission electron micrographs) . 2 5

1 .8 Light m icrograph of Chrysochromulina quadrikonta. 28

1 .9 Transmiss ion e lectron micrographs of Chrysochromulina

quadrikonta. 29

1 . 1 0 Scann ing e lectron micrographs o f Emiliania huxleyi i so lated from Big G lo ry Bay, Stewart I s l and . 3 1

1 . 1 1 Scann ing e lectron micrographs of Gephyrocapsa oceanica. 32

1 . 1 2 Light m icrograph of Pleurochrysis sp . shedding scales and e lectron micrograph of sca les . 34

1 .1 3 Light m icrographs of Syracosphaera cf. pirus iso lated from North land and scanning e lectron m icrograph of coccol iths of Umbilicosphaera sp . 34

2.1 The spine scales of Chrysochromulina ericina and C. quadrikonta fluorescing under U V l ight fol lowing the add it ion of the f luorescent dye, Ca lcofluor White. 49

xi

2.2 The thecae of d inoflagel l ates with and without the additio n . o f F I TC-tagged l ectins, photographed under b lue l ight excitat ion . 49

3.1 G ro wth rates (doubl ings per d ay) of severa l Chrysochromulina

spec ies at d i fferent temp eratures . 65

3.2 G ro wth rates (doubl ings per d ay) of several Chrysochromulina

species at d i fferent sa l in it ies . 67

3.3 Growth rates (doubl ings per day) of several Chrysochromulina

species at d i fferent p H . 68

3.4 G rowth rates of Emiliania huxleyi, Gephyrocapsa oceanica

and Pleurochrysis sp. at d ifferent temperatures. 7 2

3.5 G rowth rates of Emiliania huxleyi and Gephyrocapsa oceanica

at d i fferent sa l in ities. 73

3.6 G rowth rates of Emf/iania huxleyi, Gephyrocapsa oceanica

and Pleurochrysis sp. at d ifferent p H . 74

4. 1 M aps showing sites of sampl ing and b loom events referred to in this study . 83-4

4.2 Ce l l numbers of indiv idua l b loom species at Leigh, August 1 99 2 to January 1 9 93 and estimated ce l l volume of those s p ec ies . 88

4.3 Micrographs of the dominant species i nvolved in the N orth land bloom, spr ing-summer 1 99 2 . 92

4.4 Light micrograph of Gephyrocapsa oceanica i n sca l lop guts conta in ing domoic ac id from Rangaunu Bay, N o rth land and Pseudonitzschia australis from sea water samples . 92

4.5 D i str ibution of Emiliania huxleyi within Big G lory Bay and Paterson I n l et , 26 November 1 9 9 2 . 93

4.6 Maximum cel l numbers of Emiliania huxleyi from onset to demise of a bloom at Big G lory Bay, Stewart I s l and, October-December 1 992. 94

4.7 Cel l n u m bers of b loom s pecies at M arsden Point, O ctober to December 1 993 . 9 7

xii

5.1 U-tube a pparatus used in the screen ing for a l l elochem ica ls i n m ar ine m icroalgae. 1 08

5.2 Resu lts of dua l culture experiments . 1 1 1

5.3 The effect of addit ion of Chlamydomonas coccoides to exponent ia l phase cultures of Chattonella antiqua. 1 1 2

5 . 4 Extract ion of blood from anaesthetised sa lmon ( Oncorhynchus tshawytscha) . 1 25

5.5 Paua l a rval b ioassays . 1 25

5.6 M icroalga l b ioassays . 1 29

5 . 7 H aemolys is of sa lmon erythrocytes by m icroa lga l cu lture extracts . 1 28

5.8 Sa lmon e rythrocytes . 1 29

Tables

1

1 . 1

1 . 2

1 . 3

2 . 1

2 . 2

2 . 3

2 . 4

3.1

3 . 2

3 . 3

3 . 4

4. 1

Classificatio n of Prymnesiophyta (Chn§tiennot-D inet et al. ,

199 3 ) .

Pub l ished records of p rymnesiophytes from New Zealand coastal waters , identif ied by sca les using e lectron m icroscopy .

