Bonamia spp of the Phylum Haplosporidia are ob -ligate intrahaemocytic protistan parasites primarilyof ostreid oysters (Carnegie amp Cochennec-Laureau2004 Engelsma et al 2014 this DAO Special) Fourspecies have been described B ostreae infects Ostreaedulis (Pichot et al 1979) and possibly O angasi (seeBougrier et al 1986) O chilensis (see Grizel et al 1983)
and O puelchana (see Pascual et al 1991) It may alsoinfect Crassostrea spp (Car ne gie amp Cochennec- Laureau2004) and C gigas may act as a re servoir for B os-treae (see Lynch et al 2010) It is reported from Oedulis in Atlantic and Mediterranean Europe Mo-rocco (A Villalba pers obs http web oie int enginfo hebdo AIS_43 HTM Sec2) and western (Elstonet al 1986 Friedman et al 1989 Marty et al 2006)and eastern (Friedman amp Perkins 1994) North America
Ultrastructural comparison of Bonamia spp (Haplosporidia) infecting ostreid oysters
P M Hine16 R B Carnegie2 M A Kroeck3 A Villalba4 M Y Engelsma5 E M Burreson2
1Investigation and Diagnostic Centre Biosecurity New Zealand PO Box 40-742 Upper Hutt 6007 New Zealand 2Virginia Institute of Marine Science College of William amp Mary PO Box 1346 Gloucester Point Virginia 23062 USA
3LABPAT-IBMP (L 709) Instituto de Biologiacutea Marina y Pesquera lsquoAlte Stornirsquo Universidad Nacional del Comahue (8520) San Antonio Oeste Rio Negro Argentina
4Centro de Investigacioacutens Marintildeas Conselleriacutea do Medio Rural e do Mar Xunta de Galicia Aptdo 13 36620 Vilanova de Arousa Spain
5Central Veterinary Institute of Wageningen UR PO Box 65 8200 AB Lelystad The Netherlands
6Present address 73 rue de la Feacutee au Bois 17450 Fouras France
ABSTRACT The ultrastructure of Bonamia from Ostrea angasi from Australia Crassostrea aria -kensis from the USA O puelchana from Argentina and O edulis from Spain was compared withdescribed Bonamia spp All appear conspecific with B exitiosa The Bonamia sp from Chile hadsimilarities to the type B exitiosa from New Zealand (NZ) but less so than the other forms recog-nized as B exitiosa Two groups of ultrastructural features were identified those associated withmetabolism (mitochondrial profiles lipid droplets and endoplasmic reticulum) and those associ-ated with haplosporogenesis (Golgi indentations in the nuclear surface the putative trans-Golginetwork perinuclear granular material and haplosporosome-like bodies) Metabolic featureswere regarded as having little taxonomic value and as the process of haplosporogenesis is notunderstood only haplosporosome shape and size may be of taxonomic value However the uni-nucleate stages of spore-forming haplosporidians are poorly known and may be confused withBonamia spp uni-nucleate stages The many forms of NZ B exitiosa have not been observed inother hosts which may indicate that it has a plastic life cycle Although there are similaritiesbetween NZ B exitiosa and Chilean Bonamia in the development of a larger uni-nucleate stageand the occurrence of cylindrical confronting cisternae the clarification of the identity of ChileanBonamia must await molecular studies
Resale or republication not permitted without written consent of the publisher
Contribution to DAO Special 7 Microcell parasites of molluscs FREEREE ACCESSCCESS
Dis Aquat Org 110 55ndash63 2014
It is an internationally notifiable OIE-listed pathogenBonamia (syn Mikrocytos) roughleyi was describedfrom Saccostrea glo merata (syn commercialis) fromAustralia (Farley et al 1988 Cochennec-Laureau etal 2003) but has not been subsequently isolated andits validity as a Bo na mia species is doubtful (Hill et al2010 Carnegie et al 2014)
B exitiosa (syn exitiosus) infects O (syn Tiostrea)chilensis (Hine et al 2001 Berthe amp Hine 2003) inNew Zealand (NZ) O angasi (see Corbeil et al 2006)in Australia O puelchana in Argentina (Kroeck ampMontes 2005 Kroeck et al 2008 Kroeck 2010) Oedulis off the Atlantic (Abollo et al 2008) and theMediterranean (Narcisi et al 2010) coasts of Europeand O stentina in Tunisia (Hill et al 2010) North andSouth Carolina (USA) NZ and Argentina (Hill et al2010) It also occurs in C ariakensis at a port in NorthCarolina into which it could conceivably have beenintroduced from Australia by shipping (Burreson etal 2004 Bishop et al 2006) Like B ostreae it is anOIE-listed and internationally notifiable pathogen Bperspora infects O (syn Ostreola) stentina (synequestris) in the southeastern USA and differs fromall other reported Bonamia spp in having a sporestage (Carnegie et al 2006) B exitiosa-like isolateshave been reported from O chilensis in Chile (Bal-seiro et al 2006 Campalans amp Lohrmann 2009 Lohr -mann et al 2009)
Bonamia spp have different patterns of develop-ment and of developmental stages B perspora ap -pears to have very few uni-nucleate bi-nucleatediplokaryotic and small multi-nucleate plasmodialvegetative stages Plasmodia develop into sporontssporoblasts sporocysts and spores as do Haplo spo ri -dium spp and Minchinia spp (Carnegie et al 2006)NZ B exitiosa has many uni-nucleate fewer bi-nucle-ate and diplokaryotic stages and no sporulationstages and many uni-nucleate cells grow into largeforms (Hine 1991a Hine et al 2001) Chilean Bo na -mia sp is similar to B exitiosa in developing largeuni-nucleate stages during winter but bi-nucleatestages are rare and multi-nucleate plasmodia unre-ported (Lohrmann et al 2009) B ostreae has denseand light forms of the uni-nucleate stage which maybecome bi-nucleate or rarely diplokaryotic (Pichot etal 1979) but plasmodia are small and very rarely en -countered Large plasmodia reported from O edulis(Breheacutelin et al 1982 Vivaregraves et al 1982 Bonami et al1985) at one site in Brittany were probably vegetativestages of H armoricanum Therefore initial uni- nucleate stages may be directly compared but the de-velopment of large uni-nucleate forms in B exitiosaand the Chilean Bonamia may not be directly compa-
rable with the uni-nucleate forms of B perspora and Bostreae There are also 2 forms of bi-nucleate cellthose with separated nuclei after karyokinesis and be-fore cytokinesis common in B ostreae (see Balouet etal 1983) and those with 2 apposed nuclei in a di plo -karyon which are common in B perspora (Carnegieet al 2006) and B exitiosa (Hine 1991a Hine et al2001) but rare in B ostreae (Pichot et al 1979)
For brevity the ultrastructures of published Bo na -mia spp will not be illustrated here as they arealready well illustrated (Pichot et al 1979 Hine et al2001 Carnegie et al 2006 Lohrmann et al 2009)Also previous studies nicely illustrate important fea-tures in this study such as nuclear membrane-boundGolgi (NM-BG) indentations in the nuclear surface(INS) perinuclear granular material a reticulatedstructure which is the trans-Golgi network (TGN)and haplosporosome-like bodies (H-LBs) (Hine 1991a1992 Hine amp Wesney 1994a Hine et al 2001) Herewe compared B exitiosa from C ariakensis on the USeast coast O puelchana in Argentina and O edulisin Spain and considered what features permit theirdiscrimination from other Bonamia species Initiallyin this study all cellular features were recorded butsubsequent ana lysis of nuclear shape size and endo-plasmic reticulum failed to show any patterns andare therefore not included herein
MATERIALS AND METHODS
The fixation and processing of tissues infected withBonamia spp for transmission electron microscopy(TEM) has been reported for B exitiosa (Hine et al2001) B perspora (Carnegie et al 2006) and ChileanBonamia sp (Lohrmann et al 2009) It should benoted that there was variation in methods of fixationand processing particularly the use of reduced os-mium tetroxide (OsO4) instead of normal OsO4 in theChilean study which enhanced membranes possiblymaking NM-BG more apparent The oysters infectedwith B ostreae in our study were sampled from LakeGrevelingen The Netherlands and fixed for 1 h in25 glutaraldehyde in 022 microm filtered seawater(FSW) washed 2times in FSW post-fixed for 1 h in 1OsO4 stained en bloc with 5 uranyl acetate in 01 Msodium acetate for 45 min dehydrated through 50 to100 ethanol cut and stained with 5 uranyl acetatefor 10 min and 5 lead citrate for 5 to 6 min
Ostrea puelchana infected with B exitiosa weresampled from San Matias Gulf northern PatagoniaArgentina Hearts were fixed for 1 h in 25 glu-taraldehyde in 045 microm FSW and washed 3times in FSW
56
Hine et al Bonamia spp ultrastructure
post-fixed in 1 OsO4 buffered with 01 M sodiumcacodylate at pH 72 dehydrated through a gradedethanol series including en bloc staining with 1uranyl acetate at the 70 ethanol stage and embed-ded in Spurrrsquos resin Ultrathin (~90 nm) sections weremounted on carbon-stabilised formvar-coated 1-holegrids stained with Reynoldrsquos lead citrate and exam-ined on a Zeiss CEM 902 TEM Crassostrea ariaken-sis infected with B exitiosa from North Carolina(USA) were processed following methods used ear-lier for observation of B perspora (Carnegie et al2006) and examined on a Zeiss CEM 902 TEM
The measurements given are from sections throughthe centre of the cell in which the bilaminar nuclearand plasma membranes are clearly defined Cell sizenuclear size and the size of haplosporosomes given inthe following tables were calculated as the means ofthe longest dimension multiplied by the dimension atright angles to it The nucleuscytoplasmic (NC) ratiowas the mean of nuclear dimensions expressed as apercentage of the mean size of the cell The termlsquomulti-nucleatersquo is used for cells with more than 2nuclei rather than lsquoplasmodiumrsquo which is reservedfor a pre-sporulation stage
RESULTS
