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Algivore or Phototroph? Plakobranchus ocellatus
(Gastropoda) Continuously Acquires Kleptoplasts and
Nutrition from Multiple Algal Species in Nature
Taro M aeda1'2, Euichi Hi ro se3, Y osh ito Chikaraishi2, Masaru Kawato2, Kiyotaka Takishita2,
Takao Y osh ida1'2, H eroen V erb ru ggen 4'5, Jiro Tanaka1, Shigeru Shim am ura2, Yoshihiro Takaki2,
M asashi Tsuchiya2, Kenji Iwai6, Tadashi M aruyam a1'2*
1 G raduate School o f Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo, Japan, 2 Institute of
Biogeosciences, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka, Kanagawa, Japan, 3 D epartm ent of Chemistry, Biology and
Marine Science, Faculty o f Science, University o f th e Ryukyus, Nishihara-cho, Okinawa, Japan, 4Phycology Research Group, Ghent University, Krijgslaan 281 (S8), Ghent,
Belgium, 5 School o f Botany, The University o f Melbourne, Victoria, Australia, 6 Okinawa Prefectural Fisheries and Ocean Research Center, 1-3-1 Nishizaki, Itoman-shi,
Okinawa, Japan
Abstract
T he se a slug P lakobranchus ocellatus (S acoglossa, G a stro p o d a ) re ta in s p h o to sy n th e tlc a lly a ctiv e c h lo ro p la sts from In g e sted
a lg a e (functional k lep to p lasts) In th e ep ith elial cells o f Its d ig estiv e g la n d for u p to 10 m o n th s. W hile Its fe e d in g b e h av io r has
n o t b e e n o b se rv e d In n a tu ra l h a b ita ts, tw o h y p o th e s e s h av e b e e n p ro p o se d : 1) a d u lt P. ocellatus u ses k le p to p la s ts to o b ta in
p h o to s y n th a te s a n d nutritionally b e h a v e s as a p h o to a u to tro p h w ith o u t re p le n ish in g th e k lep to p lasts; o r 2) It b e h a v e s as a
m lx o tro p h ( p h o to a u to tro p h a n d h e rb iv o ro u s c o n su m e r) a n d re p le n ish e s k le p to p la sts c o n tin u a lly o r periodically. To a d d re ss
th e q u e s tio n o f w hich h y p o th e sis Is m o re likely, w e e x a m in e d th e so u rc e a lg a e for k le p to p la sts a n d te m p o ra l c h a n g e s In
k le p to p la st c o m p o s itio n a n d nu tritio n al c o n trib u tio n . By c h ara cte riz in g th e te m p o ra l diversity o f P. ocellatus k le p to p la sts
using rbcL s e q u e n c e s , w e fo u n d th a t P. ocellatus h a rv ests k le p to p la sts from a t least 8 d iffere n t s lp h o n o u s g re e n algal
sp ecies, th a t k le p to p la sts from m o re th a n o n e sp e c ie s a re p re s e n t In e a c h Individual se a slug, a n d th a t th e k le p to p la st
c o m p o s itio n differs tem p o rally . T h ese results s u g g e s t th a t wild P. ocellatus o fte n fe ed o n m ultip le sp e c ie s o f slp h o n o u s
a lg a e from w hich th e y c o n tin u a lly o b ta in fresh c h lo ro p lasts. By e stim a tin g th e tro p h ic p o sitio n o f wild a n d sta rv e d P.
ocellatus using th e sta b le n itro g e n Iso to p lc c o m p o s itio n o f a m in o acids, w e s h o w e d th a t d e s p ite th e a b u n d a n c e o f
k lep to p lasts, th e ir p h o to s y n th a te s d o n o t c o n trib u te g re atly to th e n u tritio n o f wild P. ocellatus, b u t th a t k le p to p la st
p h o to s y n th a te s form a significant so u rc e o f n u tritio n for sta rv ed se a slugs. T he h e rb iv o ro u s n a tu re o f wild P. ocellatus Is
c o n siste n t w ith Insights from m o lecu lar a n aly se s In dicating th a t k le p to p la sts a re fre q u e n tly re p le n ish e d from In g e sted
a lg a e, leading to th e c o n clu sio n th a t natu ral p o p u la tio n s o f P. ocellatus d o n o t rely o n p h o to sy n th e s is b u t m ainly o n th e
d ig e s tio n o f In g e ste d algae.
C itation : Maeda T, Hirose E, Chikaraishi Y, Kawato M, Takishita K, e t al. (2012) Algivore or Phototroph? Plakobranchus ocellatus (Gastropoda) Continuously
Acquires Kleptoplasts and Nutrition from Multiple Algal Species in Nature. PLoS ONE 7(7): e42024. doi:10.1371/journal.pone.0042024
Editor: Ross Frederick Waller, University o f M elbourne, Australia
Received April 11, 2012; A ccepted June 29, 2012; Published July 25, 2012
C opyrigh t: © 2012 Maeda e t al. This is an open-access article distributed under th e term s of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided th e original author and source are credited.