D ifferences i n taxonomic characteristics between Chrysochromulina ericina ( iso lated from M ar lborough Sounds, New Zeal and ) , C. ericina and C. quadrikonta ( i so lated in Ne lson, New Zeala nd ) .

D i fferences i n taxo nomic characteristics between Chrysochromulina hirta from the Mar lborough Sounds, New Zealand , from south-western Austral i a and f rom the G a lapagos I s l and s .

F ITC-conjugated l ectins used as probes .

Carbohydrates used to i nhibit b ind ing of F ITC-Iabel led l ectin .

F ITC-conjugated l ectins and Ca lcofluor used as f luorescent probes; the i r sou rce and specificity .

C arbohydrate inhibit ion of FITC-Iabe l led l ectins .

Se len ium as a l im it ing growth factor i n Chrysochromulina

species .

G rowth rates (doubl ings p e r day) of Chrysochromulina

species cu ltured with d ifferent n itrogen sources .

G rowth rates (doubl ings per day) of Chrysochromulina

s pecies cultured at d i fferent l i ght i ntensiti e s .

G rowth rates o f coccol ithophores cu ltured with d ifferent n i trogen sources .

Phytop lankton s pecies present , and their abundance, at the height of the raphidophyte dominated b loom at Leigh, 8 October 199 2 .

xiii

page

8

1 2

2 2

26

44

44

4 7-8

5 1

63

64

69

7 1

89

4.2 Comparison of ce l l numbers of dominant species at sites throughout the north-eastern coastal reg ion of New Zea land , 1 3 November 1 99 2 .

4.3 Species iso l ated, c ultured and tested for i chthyotoxic ity by the A rtemia salina bioassay during the raphidophyte-coccol ithophore b loom of 1 99 2 .

4.4 N itroge n : phosphate ( N : P) molar ratios ca lcu lated from water co lumn a n alyses of samples from within B ig G lory Bay, Stewart I s land , 1 988- 1 9 9 2 .

5 . 1 Microa lga l species i nvestigated for a l l e lopathic i nteract ions .

5.2 Microa lga l species tested in dua l culture for a l le lopathic i nteractions .

5.3 Resu lts of Artemia salina bioassays for d etection of m icroa lga l toxicity.

5.4 Resu lts of Haliotis iris bioassays for detection of m icro a lga l toxicity .

5 . 5 Evaluat ion o f Chattone/la antiqua a n d Heterocapsa triquetra

as b ioassay o rganisms by testing w ith known toxic microa lga l species.

XIV

90

90

1 00

1 07

1 1 0

1 24

1 26

1 27

Abbreviations

aff.

BSA

cf.

CHRY

ConA

d

OAPI

OMS

OTT

ECA

EOTA

FITC

GC

GP

GR

h

HPA

HPLC

MAF ORPP

N:P

Na; NaOH

PAHBAH

PEA

PHA

PWM

SBA

Se

SEM

sp.

SST

t050

tM50

TEM

TES

UEA

UV

v/v

WGA

% 0

has an affinity with

bovine serum albumin

compares with

Chrysochromufina

Concanavalin A

day

4'6-diamidino-2-phenylindole

dimethylsulphide

d ithiothreitol

Erythrina cristagalli A (coral tree) lectin

ethylenediamine tetraacetic acid

Fluorescein isothiocyanate

gas chromatography

general purpose

growth rate

hour

Helix pomatia A (snail) lectin

high performance liquid chromatography

Ministry of Agriculture and Fisheries operational research

phytoplankton programme

nitrogen: phosphate

sodium; sodium hydroxide

para hydroxy-benzoic acid hydrazide

Pisum sativum (pea) lectin

Phaseolus limensis (lima bean) lectin

Phytolacca americana (pokeweed) lectin

Glycine max (soy bean) lectin

selenium

scanning electron microscopy

species

sea surface temperature

time until 50% of bioassay organisms are dead

time until 5 0% of bioassay organisms are morbid

transmission electron microscopy

(N-tris[hydroxymethyIJ-methyl-2-aminoethane-sulfonic acid)

Ulex europaeus (gorse) lectin

ultraviolet

volume per volume

Triticum vulgaris (wheat germ) lectin

salinity (grams salt per kilogram seawater) in parts per thousand

xv