All Bonamia spp occurred as uni-nucleate and bi-nucleate forms and a multi-nucleate stage may occurin all species but was not encountered because of rar-ity NZ B exitiosa and B perspora also have a diplo -karyotic stage TEM observations on moribund Broughleyi showed a multi-nucleate stage (Cochen-nec-Laureau et al 2003) that appeared to undergoschizogony to form the uni-nucleate stage unlikeBonamia spp and other known haplosporidians (PM Hine pers obs) suggesting that it may not be aBonamia species
Uni-nucleate B exitiosa from Ostrea angasi in Aus-tralia Crassostrea ariakensis from the eastern USA(Fig 1B) O puelchana from Argentina and SpanishO edulis (Fig 1A) (Table 1) resemble NZ lsquotypersquo B exi-tiosa (Table 2) being larger than B ostreae (Fig 2)with smaller haplo spo ro somes and slightly largerthan Chilean Bonamia with larger haplosporosomesHaplosporogenesis was less often observed in B exi-tiosa from C ariakensis than in other species (Table 1)except B ostreae (Table 2) The uni-nucleate stage ofB perspora was larger but with smaller chromosomes(Table 2) (Fig 3A) than the other Bonamia spp withsubsequent multi-nucleate plasmodia (Fig 3B) devel-oping into spores (Fig 4) Few data were available on
57
Sp
ecie
s
Ori
gin
n
H
ost
S
ize
N
C
Hap
losp
orog
enes
is
Mit
och
ond
rial
L
ipid
dro
ple
ts
(
microm
2 )
()
Pre
v
N
M-B
G T
GN
INS
Hap
losp
oros
omes
H-L
Bs
p
rofi
les
N
o
Pre
v
(
)
(
)
()
(
)
Not
es
No
S
ize
(microm
2 )
N
o
R
ang
e
No
R
ang
e
(
)
B e
xiti
osa
A
ust
rali
a
45
O
stre
a an
gas
i
28
plusmn 0
4
N
R
NR
N
R
N
R
N
R
10
plusmn 4
1
56 plusmn
15
NR
4
plusmn 1
0
5 plusmn
08
30
B e
xiti
osa
A
rgen
tin
a
27
O
pu
elch
ana
3
1 plusmn
03
4
9 plusmn
6
6
1
4
3
25
3
1
Sli
gh
t
1
5 plusmn
7
148
plusmn 1
2
4
0
05
3 plusmn
08
0minus
2
3 plusmn
2
1minus
6
06
plusmn 1
1
37B
exi
tios
a
Eas
t U
SA
4
5
C a
riak
ensi
s
28
plusmn 0
4
50
plusmn 4
18
9
0
5 F
latt
ened
18 plusmn
10
15
2 plusmn
8
13
0
2 plusmn
05
3 plusmn
1
0minus
7
08
plusmn 0
9
52B
exi
tios
a
S
pai
n
4
O e
du
lis
27
(2
8
53
ndash57
50
2
5
0
2
5
17
12
1
54 plusmn
20
0
7
5
2
minus7
1
1 1
0
75
2
5 2
6 2
7)
11
7
(
n =
12)
4 2
Bon
amia
sp
Ch
ile
59
O
ch
ilen
sis
21
plusmn 0
4
58 plusmn
12
4
8
4
1
2
0
1
3 plusmn
7
138
plusmn 1
9
2
0
037
plusmn 1
06
0minus
7
2 plusmn
1
0minus
5
03
plusmn 0
4
26
Tab
le 1
Com
par
ison
of
the
un
i-n
ucl
eate
sta
ge
of B
onam
iasp
p H
aplo
spor
ogen
esis
in t
his
an
d t
he
foll
owin
g t
able
s is
cal
cula
ted
on
th
e n
um
ber
of
ind
ivid
ual
s h
avin
g n
u-
clea
r m
emb
ran
e-b
oun
d G
olg
i (N
M-B
G)
tra
ns-
Gol
gi n
etw
ork
(T
GN
) i
nd
enta
tion
s in
th
e n
ucl
ear
surf
ace
(IN
S)
or
hap
losp
oros
ome-
lik
e b
odie
s (H
-LB
s) S
ize
is le
ng
th times
w
idth
NC
nu
cleu
scy
top
lasm
rat
io P
rev
pre
vale
nce
NR
not
rec
ord
ed C
ari
aken
sis
Cra
ssos
trea
ari
aken
sis
Dat
a ar
e m
ean
s plusmn
SD
Sp
ecie
s
O
rig
in
n
Hos
t
Ref
eren
ce
Siz
e
N
C
Hap
losp
orog
enes
is
Mit
och
ond
rial
L
ipid
dro
ple
ts
(micro
m2 )
P
rev
NM
-BG
TG
N I
NS
Hap
losp
oros
omes
H
-LB
s
p
rofi
les
N
o
Pre
v
(
)
()
()
(
)
N
o
Siz
e (micro
m2 )
(
)
No
Ran
ge
N
o
R
ang
e
(
)
B o
stre
ae
Net
her
lan
ds
23
Ost
rea
edu
lis
P
ich
ot e
t al
(19
79)
2
1 plusmn
04
55
plusmn 7
14
27
0
0
9
plusmn 4
1
88 plusmn
21
26 0
30
plusmn 0
47
0minus
1
1 plusmn
1
0
minus4
04
plusmn 0
6
30
B e
xiti
osa
N
ew Z
eala
nd
60
O
ch
ilen
sis
H
ine
et a
l (
2001
)
32
plusmn 0
5
52 plusmn
6
52
11
32
5
29 plusmn
17
148
plusmn 1
1
21
04
7 plusmn
09
6
0minus3
4
plusmn 1
2minus
6
0
5 plusmn
09
3
2B
per
spor
a
Eas
t U
SA
N
G
O s
ten
tin
a
Car
neg
ie e
t al
(20
06)
41
plusmn 0
6
NG
N
G
N
G
N
G
NG
3 plusmn
3
137
plusmn 1
9
NG
6
plusmn 2
NG
NG
38
Tab
le 2
Com
par
ison
of t
he
un
i-n
ucl
eate
sta
ge
of th
e 3
des
crib
ed B
onam
iasp
p N
M-B
G n
ucl
ear
mem
bra
ne-
bou
nd
Gol
gi
TG
N t
ran
s-G
olg
i net
wor
k I
NS
in
den
tati
ons
in th
e n
ucl
ear
surf
ace
H-L
Bs
hap
losp
oros
ome-
lik
e b
odie
s N
C n
ucl
eus
cyto
pla
sm r
atio
Pre
v p
reva
len
ce N
G n
ot g
iven
Dat
a ar
e m
ean
s plusmn
SD
Dis Aquat Org 110 55ndash63 2014
diplokaryotic and bi-nucleate forms because of thedifficulty in getting central sections through bothnuclei (Table 3) The diplokaryon of NZ B exitiosawas similar in size to the bi-nucleate forms inArgen tinian oysters but had a higher NC ratiohaplospo rogenesis and lipid droplets Haplosporo-some numbers were higher in NZ and ArgentinianB exitiosa and Chilean Bonamia sp than in B os-treae but haplosporogenesis was more prevalent inNZ B exitiosa than in the other species whichshowed similar levels of haplosporogenesis (Table 3)
With increase in size each Bonamia showed ageneral increase in haplosporogenesis number ofmitochondrial profiles and lipid except in Argen-tinian B exitiosa and B exitiosa from C ariakensis(Table 4) The size groups of B exitiosa in C ariak-ensis and more so Chilean Bonamia sp and Bostreae were smaller than those of Argentinianand NZ B exitiosa In relation to seasonal differ-ences Bonamia from O chilensis in NZ and Chilesampled at different times of the year showed thatwhile NZ B exitiosa showed a great increase in allparameters Chilean Bonamia showed the sametrend but less so (Table 5)
58
Species Origin n Stage Size NC Haplosporogenesis Mitochondrial Lipid droplets (microm2) () Prev NM-BG TGN INS No of haplo- H-LBs profiles No Prev () () () () sporosomes ()
Table 3 Comparison of bi-nucleate and diplokaryotic stages of Bonamia spp NM-BG nuclear membrane-bound Golgi TGNtrans-Golgi network INS indentations in the nuclear surface H-LBs haplosporosome-like bodies NC nucleuscytoplasm
ratio Prev prevalence
Size Haplosporogenesis Mitochondrial Lipid dropletsgroup Prev No of haplo- profiles No Prev(microm) () sporosomes ()
Table 5 Comparison of Bonamia infecting Ostrea chilensis in New Zealand and Chile in relation to season Data are means plusmn SD Prev prevalence
Hine et al Bonamia spp ultrastructure
DISCUSSION
The problem with comparative ultrastructural stud-ies on congeneric undescribed species as in molecularstudies showing DNA sequence divergence amongsuch species is deciding how much difference consti-tutes a separate species While molecular studies re-veal genotypes and are quantifiable in base transi-tions ultrastructural studies reveal phenotypes whichmay be subject to many variables These include hostfactors such as the species concerned its age physio-logical state reproductive state and the presence ofother infections (Hine 2002) environmental factorssuch as water temperature and salinity (Audemard etal 2008 Carnegie et al 2008) and methods of sam-
59
Fig 1 Uni-nucleate stages of Bonamia exitiosa in (A) Ostreaedulis from Spain and (B) Crassostrea ariakensis from theUSA Host haemocyte cytoplasm indicated in both cases (H)to mark the exterior of the B exitiosa cells Note the widelyvarying abundance of haplosporosomes (arrows) and theparallel arrays of smooth endoplasmic reticulum (arrow-
head) in (A) Both scale bars = 1 microm
Fig 2 Bonamia ostreae uni-nucleate stages in Ostrea edulis(A) Low-power image of 2 cells infecting a single haemo-cyte Note the displacement of B ostreae nucleus to cell periphery a common observation and distinct trait of thisBonamia species Note also an extracellular haplosporosome(arrow) present along with numerous cytoplasmic haplo -sporosomes Scale bar = 1 microm (B) High-power image of asingle B ostreae cell Note again the abundant haplosporo-somes with smooth endoplasmic reticulum also displayedalong with an intranuclear microtubule (arrow) Scale bar =
05 microm
Dis Aquat Org 110 55ndash63 2014
pling fixation and post-fixation Parasite factors suchas the stages present at the time of sampling whichmay be seasonal (Hine 1991ab) the physiologicalstate of the parasite and possible plasticity of life cy-cles have also to be considered In the latter case al-though a spore stage is not known for Bo na mia exi-tiosa it does not necessarily mean that spores may notbe formed under certain environmental conditions
Interpretation of the ultrastructural features ofBonamia spp must take these variables into accountThese features fall into 3 groups parasite metabo-lism haplosporogenesis and sporogony the latter
only being known in B perspora The metabolic stateof the parasite is indicated by the mitochondria lipiddroplets and endoplasmic reticulum which are mostobvious in the development from a small intracellularuni-nucleate early stage to the large extracellularamoeboid feeding stage with large amounts of lipidmitochondria and parallel arrays of endoplasmic re ti -culum in NZ B exitiosa (see Hine amp Wesney 1994a)High lipid content was linked to season and develop-ment in the lipid-rich ovary in autumn to early winter(April to June Hine 1991b) Seasonal annual devel-opment may also be linked to temperature in C ari-akensis infections (Carnegie et al 2008) The produc-tion of lipid vesicles from and acid hydrolases inlipid droplets are associated with formation of a par-asitophorous vacuole (PV Hine amp Wesney 1994b)and 36 of Bonamia sp in Crassostrea ariakensiswere in PVs Therefore features reflecting metabo-lism are of little taxonomic value unless multiple vari-ables are taken into account
The taxonomic significance of haplosporogenesisas indicated by NM-BG TGN INS perinuclear gran-ular material and H-LBs (Hine amp Wesney 1992) isdifficult to assess as the function of haplosporosomesis unknown Whilst it has been suggested that theirglycoprotein coat and putative DNA core resemblesthe structure of viruses their release associated withlysis of surrounding cells suggests a lytic function(Hine et al 2002) but subsequent re-entry of cores in
60