F u n d in g : The present research was supported by a grant from th e Mikimoto Fund for Marine Ecology, a Grant-in-Aid for a Japan Society for th e Promotion of
Science Fellow (No. 20234567 to T. Maeda), a grant from th e Research Foundation Flanders, a grant from the Australian Research Council (FT110100585 to H.
Verbruggen), and a grant from th e Okinawa Intellectual Cluster Program (to T. Maruyama). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation o f th e manuscript.
C om peting Interests: The authors have declared th a t no com peting interests exist.
* E-mail: tadashim@ jamstec.go.jp
feed o n Halimeda tuna [6—8] a n d algae ingested b y Oxynoe antillarum
a re lim ited to 2 species: Caulerpa racemosa a n d C. sertularioides [7].
Kleptoplasts retain p hoto synthetic activity for a few days to several
m onths in some sacoglossan species [10,11], L aboratory experim ents
showed that the photo synthetic products o f kleptoplasts (e.g., sugars
a n d am ino acids) are transferred to a n d used by sea slugs w hen they
are starved [12-14], This “functional kleptoplasty” [15] perm its sea
slugs to have a m ixotrophic lifestyle involving photosynthesis via
kleptoplasts a n d hetero trophy by feeding on algae [16]. How ever, the
relative im portance o f these feeding m echanism s un d er natural
conditions has not been characterized in detail.
T h e reten tio n p e rio d o f functional kleptoplasts in the tropical
Indo-Pacific species Plakobranchus ocellatus v a n H asselt, 1824 has
Introduction
Since the discovery o f “ chloroplast reten tio n ” in Elysia atroviridis
by K aw aguti & Y am asu [1], it has b e en w idely accepted th a t m any
species o f sacoglossan sea slugs (Sacoglossa, G astropoda, M ollusca)
retain chloroplasts o f ingested algae in digestive gland cells [2]. A
sequestered chloroplast is called a “kleptoplast” [3], T h e klepto­
plasts are not passed o n to progeny, so new kleptoplasts m ust be
a cquired in each g eneration [4-7]. Food algae o f sacoglossan species
have b e e n studied in lab o rato ry feeding experim ents. M ost
sacoglossan species have a fairly high feeding preference for one
o r a few algal species [7-9], a n d hence their kleptoplasts com e from
a lim ited n u m b er o f source algae. F o r exam ple, Bosellia mimetica only
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K le p to p la s ty in Plakobranchus ocellatus
b e en estim ated to b e u p to 10 m o n th s from lin ear ex trapolation o f
the photosynthetic activity m easu red by pulse am plitude m o d u ­
lation (PAM) fluorom etry [10,17]. T h e n a tu ra l algal source species
for kleptoplasts in P. ocellatus have n o t b e en d e term in ed [17,18],
b u t P. ocellatus are know n to feed o n m ultiple siphonous green algae
o f the o rd e r Bryopsidales u n d e r artificial conditions, i.e.,
Chlorodesmis hildebrandtii, Rhipidosiphon javensis , Caulerpella ambigua ,
a n d Bryopsis sp. [18-20], It has b e en p roposed th a t once the sea
slug acquires functional kleptoplasts, they are not replenished [21].
T his hypothesis is based o n the know ledge th a t the chlorophyll
contents o f kleptoplasts re m a in unaffected in starved P. ocellatus for
27 days u n d e r 500 ft-candle lighting (= 1 0 9 .5 pinoi p hotons
m ~ s 1 equivalent o f incandescent light) [10], th a t P. ocellatus
are m ostly found o n sandy beaches w here poten tial food algae are
ra re [21], a n d th a t feeding b e h av io r has never b e en observed in
th eir n a tu ra l habitats [21]. H ow ever, D u n lap [20] re p o rte d th at
the chlorophyll c ontent a n d 14C -inorganic carb o n fixation rate
dim inished after 2 m onths o f incubation u n d e r o u td o o r lighting
conditions, a n d concluded th a t kleptoplasts in P. ocellatus m ust be
replaced continually o r at periodic intervals to replace those th at
have degenerated. T hese two hypotheses re m a in to b e exam ined.
Before addressing w hich hypothesis is m ore likely, a n u m b e r o f
questions m ust b e answ ered. First, w hich algae a re the sources o f
kleptoplasts for the sea slugs? Second, do kleptoplasts in each P.
ocellatus individual derive from a single or m ultiple algal species?
Finally, to w h at degree does the sea slug’s n u tritio n d e p en d on
kleptoplast photosynthesis a n d o n algivory?
T o address these questions, w e identified the source algae o f
kleptoplasts based o n sequences o f the R u B isC O (ribulose 1,5bisphosphate carboxylase/oxygenase) large subunit gene (rbcL),
w hich is en co d ed in the chloroplast genom e [9,22,23] a n d has
b e en sequenced for a w ide range o f siphonous green algae [22].