Fig 3 Vegetative stages of Bonamia perspora in Ostreastentina from the USA (A) Uni-nucleate stage (B) Smalltrinucleate plasmodium Note the abundant mitochondriaparticularly closely apposed to the nucleus in (A) the longstretch of endoplasmic reticulum head (arrows) in (B) andthe general paucity of haplosporosomes Dark bodies in (A)
are lipid bodies Both scale bars = 1 microm
Fig 4 Prespore of Bonamia perspora in Ostrea stentina fromthe USA Note the developing wall (arrow) with episporecytoplasm (ES) still present and the well-defined lid (L)
nucleus (N) and spherule (S) Scale bar = 1 microm
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Dis Aquat Org 110 55ndash63 2014
It is an internationally notifiable OIE-listed pathogenBonamia (syn Mikrocytos) roughleyi was describedfrom Saccostrea glo merata (syn commercialis) fromAustralia (Farley et al 1988 Cochennec-Laureau etal 2003) but has not been subsequently isolated andits validity as a Bo na mia species is doubtful (Hill et al2010 Carnegie et al 2014)
B exitiosa (syn exitiosus) infects O (syn Tiostrea)chilensis (Hine et al 2001 Berthe amp Hine 2003) inNew Zealand (NZ) O angasi (see Corbeil et al 2006)in Australia O puelchana in Argentina (Kroeck ampMontes 2005 Kroeck et al 2008 Kroeck 2010) Oedulis off the Atlantic (Abollo et al 2008) and theMediterranean (Narcisi et al 2010) coasts of Europeand O stentina in Tunisia (Hill et al 2010) North andSouth Carolina (USA) NZ and Argentina (Hill et al2010) It also occurs in C ariakensis at a port in NorthCarolina into which it could conceivably have beenintroduced from Australia by shipping (Burreson etal 2004 Bishop et al 2006) Like B ostreae it is anOIE-listed and internationally notifiable pathogen Bperspora infects O (syn Ostreola) stentina (synequestris) in the southeastern USA and differs fromall other reported Bonamia spp in having a sporestage (Carnegie et al 2006) B exitiosa-like isolateshave been reported from O chilensis in Chile (Bal-seiro et al 2006 Campalans amp Lohrmann 2009 Lohr -mann et al 2009)
Bonamia spp have different patterns of develop-ment and of developmental stages B perspora ap -pears to have very few uni-nucleate bi-nucleatediplokaryotic and small multi-nucleate plasmodialvegetative stages Plasmodia develop into sporontssporoblasts sporocysts and spores as do Haplo spo ri -dium spp and Minchinia spp (Carnegie et al 2006)NZ B exitiosa has many uni-nucleate fewer bi-nucle-ate and diplokaryotic stages and no sporulationstages and many uni-nucleate cells grow into largeforms (Hine 1991a Hine et al 2001) Chilean Bo na -mia sp is similar to B exitiosa in developing largeuni-nucleate stages during winter but bi-nucleatestages are rare and multi-nucleate plasmodia unre-ported (Lohrmann et al 2009) B ostreae has denseand light forms of the uni-nucleate stage which maybecome bi-nucleate or rarely diplokaryotic (Pichot etal 1979) but plasmodia are small and very rarely en -countered Large plasmodia reported from O edulis(Breheacutelin et al 1982 Vivaregraves et al 1982 Bonami et al1985) at one site in Brittany were probably vegetativestages of H armoricanum Therefore initial uni- nucleate stages may be directly compared but the de-velopment of large uni-nucleate forms in B exitiosaand the Chilean Bonamia may not be directly compa-
rable with the uni-nucleate forms of B perspora and Bostreae There are also 2 forms of bi-nucleate cellthose with separated nuclei after karyokinesis and be-fore cytokinesis common in B ostreae (see Balouet etal 1983) and those with 2 apposed nuclei in a di plo -karyon which are common in B perspora (Carnegieet al 2006) and B exitiosa (Hine 1991a Hine et al2001) but rare in B ostreae (Pichot et al 1979)
For brevity the ultrastructures of published Bo na -mia spp will not be illustrated here as they arealready well illustrated (Pichot et al 1979 Hine et al2001 Carnegie et al 2006 Lohrmann et al 2009)Also previous studies nicely illustrate important fea-tures in this study such as nuclear membrane-boundGolgi (NM-BG) indentations in the nuclear surface(INS) perinuclear granular material a reticulatedstructure which is the trans-Golgi network (TGN)and haplosporosome-like bodies (H-LBs) (Hine 1991a1992 Hine amp Wesney 1994a Hine et al 2001) Herewe compared B exitiosa from C ariakensis on the USeast coast O puelchana in Argentina and O edulisin Spain and considered what features permit theirdiscrimination from other Bonamia species Initiallyin this study all cellular features were recorded butsubsequent ana lysis of nuclear shape size and endo-plasmic reticulum failed to show any patterns andare therefore not included herein
MATERIALS AND METHODS
The fixation and processing of tissues infected withBonamia spp for transmission electron microscopy(TEM) has been reported for B exitiosa (Hine et al2001) B perspora (Carnegie et al 2006) and ChileanBonamia sp (Lohrmann et al 2009) It should benoted that there was variation in methods of fixationand processing particularly the use of reduced os-mium tetroxide (OsO4) instead of normal OsO4 in theChilean study which enhanced membranes possiblymaking NM-BG more apparent The oysters infectedwith B ostreae in our study were sampled from LakeGrevelingen The Netherlands and fixed for 1 h in25 glutaraldehyde in 022 microm filtered seawater(FSW) washed 2times in FSW post-fixed for 1 h in 1OsO4 stained en bloc with 5 uranyl acetate in 01 Msodium acetate for 45 min dehydrated through 50 to100 ethanol cut and stained with 5 uranyl acetatefor 10 min and 5 lead citrate for 5 to 6 min
Ostrea puelchana infected with B exitiosa weresampled from San Matias Gulf northern PatagoniaArgentina Hearts were fixed for 1 h in 25 glu-taraldehyde in 045 microm FSW and washed 3times in FSW
56
Hine et al Bonamia spp ultrastructure
post-fixed in 1 OsO4 buffered with 01 M sodiumcacodylate at pH 72 dehydrated through a gradedethanol series including en bloc staining with 1uranyl acetate at the 70 ethanol stage and embed-ded in Spurrrsquos resin Ultrathin (~90 nm) sections weremounted on carbon-stabilised formvar-coated 1-holegrids stained with Reynoldrsquos lead citrate and exam-ined on a Zeiss CEM 902 TEM Crassostrea ariaken-sis infected with B exitiosa from North Carolina(USA) were processed following methods used ear-lier for observation of B perspora (Carnegie et al2006) and examined on a Zeiss CEM 902 TEM
The measurements given are from sections throughthe centre of the cell in which the bilaminar nuclearand plasma membranes are clearly defined Cell sizenuclear size and the size of haplosporosomes given inthe following tables were calculated as the means ofthe longest dimension multiplied by the dimension atright angles to it The nucleuscytoplasmic (NC) ratiowas the mean of nuclear dimensions expressed as apercentage of the mean size of the cell The termlsquomulti-nucleatersquo is used for cells with more than 2nuclei rather than lsquoplasmodiumrsquo which is reservedfor a pre-sporulation stage
RESULTS
All Bonamia spp occurred as uni-nucleate and bi-nucleate forms and a multi-nucleate stage may occurin all species but was not encountered because of rar-ity NZ B exitiosa and B perspora also have a diplo -karyotic stage TEM observations on moribund Broughleyi showed a multi-nucleate stage (Cochen-nec-Laureau et al 2003) that appeared to undergoschizogony to form the uni-nucleate stage unlikeBonamia spp and other known haplosporidians (PM Hine pers obs) suggesting that it may not be aBonamia species
Uni-nucleate B exitiosa from Ostrea angasi in Aus-tralia Crassostrea ariakensis from the eastern USA(Fig 1B) O puelchana from Argentina and SpanishO edulis (Fig 1A) (Table 1) resemble NZ lsquotypersquo B exi-tiosa (Table 2) being larger than B ostreae (Fig 2)with smaller haplo spo ro somes and slightly largerthan Chilean Bonamia with larger haplosporosomesHaplosporogenesis was less often observed in B exi-tiosa from C ariakensis than in other species (Table 1)except B ostreae (Table 2) The uni-nucleate stage ofB perspora was larger but with smaller chromosomes(Table 2) (Fig 3A) than the other Bonamia spp withsubsequent multi-nucleate plasmodia (Fig 3B) devel-oping into spores (Fig 4) Few data were available on
57
Sp
ecie
s
Ori
gin
n
H
ost
S
ize
N
C
Hap
losp
orog
enes
is
Mit
och
ond
rial
L
ipid
dro
ple
ts
(
microm
2 )
()
Pre
v
N
M-B
G T
GN
INS
Hap
losp
oros
omes
H-L
Bs
p
rofi
les
N
o
Pre
v
(
)
(
)
()
(
)
Not
es
No
S
ize
(microm
2 )
N
o
R
ang
e
No
R
ang
e
(
)
B e
xiti
osa
A
ust
rali
a
45
O
stre
a an
gas
i
28
plusmn 0
4
N
R
NR
N
R
N
R
N
R
10
plusmn 4
1
56 plusmn
15
NR
4
plusmn 1
0
5 plusmn
08
30
B e
xiti
osa
A
rgen
tin
a
27
O
pu
elch
ana
3
1 plusmn
03
4
9 plusmn
6
6
1
4
3
25
3
1
Sli
gh
t
1
5 plusmn
7
148
plusmn 1
2
4
0
05
3 plusmn
08
0minus
2
3 plusmn
2
1minus
6
06
plusmn 1
1
37B
exi
tios
a
Eas
t U
SA
4
5
C a
riak
ensi
s
28
plusmn 0
4
50
plusmn 4
18
9
0
5 F
latt
ened
18 plusmn
10
15
2 plusmn
8
13
0
2 plusmn
05
3 plusmn
1
0minus
7
08
plusmn 0
9
52B
exi
tios
a
S
pai
n
4
O e
du
lis
27
(2
8
53
ndash57
50
2
5
0
2
5
17
12
1
54 plusmn
20
0
7
5
2
minus7
1
1 1
0
75
2
5 2
6 2
7)
11
7
(
n =
12)
4 2
Bon
amia
sp
Ch
ile
59
O
ch
ilen
sis
21
plusmn 0
4
58 plusmn
12
4
8
4
1
2
0
1
3 plusmn
7
138
plusmn 1
9
2
0
037
plusmn 1
06
0minus
7
2 plusmn
1
0minus
5
03
plusmn 0
4
26
Tab
le 1
Com
par
ison
of
the
un
i-n
ucl
eate
sta
ge
of B
onam
iasp
p H
aplo
spor
ogen
esis
in t
his
an
d t
he
foll
owin
g t
able
s is
cal
cula
ted
on
th
e n
um
ber
of
ind
ivid
ual
s h
avin
g n
u-
clea
r m
emb
ran
e-b
oun
d G
olg
i (N
M-B
G)
tra
ns-
Gol
gi n
etw
ork
(T
GN
) i
nd
enta
tion
s in
th
e n
ucl
ear
surf
ace
(IN
S)
or
hap
losp
oros
ome-
lik
e b
odie
s (H
-LB
s) S
ize
is le
ng
th times
w
idth
NC
nu
cleu
scy
top
lasm
rat
io P
rev
pre
vale
nce
NR
not
rec
ord
ed C
ari
aken
sis
Cra
ssos
trea
ari
aken
sis
Dat
a ar
e m
ean
s plusmn
SD
Sp
ecie
s
O
rig
in
n
Hos
t
Ref
eren
ce
Siz
e
N
C
Hap
losp
orog
enes
is
Mit
och
ond
rial
L
ipid
dro
ple
ts
(micro
m2 )
P
rev
NM
-BG
TG
N I
NS
Hap
losp
oros
omes
H
-LB
s
p
rofi
les
N
o
Pre
v
(
)
()
()
(
)
N
o
Siz
e (micro
m2 )
(
)
No
Ran
ge
N
o
R
ang
e
(
)
B o
stre
ae
Net
her
lan
ds
23
Ost
rea
edu
lis
P
ich
ot e
t al
(19
79)
2
1 plusmn
04
55
plusmn 7
14
27
0
0
9
plusmn 4
1
88 plusmn
21
26 0
30
plusmn 0
47
0minus
1
1 plusmn
1
0
minus4
04
plusmn 0
6
30
B e
xiti
osa
N
ew Z
eala
nd
60
O
ch
ilen
sis
H
ine
et a
l (
2001
)
32
plusmn 0
5
52 plusmn
6
52
11
32
5
29 plusmn
17
148
plusmn 1
1
21
04
7 plusmn
09
6
0minus3
4
plusmn 1
2minus
6
0
5 plusmn
09
3
2B
per
spor
a
Eas
t U
SA
N
G
O s
ten
tin
a
Car
neg
ie e
t al
(20
06)
41
plusmn 0
6
NG
N
G
N
G
N
G
NG
3 plusmn
3
137
plusmn 1
9
NG
6
plusmn 2
NG
NG
38
Tab
le 2
Com
par
ison
of t
he
un
i-n
ucl
eate
sta
ge
of th
e 3
des
crib
ed B
onam
iasp
p N
M-B
G n
ucl
ear
mem
bra
ne-
bou
nd
Gol
gi
TG
N t
ran
s-G
olg
i net
wor
k I
NS
in
den
tati
ons
in th
e n
ucl
ear
surf
ace
H-L
Bs
hap
losp
oros
ome-
lik
e b
odie
s N
C n
ucl
eus
cyto
pla
sm r
atio
Pre
v p
reva
len
ce N
G n
ot g
iven
Dat
a ar
e m
ean
s plusmn
SD
Dis Aquat Org 110 55ndash63 2014
diplokaryotic and bi-nucleate forms because of thedifficulty in getting central sections through bothnuclei (Table 3) The diplokaryon of NZ B exitiosawas similar in size to the bi-nucleate forms inArgen tinian oysters but had a higher NC ratiohaplospo rogenesis and lipid droplets Haplosporo-some numbers were higher in NZ and ArgentinianB exitiosa and Chilean Bonamia sp than in B os-treae but haplosporogenesis was more prevalent inNZ B exitiosa than in the other species whichshowed similar levels of haplosporogenesis (Table 3)
With increase in size each Bonamia showed ageneral increase in haplosporogenesis number ofmitochondrial profiles and lipid except in Argen-tinian B exitiosa and B exitiosa from C ariakensis(Table 4) The size groups of B exitiosa in C ariak-ensis and more so Chilean Bonamia sp and Bostreae were smaller than those of Argentinianand NZ B exitiosa In relation to seasonal differ-ences Bonamia from O chilensis in NZ and Chilesampled at different times of the year showed thatwhile NZ B exitiosa showed a great increase in allparameters Chilean Bonamia showed the sametrend but less so (Table 5)
58
Species Origin n Stage Size NC Haplosporogenesis Mitochondrial Lipid droplets (microm2) () Prev NM-BG TGN INS No of haplo- H-LBs profiles No Prev () () () () sporosomes ()
Table 3 Comparison of bi-nucleate and diplokaryotic stages of Bonamia spp NM-BG nuclear membrane-bound Golgi TGNtrans-Golgi network INS indentations in the nuclear surface H-LBs haplosporosome-like bodies NC nucleuscytoplasm
ratio Prev prevalence
Size Haplosporogenesis Mitochondrial Lipid dropletsgroup Prev No of haplo- profiles No Prev(microm) () sporosomes ()
Table 5 Comparison of Bonamia infecting Ostrea chilensis in New Zealand and Chile in relation to season Data are means plusmn SD Prev prevalence
Hine et al Bonamia spp ultrastructure
DISCUSSION
The problem with comparative ultrastructural stud-ies on congeneric undescribed species as in molecularstudies showing DNA sequence divergence amongsuch species is deciding how much difference consti-tutes a separate species While molecular studies re-veal genotypes and are quantifiable in base transi-tions ultrastructural studies reveal phenotypes whichmay be subject to many variables These include hostfactors such as the species concerned its age physio-logical state reproductive state and the presence ofother infections (Hine 2002) environmental factorssuch as water temperature and salinity (Audemard etal 2008 Carnegie et al 2008) and methods of sam-
59
Fig 1 Uni-nucleate stages of Bonamia exitiosa in (A) Ostreaedulis from Spain and (B) Crassostrea ariakensis from theUSA Host haemocyte cytoplasm indicated in both cases (H)to mark the exterior of the B exitiosa cells Note the widelyvarying abundance of haplosporosomes (arrows) and theparallel arrays of smooth endoplasmic reticulum (arrow-
head) in (A) Both scale bars = 1 microm
Fig 2 Bonamia ostreae uni-nucleate stages in Ostrea edulis(A) Low-power image of 2 cells infecting a single haemo-cyte Note the displacement of B ostreae nucleus to cell periphery a common observation and distinct trait of thisBonamia species Note also an extracellular haplosporosome(arrow) present along with numerous cytoplasmic haplo -sporosomes Scale bar = 1 microm (B) High-power image of asingle B ostreae cell Note again the abundant haplosporo-somes with smooth endoplasmic reticulum also displayedalong with an intranuclear microtubule (arrow) Scale bar =
05 microm
Dis Aquat Org 110 55ndash63 2014
pling fixation and post-fixation Parasite factors suchas the stages present at the time of sampling whichmay be seasonal (Hine 1991ab) the physiologicalstate of the parasite and possible plasticity of life cy-cles have also to be considered In the latter case al-though a spore stage is not known for Bo na mia exi-tiosa it does not necessarily mean that spores may notbe formed under certain environmental conditions
Interpretation of the ultrastructural features ofBonamia spp must take these variables into accountThese features fall into 3 groups parasite metabo-lism haplosporogenesis and sporogony the latter
only being known in B perspora The metabolic stateof the parasite is indicated by the mitochondria lipiddroplets and endoplasmic reticulum which are mostobvious in the development from a small intracellularuni-nucleate early stage to the large extracellularamoeboid feeding stage with large amounts of lipidmitochondria and parallel arrays of endoplasmic re ti -culum in NZ B exitiosa (see Hine amp Wesney 1994a)High lipid content was linked to season and develop-ment in the lipid-rich ovary in autumn to early winter(April to June Hine 1991b) Seasonal annual devel-opment may also be linked to temperature in C ari-akensis infections (Carnegie et al 2008) The produc-tion of lipid vesicles from and acid hydrolases inlipid droplets are associated with formation of a par-asitophorous vacuole (PV Hine amp Wesney 1994b)and 36 of Bonamia sp in Crassostrea ariakensiswere in PVs Therefore features reflecting metabo-lism are of little taxonomic value unless multiple vari-ables are taken into account
The taxonomic significance of haplosporogenesisas indicated by NM-BG TGN INS perinuclear gran-ular material and H-LBs (Hine amp Wesney 1992) isdifficult to assess as the function of haplosporosomesis unknown Whilst it has been suggested that theirglycoprotein coat and putative DNA core resemblesthe structure of viruses their release associated withlysis of surrounding cells suggests a lytic function(Hine et al 2002) but subsequent re-entry of cores in
60
Fig 3 Vegetative stages of Bonamia perspora in Ostreastentina from the USA (A) Uni-nucleate stage (B) Smalltrinucleate plasmodium Note the abundant mitochondriaparticularly closely apposed to the nucleus in (A) the longstretch of endoplasmic reticulum head (arrows) in (B) andthe general paucity of haplosporosomes Dark bodies in (A)
are lipid bodies Both scale bars = 1 microm
Fig 4 Prespore of Bonamia perspora in Ostrea stentina fromthe USA Note the developing wall (arrow) with episporecytoplasm (ES) still present and the well-defined lid (L)
nucleus (N) and spherule (S) Scale bar = 1 microm
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Hine et al Bonamia spp ultrastructure
post-fixed in 1 OsO4 buffered with 01 M sodiumcacodylate at pH 72 dehydrated through a gradedethanol series including en bloc staining with 1uranyl acetate at the 70 ethanol stage and embed-ded in Spurrrsquos resin Ultrathin (~90 nm) sections weremounted on carbon-stabilised formvar-coated 1-holegrids stained with Reynoldrsquos lead citrate and exam-ined on a Zeiss CEM 902 TEM Crassostrea ariaken-sis infected with B exitiosa from North Carolina(USA) were processed following methods used ear-lier for observation of B perspora (Carnegie et al2006) and examined on a Zeiss CEM 902 TEM
The measurements given are from sections throughthe centre of the cell in which the bilaminar nuclearand plasma membranes are clearly defined Cell sizenuclear size and the size of haplosporosomes given inthe following tables were calculated as the means ofthe longest dimension multiplied by the dimension atright angles to it The nucleuscytoplasmic (NC) ratiowas the mean of nuclear dimensions expressed as apercentage of the mean size of the cell The termlsquomulti-nucleatersquo is used for cells with more than 2nuclei rather than lsquoplasmodiumrsquo which is reservedfor a pre-sporulation stage
RESULTS
All Bonamia spp occurred as uni-nucleate and bi-nucleate forms and a multi-nucleate stage may occurin all species but was not encountered because of rar-ity NZ B exitiosa and B perspora also have a diplo -karyotic stage TEM observations on moribund Broughleyi showed a multi-nucleate stage (Cochen-nec-Laureau et al 2003) that appeared to undergoschizogony to form the uni-nucleate stage unlikeBonamia spp and other known haplosporidians (PM Hine pers obs) suggesting that it may not be aBonamia species
Uni-nucleate B exitiosa from Ostrea angasi in Aus-tralia Crassostrea ariakensis from the eastern USA(Fig 1B) O puelchana from Argentina and SpanishO edulis (Fig 1A) (Table 1) resemble NZ lsquotypersquo B exi-tiosa (Table 2) being larger than B ostreae (Fig 2)with smaller haplo spo ro somes and slightly largerthan Chilean Bonamia with larger haplosporosomesHaplosporogenesis was less often observed in B exi-tiosa from C ariakensis than in other species (Table 1)except B ostreae (Table 2) The uni-nucleate stage ofB perspora was larger but with smaller chromosomes(Table 2) (Fig 3A) than the other Bonamia spp withsubsequent multi-nucleate plasmodia (Fig 3B) devel-oping into spores (Fig 4) Few data were available on
57
Sp
ecie
s
Ori
gin
n
H
ost
S
ize
N
C
Hap
losp
orog
enes
is
Mit
och
ond
rial
L
ipid
dro
ple
ts
(
microm
2 )
()
Pre
v
N
M-B
G T
GN
INS
Hap
losp
oros
omes
H-L
Bs
p
rofi
les
N
o
Pre
v
(
)
(
)
()
(
)
Not
es
No
S
ize
(microm
2 )
N
o
R
ang
e
No
R
ang
e
(
)
B e
xiti
osa
A
ust
rali
a
45
O
stre
a an
gas
i
28
plusmn 0
4
N
R
NR
N
R
N
R
N
R
10
plusmn 4
1
56 plusmn
15
NR
4
plusmn 1
0
5 plusmn
08
30
B e
xiti
osa
A
rgen
tin
a
27
O
pu
elch
ana
3
1 plusmn
03
4
9 plusmn
6
6
1
4
3
25
3
1
Sli
gh
t
1
5 plusmn
7
148
plusmn 1
2
4
0
05
3 plusmn
08
0minus
2
3 plusmn
2
1minus
6
06
plusmn 1
1
37B
exi
tios
a
Eas
t U
SA
4
5
C a
riak
ensi
s
28
plusmn 0
4
50
plusmn 4
18
9
0
5 F
latt
ened
18 plusmn
10
15
2 plusmn
8
13
0
2 plusmn
05
3 plusmn
1
0minus
7
08
plusmn 0
9
52B
exi
tios
a
S
pai
n
4
O e
du
lis
27
(2
8
53
ndash57
50
2
5
0
2
5
17
12
1
54 plusmn
20
0
7
5
2
minus7
1
1 1
0
75
2
5 2
6 2
7)
11
7
(
n =
12)
4 2
Bon
amia
sp
Ch
ile
59
O
ch
ilen
sis
21
plusmn 0
4
58 plusmn
12
4
8
4
1
2
0
1
3 plusmn
7
138
plusmn 1
9
2
0
037
plusmn 1
06
0minus
7
2 plusmn
1
0minus
5
03
plusmn 0
4
26
Tab
le 1
Com
par
ison
of
the
un
i-n
ucl
eate
sta
ge
of B
onam
iasp
p H
aplo
spor
ogen
esis
in t
his
an
d t
he
foll
owin
g t
able
s is
cal
cula
ted
on
th
e n
um
ber
of
ind
ivid
ual
s h
avin
g n
u-
clea
r m
emb
ran
e-b
oun
d G
olg
i (N
M-B
G)
tra
ns-
Gol
gi n
etw
ork
(T
GN
) i
nd
enta
tion
s in
th
e n
ucl
ear
surf
ace
(IN
S)
or
hap
losp
oros
ome-
lik
e b
odie
s (H
-LB
s) S
ize
is le
ng
th times
w
idth
NC
nu
cleu
scy
top
lasm
rat
io P
rev
pre
vale
nce
NR
not
rec
ord
ed C
ari
aken
sis
Cra
ssos
trea
ari
aken
sis
Dat
a ar
e m
ean
s plusmn
SD
Sp
ecie
s
O
rig
in
n
Hos
t
Ref
eren
ce
Siz
e
N
C
Hap
losp
orog
enes
is
Mit
och
ond
rial
L
ipid
dro
ple
ts
(micro
m2 )
P
rev
NM
-BG
TG
N I
NS
Hap
losp
oros
omes
H
-LB
s
p
rofi
les
N
o
Pre
v
(
)
()
()
(
)
N
o
Siz
e (micro
m2 )
(
)
No
Ran
ge
N
o
R
ang
e
(
)
B o
stre
ae
Net
her
lan
ds
23
Ost
rea
edu
lis
P
ich
ot e
t al
(19
79)
2
1 plusmn
04
55
plusmn 7
14
27
0
0
9
plusmn 4
1
88 plusmn
21
26 0
30
plusmn 0
47
0minus
1
1 plusmn
1
0
minus4
04
plusmn 0
6
30
B e
xiti
osa
N
ew Z
eala
nd
60
O
ch
ilen
sis
H
ine
et a
l (
2001
)
32
plusmn 0
5
52 plusmn
6
52
11
32
5
29 plusmn
17
148
plusmn 1
1
21
04
7 plusmn
09
6
0minus3
4
plusmn 1
2minus
6
0
5 plusmn
09
3
2B
per
spor
a
Eas
t U
SA
N
G
O s
ten
tin
a
Car
neg
ie e
t al
(20
06)
41
plusmn 0
6
NG
N
G
N
G
N
G
NG
3 plusmn
3
137
plusmn 1
9
NG
6
plusmn 2
NG
NG
38
Tab
le 2
Com
par
ison
of t
he
un
i-n
ucl
eate
sta
ge
of th
e 3
des
crib
ed B
onam
iasp
p N
M-B
G n
ucl
ear
mem
bra
ne-
bou
nd
Gol
gi
TG
N t
ran
s-G
olg
i net
wor
k I
NS
in
den
tati
ons
in th
e n
ucl
ear
surf
ace
H-L
Bs
hap
losp
oros
ome-
lik
e b
odie
s N
C n
ucl
eus
cyto
pla
sm r
atio
Pre
v p
reva
len
ce N
G n
ot g
iven
Dat
a ar
e m
ean
s plusmn
SD
Dis Aquat Org 110 55ndash63 2014
diplokaryotic and bi-nucleate forms because of thedifficulty in getting central sections through bothnuclei (Table 3) The diplokaryon of NZ B exitiosawas similar in size to the bi-nucleate forms inArgen tinian oysters but had a higher NC ratiohaplospo rogenesis and lipid droplets Haplosporo-some numbers were higher in NZ and ArgentinianB exitiosa and Chilean Bonamia sp than in B os-treae but haplosporogenesis was more prevalent inNZ B exitiosa than in the other species whichshowed similar levels of haplosporogenesis (Table 3)
With increase in size each Bonamia showed ageneral increase in haplosporogenesis number ofmitochondrial profiles and lipid except in Argen-tinian B exitiosa and B exitiosa from C ariakensis(Table 4) The size groups of B exitiosa in C ariak-ensis and more so Chilean Bonamia sp and Bostreae were smaller than those of Argentinianand NZ B exitiosa In relation to seasonal differ-ences Bonamia from O chilensis in NZ and Chilesampled at different times of the year showed thatwhile NZ B exitiosa showed a great increase in allparameters Chilean Bonamia showed the sametrend but less so (Table 5)
58
Species Origin n Stage Size NC Haplosporogenesis Mitochondrial Lipid droplets (microm2) () Prev NM-BG TGN INS No of haplo- H-LBs profiles No Prev () () () () sporosomes ()
Table 3 Comparison of bi-nucleate and diplokaryotic stages of Bonamia spp NM-BG nuclear membrane-bound Golgi TGNtrans-Golgi network INS indentations in the nuclear surface H-LBs haplosporosome-like bodies NC nucleuscytoplasm
ratio Prev prevalence
Size Haplosporogenesis Mitochondrial Lipid dropletsgroup Prev No of haplo- profiles No Prev(microm) () sporosomes ()
Table 5 Comparison of Bonamia infecting Ostrea chilensis in New Zealand and Chile in relation to season Data are means plusmn SD Prev prevalence
Hine et al Bonamia spp ultrastructure
DISCUSSION
The problem with comparative ultrastructural stud-ies on congeneric undescribed species as in molecularstudies showing DNA sequence divergence amongsuch species is deciding how much difference consti-tutes a separate species While molecular studies re-veal genotypes and are quantifiable in base transi-tions ultrastructural studies reveal phenotypes whichmay be subject to many variables These include hostfactors such as the species concerned its age physio-logical state reproductive state and the presence ofother infections (Hine 2002) environmental factorssuch as water temperature and salinity (Audemard etal 2008 Carnegie et al 2008) and methods of sam-
59
Fig 1 Uni-nucleate stages of Bonamia exitiosa in (A) Ostreaedulis from Spain and (B) Crassostrea ariakensis from theUSA Host haemocyte cytoplasm indicated in both cases (H)to mark the exterior of the B exitiosa cells Note the widelyvarying abundance of haplosporosomes (arrows) and theparallel arrays of smooth endoplasmic reticulum (arrow-
head) in (A) Both scale bars = 1 microm
Fig 2 Bonamia ostreae uni-nucleate stages in Ostrea edulis(A) Low-power image of 2 cells infecting a single haemo-cyte Note the displacement of B ostreae nucleus to cell periphery a common observation and distinct trait of thisBonamia species Note also an extracellular haplosporosome(arrow) present along with numerous cytoplasmic haplo -sporosomes Scale bar = 1 microm (B) High-power image of asingle B ostreae cell Note again the abundant haplosporo-somes with smooth endoplasmic reticulum also displayedalong with an intranuclear microtubule (arrow) Scale bar =
05 microm
Dis Aquat Org 110 55ndash63 2014
pling fixation and post-fixation Parasite factors suchas the stages present at the time of sampling whichmay be seasonal (Hine 1991ab) the physiologicalstate of the parasite and possible plasticity of life cy-cles have also to be considered In the latter case al-though a spore stage is not known for Bo na mia exi-tiosa it does not necessarily mean that spores may notbe formed under certain environmental conditions
Interpretation of the ultrastructural features ofBonamia spp must take these variables into accountThese features fall into 3 groups parasite metabo-lism haplosporogenesis and sporogony the latter
only being known in B perspora The metabolic stateof the parasite is indicated by the mitochondria lipiddroplets and endoplasmic reticulum which are mostobvious in the development from a small intracellularuni-nucleate early stage to the large extracellularamoeboid feeding stage with large amounts of lipidmitochondria and parallel arrays of endoplasmic re ti -culum in NZ B exitiosa (see Hine amp Wesney 1994a)High lipid content was linked to season and develop-ment in the lipid-rich ovary in autumn to early winter(April to June Hine 1991b) Seasonal annual devel-opment may also be linked to temperature in C ari-akensis infections (Carnegie et al 2008) The produc-tion of lipid vesicles from and acid hydrolases inlipid droplets are associated with formation of a par-asitophorous vacuole (PV Hine amp Wesney 1994b)and 36 of Bonamia sp in Crassostrea ariakensiswere in PVs Therefore features reflecting metabo-lism are of little taxonomic value unless multiple vari-ables are taken into account
The taxonomic significance of haplosporogenesisas indicated by NM-BG TGN INS perinuclear gran-ular material and H-LBs (Hine amp Wesney 1992) isdifficult to assess as the function of haplosporosomesis unknown Whilst it has been suggested that theirglycoprotein coat and putative DNA core resemblesthe structure of viruses their release associated withlysis of surrounding cells suggests a lytic function(Hine et al 2002) but subsequent re-entry of cores in
60
Fig 3 Vegetative stages of Bonamia perspora in Ostreastentina from the USA (A) Uni-nucleate stage (B) Smalltrinucleate plasmodium Note the abundant mitochondriaparticularly closely apposed to the nucleus in (A) the longstretch of endoplasmic reticulum head (arrows) in (B) andthe general paucity of haplosporosomes Dark bodies in (A)
are lipid bodies Both scale bars = 1 microm
Fig 4 Prespore of Bonamia perspora in Ostrea stentina fromthe USA Note the developing wall (arrow) with episporecytoplasm (ES) still present and the well-defined lid (L)
nucleus (N) and spherule (S) Scale bar = 1 microm
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Dis Aquat Org 110 55ndash63 2014
diplokaryotic and bi-nucleate forms because of thedifficulty in getting central sections through bothnuclei (Table 3) The diplokaryon of NZ B exitiosawas similar in size to the bi-nucleate forms inArgen tinian oysters but had a higher NC ratiohaplospo rogenesis and lipid droplets Haplosporo-some numbers were higher in NZ and ArgentinianB exitiosa and Chilean Bonamia sp than in B os-treae but haplosporogenesis was more prevalent inNZ B exitiosa than in the other species whichshowed similar levels of haplosporogenesis (Table 3)
With increase in size each Bonamia showed ageneral increase in haplosporogenesis number ofmitochondrial profiles and lipid except in Argen-tinian B exitiosa and B exitiosa from C ariakensis(Table 4) The size groups of B exitiosa in C ariak-ensis and more so Chilean Bonamia sp and Bostreae were smaller than those of Argentinianand NZ B exitiosa In relation to seasonal differ-ences Bonamia from O chilensis in NZ and Chilesampled at different times of the year showed thatwhile NZ B exitiosa showed a great increase in allparameters Chilean Bonamia showed the sametrend but less so (Table 5)
58
Species Origin n Stage Size NC Haplosporogenesis Mitochondrial Lipid droplets (microm2) () Prev NM-BG TGN INS No of haplo- H-LBs profiles No Prev () () () () sporosomes ()
Table 3 Comparison of bi-nucleate and diplokaryotic stages of Bonamia spp NM-BG nuclear membrane-bound Golgi TGNtrans-Golgi network INS indentations in the nuclear surface H-LBs haplosporosome-like bodies NC nucleuscytoplasm
ratio Prev prevalence
Size Haplosporogenesis Mitochondrial Lipid dropletsgroup Prev No of haplo- profiles No Prev(microm) () sporosomes ()
Table 5 Comparison of Bonamia infecting Ostrea chilensis in New Zealand and Chile in relation to season Data are means plusmn SD Prev prevalence
Hine et al Bonamia spp ultrastructure
DISCUSSION
The problem with comparative ultrastructural stud-ies on congeneric undescribed species as in molecularstudies showing DNA sequence divergence amongsuch species is deciding how much difference consti-tutes a separate species While molecular studies re-veal genotypes and are quantifiable in base transi-tions ultrastructural studies reveal phenotypes whichmay be subject to many variables These include hostfactors such as the species concerned its age physio-logical state reproductive state and the presence ofother infections (Hine 2002) environmental factorssuch as water temperature and salinity (Audemard etal 2008 Carnegie et al 2008) and methods of sam-
59
Fig 1 Uni-nucleate stages of Bonamia exitiosa in (A) Ostreaedulis from Spain and (B) Crassostrea ariakensis from theUSA Host haemocyte cytoplasm indicated in both cases (H)to mark the exterior of the B exitiosa cells Note the widelyvarying abundance of haplosporosomes (arrows) and theparallel arrays of smooth endoplasmic reticulum (arrow-
head) in (A) Both scale bars = 1 microm
Fig 2 Bonamia ostreae uni-nucleate stages in Ostrea edulis(A) Low-power image of 2 cells infecting a single haemo-cyte Note the displacement of B ostreae nucleus to cell periphery a common observation and distinct trait of thisBonamia species Note also an extracellular haplosporosome(arrow) present along with numerous cytoplasmic haplo -sporosomes Scale bar = 1 microm (B) High-power image of asingle B ostreae cell Note again the abundant haplosporo-somes with smooth endoplasmic reticulum also displayedalong with an intranuclear microtubule (arrow) Scale bar =
05 microm
Dis Aquat Org 110 55ndash63 2014
pling fixation and post-fixation Parasite factors suchas the stages present at the time of sampling whichmay be seasonal (Hine 1991ab) the physiologicalstate of the parasite and possible plasticity of life cy-cles have also to be considered In the latter case al-though a spore stage is not known for Bo na mia exi-tiosa it does not necessarily mean that spores may notbe formed under certain environmental conditions
Interpretation of the ultrastructural features ofBonamia spp must take these variables into accountThese features fall into 3 groups parasite metabo-lism haplosporogenesis and sporogony the latter
only being known in B perspora The metabolic stateof the parasite is indicated by the mitochondria lipiddroplets and endoplasmic reticulum which are mostobvious in the development from a small intracellularuni-nucleate early stage to the large extracellularamoeboid feeding stage with large amounts of lipidmitochondria and parallel arrays of endoplasmic re ti -culum in NZ B exitiosa (see Hine amp Wesney 1994a)High lipid content was linked to season and develop-ment in the lipid-rich ovary in autumn to early winter(April to June Hine 1991b) Seasonal annual devel-opment may also be linked to temperature in C ari-akensis infections (Carnegie et al 2008) The produc-tion of lipid vesicles from and acid hydrolases inlipid droplets are associated with formation of a par-asitophorous vacuole (PV Hine amp Wesney 1994b)and 36 of Bonamia sp in Crassostrea ariakensiswere in PVs Therefore features reflecting metabo-lism are of little taxonomic value unless multiple vari-ables are taken into account
The taxonomic significance of haplosporogenesisas indicated by NM-BG TGN INS perinuclear gran-ular material and H-LBs (Hine amp Wesney 1992) isdifficult to assess as the function of haplosporosomesis unknown Whilst it has been suggested that theirglycoprotein coat and putative DNA core resemblesthe structure of viruses their release associated withlysis of surrounding cells suggests a lytic function(Hine et al 2002) but subsequent re-entry of cores in
60
Fig 3 Vegetative stages of Bonamia perspora in Ostreastentina from the USA (A) Uni-nucleate stage (B) Smalltrinucleate plasmodium Note the abundant mitochondriaparticularly closely apposed to the nucleus in (A) the longstretch of endoplasmic reticulum head (arrows) in (B) andthe general paucity of haplosporosomes Dark bodies in (A)
are lipid bodies Both scale bars = 1 microm
Fig 4 Prespore of Bonamia perspora in Ostrea stentina fromthe USA Note the developing wall (arrow) with episporecytoplasm (ES) still present and the well-defined lid (L)
nucleus (N) and spherule (S) Scale bar = 1 microm
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Hine et al Bonamia spp ultrastructure
DISCUSSION
The problem with comparative ultrastructural stud-ies on congeneric undescribed species as in molecularstudies showing DNA sequence divergence amongsuch species is deciding how much difference consti-tutes a separate species While molecular studies re-veal genotypes and are quantifiable in base transi-tions ultrastructural studies reveal phenotypes whichmay be subject to many variables These include hostfactors such as the species concerned its age physio-logical state reproductive state and the presence ofother infections (Hine 2002) environmental factorssuch as water temperature and salinity (Audemard etal 2008 Carnegie et al 2008) and methods of sam-
59
Fig 1 Uni-nucleate stages of Bonamia exitiosa in (A) Ostreaedulis from Spain and (B) Crassostrea ariakensis from theUSA Host haemocyte cytoplasm indicated in both cases (H)to mark the exterior of the B exitiosa cells Note the widelyvarying abundance of haplosporosomes (arrows) and theparallel arrays of smooth endoplasmic reticulum (arrow-
head) in (A) Both scale bars = 1 microm
Fig 2 Bonamia ostreae uni-nucleate stages in Ostrea edulis(A) Low-power image of 2 cells infecting a single haemo-cyte Note the displacement of B ostreae nucleus to cell periphery a common observation and distinct trait of thisBonamia species Note also an extracellular haplosporosome(arrow) present along with numerous cytoplasmic haplo -sporosomes Scale bar = 1 microm (B) High-power image of asingle B ostreae cell Note again the abundant haplosporo-somes with smooth endoplasmic reticulum also displayedalong with an intranuclear microtubule (arrow) Scale bar =
05 microm
Dis Aquat Org 110 55ndash63 2014
pling fixation and post-fixation Parasite factors suchas the stages present at the time of sampling whichmay be seasonal (Hine 1991ab) the physiologicalstate of the parasite and possible plasticity of life cy-cles have also to be considered In the latter case al-though a spore stage is not known for Bo na mia exi-tiosa it does not necessarily mean that spores may notbe formed under certain environmental conditions
Interpretation of the ultrastructural features ofBonamia spp must take these variables into accountThese features fall into 3 groups parasite metabo-lism haplosporogenesis and sporogony the latter
only being known in B perspora The metabolic stateof the parasite is indicated by the mitochondria lipiddroplets and endoplasmic reticulum which are mostobvious in the development from a small intracellularuni-nucleate early stage to the large extracellularamoeboid feeding stage with large amounts of lipidmitochondria and parallel arrays of endoplasmic re ti -culum in NZ B exitiosa (see Hine amp Wesney 1994a)High lipid content was linked to season and develop-ment in the lipid-rich ovary in autumn to early winter(April to June Hine 1991b) Seasonal annual devel-opment may also be linked to temperature in C ari-akensis infections (Carnegie et al 2008) The produc-tion of lipid vesicles from and acid hydrolases inlipid droplets are associated with formation of a par-asitophorous vacuole (PV Hine amp Wesney 1994b)and 36 of Bonamia sp in Crassostrea ariakensiswere in PVs Therefore features reflecting metabo-lism are of little taxonomic value unless multiple vari-ables are taken into account
The taxonomic significance of haplosporogenesisas indicated by NM-BG TGN INS perinuclear gran-ular material and H-LBs (Hine amp Wesney 1992) isdifficult to assess as the function of haplosporosomesis unknown Whilst it has been suggested that theirglycoprotein coat and putative DNA core resemblesthe structure of viruses their release associated withlysis of surrounding cells suggests a lytic function(Hine et al 2002) but subsequent re-entry of cores in
60
Fig 3 Vegetative stages of Bonamia perspora in Ostreastentina from the USA (A) Uni-nucleate stage (B) Smalltrinucleate plasmodium Note the abundant mitochondriaparticularly closely apposed to the nucleus in (A) the longstretch of endoplasmic reticulum head (arrows) in (B) andthe general paucity of haplosporosomes Dark bodies in (A)
are lipid bodies Both scale bars = 1 microm
Fig 4 Prespore of Bonamia perspora in Ostrea stentina fromthe USA Note the developing wall (arrow) with episporecytoplasm (ES) still present and the well-defined lid (L)
nucleus (N) and spherule (S) Scale bar = 1 microm
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Dis Aquat Org 110 55ndash63 2014
pling fixation and post-fixation Parasite factors suchas the stages present at the time of sampling whichmay be seasonal (Hine 1991ab) the physiologicalstate of the parasite and possible plasticity of life cy-cles have also to be considered In the latter case al-though a spore stage is not known for Bo na mia exi-tiosa it does not necessarily mean that spores may notbe formed under certain environmental conditions
Interpretation of the ultrastructural features ofBonamia spp must take these variables into accountThese features fall into 3 groups parasite metabo-lism haplosporogenesis and sporogony the latter
only being known in B perspora The metabolic stateof the parasite is indicated by the mitochondria lipiddroplets and endoplasmic reticulum which are mostobvious in the development from a small intracellularuni-nucleate early stage to the large extracellularamoeboid feeding stage with large amounts of lipidmitochondria and parallel arrays of endoplasmic re ti -culum in NZ B exitiosa (see Hine amp Wesney 1994a)High lipid content was linked to season and develop-ment in the lipid-rich ovary in autumn to early winter(April to June Hine 1991b) Seasonal annual devel-opment may also be linked to temperature in C ari-akensis infections (Carnegie et al 2008) The produc-tion of lipid vesicles from and acid hydrolases inlipid droplets are associated with formation of a par-asitophorous vacuole (PV Hine amp Wesney 1994b)and 36 of Bonamia sp in Crassostrea ariakensiswere in PVs Therefore features reflecting metabo-lism are of little taxonomic value unless multiple vari-ables are taken into account
The taxonomic significance of haplosporogenesisas indicated by NM-BG TGN INS perinuclear gran-ular material and H-LBs (Hine amp Wesney 1992) isdifficult to assess as the function of haplosporosomesis unknown Whilst it has been suggested that theirglycoprotein coat and putative DNA core resemblesthe structure of viruses their release associated withlysis of surrounding cells suggests a lytic function(Hine et al 2002) but subsequent re-entry of cores in
60
Fig 3 Vegetative stages of Bonamia perspora in Ostreastentina from the USA (A) Uni-nucleate stage (B) Smalltrinucleate plasmodium Note the abundant mitochondriaparticularly closely apposed to the nucleus in (A) the longstretch of endoplasmic reticulum head (arrows) in (B) andthe general paucity of haplosporosomes Dark bodies in (A)
are lipid bodies Both scale bars = 1 microm
Fig 4 Prespore of Bonamia perspora in Ostrea stentina fromthe USA Note the developing wall (arrow) with episporecytoplasm (ES) still present and the well-defined lid (L)
nucleus (N) and spherule (S) Scale bar = 1 microm
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Hine et al Bonamia spp ultrastructure
a haplosporidian infecting abalone (Hine et al 2002)could represent horizontal gene transfer (Keeling ampPalmer 2008) The size and shape of haplosporosomesmay be taxonomically important but their plasticityin size and shape in relation to fixation is unknownHowever as haplosporosomes are the only structuresquantifiable in size and number they may be taxo-nomically important If so the uni-nucleate stages ofNZ Australian C ariakensis Argentinian and Span-ish B exitiosa and Chilean Bonamia are distinct fromB ostreae and B perspora These relationships arereflected in molecular phylogenies with NZ Aus-tralian C ariakensis Argentinian and European Bexitiosa closely related to the Chilean Bona mia (Hillet al 2014 this DAO Special) Therefore haplosporo-some shape and size may be taxonomically impor-tant but not haplosporosome number as that isrelated to the size or developmental stage of the par-asite However studies on Marteilia spp (Para my -xea) (Villalba et al 1993 Longshaw et al 2001) sug-gest that haplosporosome shape and size may not bereliable as a taxonomic feature Also asporous Bo na -mia spp are usually uni-nucleate and may be con-fused with the uni-nucleate stage of other haplo spor -idians but most haplosporidian TEM studies havebeen on spo ru lation and the uni-nucleate stage israrely de scribed The intra-haemocytic location ofasporous Bonamia spp is also unreliable because thevegetative stages of other haplosporidians may bephagocytosed by host haemocytes
Uni- bi- and multi-nucleate stages are known fromC ariakensis and probably Argentinian and SpanishB exitiosa and visually they were indistinguishablefrom each other and Australian B exitiosa ChileanBonamia could be distinguished by the more osmio-philic membranes and NM-BG but this could havebeen due to fixation andor processing NZ B exitiosaalso has a diplokaryotic stage as does B persporaand the diplokaryon is a feature of multi-nucleateplasmodia of spore-forming haplosporidians such asUrosporidium crescens Haplosporidium louisianaH nelsoni and H costale (Hine et al 2009) Large NZB exitiosa in autumn and winter also contain cylin-drical confronting cisternae (Hine amp Wesney 1992)as do Chilean Bonamia (Lohrmann et al 2009) whichhave been attributed to an underlying viral infection(Hine amp Wesney 1992) As the NZ and Chilean Bona -mia infect the same host (O chilensis) but ChileanBonamia are smaller with fewer smaller haplosporo-somes (Tables 1 amp 2) Chilean Bonamia may havederived from NZ B exitiosa but have been geograph-ically isolated for sufficient time for the ChileanBonamia to emerge as a distinct species The Bona -
mia from Australia (Corbeil et al 2006) C ariakensis(see Carnegie et al 2006) Chile (Balseiro et al 2006)and Spain (Abollo et al 2008) have all been reportedas B exitiosa but ultrastructurally the Chi lean Bona -mia is similar to but different from B exitiosa Ultra-structure supports the identification of Australian Cariakensis Argentinian and Spanish forms as B exi-tiosa as in molecular phylogenies (Hill et al 20102014) They appear more similar to each other in thecycling of uni-nucleate and bi-nucleate stages thanto the multiple forms of NZ B exitiosa but this maybe due to the smaller sample sizes taken on only 1occasion There is a possibility however that thetype species NZ B exitiosa is atypical of the speciesor that it has an underlying viral infection indicatedby its ultrastructure (Hine amp Wesney 1992)
When studying phenotypic traits of an intracellularprotozoan parasite it is necessary to consider that theultrastructure observed may be mediated by the hostas is histopathology and possibly life cycles Organ-isms are parsimonious and do not evolve to produceforms or stages that are of no benefit or waste energyreserves Bonamia spp may simply normally cyclethrough uni-nucleate and bi-nucleate stages as seenin B ostreae and the C ariakensis Argentinian andSpanish B exitiosa NZ B exitiosa targets abundanthost ovarian lipids during the female O chilensisspawning cycle to form a large actively feeding uni-nucleate stage not reported from elsewhere NZ Bexitiosa also forms diplokarya which are only knownfrom spore-forming species and therefore NZ B exi-tiosa may sporulate as a survival strategy under lim-iting conditions suggesting that Bonamia spp mayhave plastic life cycles For example the spore- formingspecies H armoricanum may be able to go throughits life cycle without sporulating as suggested by thelarge plasmodia but absence of spores in O edulisfrom St Philibert Brittany (Breheacutelin et al 1982 Viva -regraves et al 1982 Bonami et al 1985)
In conclusion the interpretation of ultrastructurehas to take into account multiple variables mediatedby the parasite the host and the environment Whilethis study on ultrastructure supports molecular phy-logenies in that B exitiosa is ultrastructurally distinctfrom B ostreae and B perspora it shows consider-able variability in the ultrastructure of B exitiosa indifferent host species These phenotypic differencesreflect not only the Bonamia species concerned butalso the inter-relationships of the parasites with theirhosts and the circumstances under which they werefixed and variables in fixation In comparison withthe precise genotypic characterisation of organismsin molecular phylogenies TEM studies are impre-
61
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Dis Aquat Org 110 55ndash63 2014
cise and because of the many variables affecting theparasite they are not often useful in identifying spe-cies The ultrastructure of the Chilean Bonamia inparticular does not adequately distinguish it fromNZ B exitiosa However while molecular studies canbe used to classify the parasite and establish its phy-logenetic affinities they give no information on thestructure of the parasite its developmental stagephysiological state and its interactions with the hostDiagnosis should combine molecular techniques within the case of protists ultrastructural characterisa-tion The latter must identify ultrastructural featuresof taxonomic rather than physiological or develop-mental importance Consequently for spore-forminghaplosporidians spore structure is most useful butthere is less certainty regarding non-sporous or pre-spore stages
Acknowledgements This study was partially funded bygrant PICT-2007-1338 (ANPCyT-Argentina) This is VirginiaInstitute of Marine Science contribution number 3385
LITERATURE CITED
Abollo E Ramilo A Casas SM Comesantildea P Cao A CarballalMJ Villalba A (2008) First detection of the protozoanparasite Bonamia exitiosa (Haplosporidia) infecting flatoyster Ostrea edulis grown in European waters Aqua-culture 274 201minus207
Audemard C Carnegie RB Stokes NA Bishop MJ PetersonCH Burreson EM (2008) Effects of salinity on Bonamiasp survival in the Asian oyster Crassostrea ariakensisJ Shellfish Res 27 535minus540
Balouet G Poder M Cahour A (1983) Haemocytic parasito-sis morphology and pathology of lesions in the Frenchflat oyster Ostrea edulis L Aquaculture 34 1minus14
Balseiro P Conchas RF Montes J Goacutemez-Leoacuten J Novoa BFigueras A (2006) Comparison of diagnostic techniquesfor the protozoan parasite Bonamia ostreae in flat oysterOstrea edulis Aquaculture 261 1135minus1143
Berthe FCJ Hine PM (2003) Bonamia exitiosa Hine et al2001 is proposed instead of B exitiosus as the valid nameof Bonamia sp infecting flat oysters Ostrea chilensis inNew Zealand Dis Aquat Org 57 181
Bishop MJ Carnegie RB Stokes NA Peterson CH BurresonEM (2006) Complications of a non-native oyster intro-duction facilitation of a local parasite Mar Ecol Prog Ser325 145minus152
Bonami JR Vivaregraves CP Breheacutelin M (1985) Eacutetude drsquoune nou-velle haplosporidie parasite de lrsquohuicirctre plate Ostreaedulis L morphologie et cytologie de diffeacuterents stadesProtistologica 21 161minus173
Bougrier S Tigeacute G Bachegravere E Grizel H (1986) Ostreaangasi acclimatization to French coasts Aquaculture 58 151minus154
Breheacutelin M Bonami JR Cousserans F Vivaregraves CP (1982)Existence de formes plasmodiales vraies chez Bonamiaostreae parasite de lrsquohuicirctre plate Ostrea edulis CR HebdSeances Acad Sci 295 45minus48
Burreson EM Stokes NA Carnegie RB Bishop MJ (2004)Bonamia sp (Haplosporidia) found in non-native oystersCrassostrea ariakensis in Bogue Sound North CarolinaJ Aquat Anim Health 16 1minus6
Campalans M Lohrmann KB (2009) Histological survey offour species of cultivated molluscs in Chile susceptible toOIE notifiable diseases Rev Biol Mar Oceanogr 44 561minus569
Carnegie RB Cochennec-Laureau N (2004) Microcell para-sites of oysters recent insights and future trends AquatLiving Resour 17 519minus528
Carnegie RB Burreson EM Hine PM Stokes NA Aude-mard C Bishop MJ Peterson PH (2006) Bonamia per-spora n sp (Haplosporidia) a parasite of the oysterOstreola equestris is the first Bonamia species known toproduce spores J Eukaryot Microbiol 53 232minus245
Carnegie RB Stokes NA Audemard C Bishop MJ and oth-ers (2008) Strong seasonality of Bonamia sp infectionand induced Crassostrea ariakensis mortality in Bogueand Masonboro Sounds North Carolina USA J Inver-tebr Pathol 98 335minus343
Carnegie RB Hill KM Stokes NA Burreson EM (2014) Thehaplosporidian Bonamia exitiosa is present in Australiabut the identity of the parasite described as Bonamia (for-merly Mikrocytos) roughleyi is uncertain J InvertebrPathol 115 33minus40
Cochennec-Laureau N Reece KS Berthe FCJ Hine PM(2003) Mikrocytos roughleyi taxonomic affiliation leadsto the genus Bonamia (Haplosporidia) Dis Aquat Org 54 209minus217
Corbeil S Arzul I Robert M Berthe FCJ Besnard-Cochen-nec N Crane MSJ (2006) Molecular characterisation ofan Australian isolate of Bonamia exitiosa Dis Aquat Org71 81minus85
Elston RA Farley CA Kent ML (1986) Occurrence and sig-nificance of bonamiasis in European flat oysters Ostreaedulis in North America Dis Aquat Org 2 49minus54
Engelsma MY Culloty SC Lynch SA Arzul I Carnegie RB(2014) Bonamia parasites a rapidly changing perspec-tive on a genus of important molluscan pathogens DisAquat Org 110 5minus23
Farley CA Wolf PH Elston RA (1988) A long-term study oflsquomicrocellrsquo disease in oysters with a description of a newgenus Mikrocytos (gn) and two new species Mikrocy-tos mackini (spn) and Mikrocytos roughleyi (spn) FishBull 86 581minus593
Friedman CS Perkins FO (1994) Range extension of Bona miaostreae to Maine USA J Invertebr Pathol 64 179minus181
Friedman CS McDowell T Groff JM Hollibaugh JT Man zerD Hedrick RP (1989) Presence of Bonamia ostreae amongpopulations of the European flat oyster Ostrea edulisLinneacute in California USA J Shellfish Res 8 133minus137
Grizel H Comps M Raguenes D Leborgne Y Tigeacute G Mar-tin AG (1983) Bilan des essais drsquoacclimatation drsquoOstreachilensis sur les cocirctes de Bretagne Rev Trav Inst PecircchesMarit 46 209minus225
Hill KM Carnegie RB Aloui-Bejaoui N Gharsalli RE WhiteDM Stokes NA Burreson EM (2010) Observation of aBonamia sp infecting the oyster Ostrea stentina inTunisia and a consideration of its phylogenetic affinitiesJ Invertebr Pathol 103 179minus185
Hill KM Stokes NA Webb SC Hine PM and others (2014)Phylogenetics of Bonamia parasites based on small-sub-unit and internal transcribed spacer region ribosomalDNA sequence data Dis Aquat Org 110 33minus54
62
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
Dis Aquat Org 110 55ndash63 201464
Hine et al Bonamia spp ultrastructure
Hine PM (1991a) Ultrastructural observations on the annualinfection pattern of Bonamia sp in flat oysters Tiostreachilensis Dis Aquat Org 11 163minus171
Hine PM (1991b) The annual pattern of infection by Bona miasp in New Zealand flat oysters Tiostrea chilensis Aqua-culture 93 241minus251
Hine PM (1992) Ultrastructural and enzyme cytochemicalobservations on Bonamia sp in oysters (Tiostrea chilen-sis) with a consideration of organelle function Aquacul-ture 107 175minus183
Hine PM (2002) Severe apicomplexan infection in the oysterOstrea chilensis a possible predisposing factor in bon a -miosis Dis Aquat Org 51 49minus60
Hine PM Wesney B (1992) Interrelationships of cytoplasmicstructures in Bonamia sp (Haplosporidia) infecting oys-ters Tiostrea chilensis an interpretation Dis Aquat Org14 59minus68
Hine PM Wesney B (1994a) The functional cytology ofBonamia sp (Haplosporidia) infecting oysters Tiostreachilensis an ultracytochemical study Dis Aquat Org 20 207minus217
Hine PM Wesney B (1994b) Interaction of phagocytosedBonamia sp (Haplosporidia) with haemocytes of oystersTiostrea chilensis Dis Aquat Org 20 219minus229
Hine PM Cochennec-Laureau N Berthe FCJ (2001) Bona -mia exitiosus n sp (Haplosporidia) infecting flat oystersOstrea chilensis in New Zealand Dis Aquat Org 47 63minus72
Hine PM Wakefield S Diggles BK Webb VL Maas EW(2002) Ultrastructure of a haplosporidian containingRickettsiae associated with mortalities among culturedpaua Haliotis iris Dis Aquat Org 49 207minus219
Hine PM Carnegie RB Burreson EM Engelsma MY (2009)Inter-relationships of haplosporidians deduced fromultrastructural studies Dis Aquat Org 83 247minus256
Kroeck MA (2010) Gross signs and histopathology of Ostreapuelchana infected by a Bonamia exitiosa-like parasite(Haplosporidia) Dis Aquat Org 89 229minus236
Kroeck MA Montes J (2005) Occurrence of the haemocyteparasite Bonamia sp in flat oysters Ostrea puelchanafarmed in San Antonio Bay (Argentina) Dis Aquat Org63 231minus235
Kroeck MA Semenas L Morsan EM (2008) Epidemiologicalstudy of Bonamia sp in the native flat oyster Ostreapuelchana from San Matiacuteas Gulf (NW PatagoniaArgentina) Aquaculture 276 5minus13
Lohrmann KB Hine PM Campalans M (2009) Ultrastructureof Bonamia sp in Ostrea chilensis in Chile Dis AquatOrg 85 199minus208
Longshaw M Feist SW Matthews RA Figueras A (2001)Ultrastructural characterisation of Marteilia species(Paramyxea) from Ostrea edulis Mytilus edulis andMytilus galloprovincialis in Europe Dis Aquat Org 44 137minus142
Lynch SA Abollo E Ramilo A Cao A Culloty SC Villalba A(2010) Observations raise the question if the Pacific oys-ter Crassostrea gigas can act as either a carrier or a re -servoir for Bonamia ostreae or Bonamia exitiosa Para-sitology 137 1515minus1526
Marty GD Bower SM Clarke KR Meyer G and others(2006) Histopathology and real-time PCR for detection ofBonamia ostreae in Ostrea edulis cultured in westernCanada Aquaculture 261 33minus42
Narcisi V Arzul I Cargini D Mosca F and others (2010)Detection of Bonamia ostreae and B exitiosa (Haplo -sporidia) in Ostrea edulis from the Adriatic Sea (Italy)Dis Aquat Org 89 79minus85
Pascual M Martin AG Zampatti E Coatanea D Defossez JRobert R (1991) Testing of the Argentina oyster Ostreapuelchana in several French oyster farming sites ICESCouncil Meeting Papers ICES CM 1991K 30 ICESCopenhagen
Pichot Y Comps M Tigeacute G Grizel H Rabouin MA (1979)Recherches sur Bonamia ostreae gen n sp n parasitenouveau de lrsquohuicirctre plate Ostrea edulis Rev Trav InstPecircches Marit 43 131minus140
Villalba A Mourelle SG Loacutepez MC Carballal MJAzevedo C (1993) Marteiliasis affecting cultured mus-sels Mytilus galloprovincialis of Galicia (NW Spain) IEtiology phases of the infection and temporal andspatial variability in prevalence Dis Aquat Org 16 61minus72
Vivaregraves CP Breheacutelin M Cousserans F Bonami JR (1982)Mise en evidence drsquoune nouvelle Haplosporidie parasitede lrsquohuitre plate Ostrea edulis L CR Acad Sci 295 127minus130
63
Editorial responsibility Stephen Feist Weymouth UK
Submitted April 4 2013 Accepted March 20 2014Proofs received from author(s) July 11 2014