W e assessed the tem p o ral kleptoplast com position in field-collected
P. ocellatus to determ in e w h eth er the sea slugs acquire kleptoplasts
only once o r repeatedly a n d estim ated the trophic positions o f the
sea slugs in th eir n a tu ra l h a b ita t a n d d u rin g starvation in the
lab o ra to ry based o n the n itrogen isotopic com position o f glutam ic
acid a n d phenylalanine [24,25], w hich can b e expected to serve as
a proxy o f the n utritional con trib u tio n o f functional kleptoplasts to
the sea slugs. B ased on this com b in atio n o f techniques, we aim ed
to gain insight into the ecological role o f functional kleptoplasty in
n a tu ra l circum stances.
T a b le
1.
M aterials and M ethods
Sampling and DNA extraction o f P. ocellatus
W e collected P. ocellatus for D N A analyses b y snorkeling or scuba
diving in a 2 0 0 -m x 2 0 0 -m near-shore a rea off T enija, O kinaw a,
J a p a n (26U3 3 'N 128"08'E). N o perm ission was req u ired for sam ple
collection. Sam ples w ere taken each m o n th from A pril to D ecem ber
2005, a n d from M ay 2007 to A pril 2008. H ow ever, the sea slug was
seldom found in w inter (D ecem ber 2005 a n d D ecem b er 2007 to
A pril 2008). C ollected sea slugs w ere fixed in 99% ethanol at room
tem p eratu re a n d preserved a t —30"C until use.
W ith a D N easy blood a n d tissue kit (Qiagen, H ilden, G erm any),
the D N A o f P. ocellatus was extracted from p a rt o f the parapodial
tissue (about 5 x 5 x 2 nini) (Figure SI), w hich included the digestive
gland (retaining kleptoplasts) b u t not the stom ach [26], A lthough a
previous study show ed chloroplasts on the outside o f the stom ach cells
o f P. ocellatus, no such “ extracellular” chloroplasts were observed in
the cavity o f the parapodial digestive gland [26], a n d hence we
believe th at the sequences obtained did not include contam inant
D N A from extracellular chloroplasts in the digestive tract.
Genetic analysis o f P. ocellatus
Because it has b e en p ro p o sed th a t P. ocellatus is a cryptic species
com plex [19,27], we checked the genetic hom ogeneity o f the
collected individuals by sequencing the m itochondrial 16S rR N A
gene (m tl6 S rD N A ). T o identify the source algae o f the
kleptoplasts, rbcL in 7 individuals o f P. ocellatus collected in
nonconsecutive m onths w ere also sequenced. F ragm ents o f rbcL
a n d o f m tl6 S rD N A w ere am plified b y P C R w ith th eir respective
p rim e r sets (T able 1).
T h e P C R m ixture (50 pi) c o n ta in ed 5 pi o f 10 x E x T a q Buffer
(T akara Bio, O tsu, Ja p a n ), 4 pi o f d N T P m ixture (10 m M ), 0.2 pi
o f T a K a R a E x T a q (T akara Bio), 1 pi o f the tem plate D N A , a n d
39.8 pi o f H oO . T h e th erm al cycle was com pleted u n d e r the
follow ing conditions: initial d é n atu ratio n at 96"C for 1 m in
follow ed b y 35 cycles o f 20 s at 96"C , 45 s at 5 0 -5 5 "C , a n d
1 m in 45 s—2 m in a t 72"C. A m plicons o f rbcL a n d m tl6 S rD N A
w ere purified using a W izard SV G el a n d a P C R Clean-ETp
System (Prom ega, H eidelberg, G erm any).
Purified am plicons o f the rbcL genes w ere cloned using a T O P O
T A C loning K it w ith T o p 10 E . coli (Invitrogen, C arlsbad, CIA)
according to the m an u fa ctu re r’s instructions. T h e insert size was
checked w ith P C R , a n d am plicons w ith the expected size w ere
Prim er list for s e q u e n c in g a n d T-RFLP.
Gene
P rim er nam e
Sequences (5 - 3 )
Reference
16S rD N A (on P. ocellatus (mitochondria)
16sar-F*f
CGC CTG TTT ATC AAA AAC AT
Palumbi e t al. [56]
16sbr-H*f
CCG GTC TGA ACT CAG ATC ACG T
Palumbi e t al. [56]
rbc1*f
CCA MAA ACW GAA ACW AAA GC
Hanyuda e t al. [57]
U3-2*f
TCT TTC CAA ACT TCA CAA GC
Hanyuda e t al. [57]
2 if
TTG GTW ACW GAA CCT TCT TC
Hanyuda e t al. [57]
rbc5f
GCT TGW GMT TTR TAR ATW GCT TC
Hanyuda e t al. [57]
trbcL-F*
CTK GCD GYD YTT MGD ATG ACA C
This study
trbcL-R*
MRG CWA RWG AAC GTC CTT CAT T
This study
rbcL (on kleptoplasts and chloroplasts)
Prim ers used fo r PCR and sequencing o f m ito c h o n d ria l 16S rD N A and k le p to p la st (chlo ro plast) rbcL genes, and fo r T-RFLP analysis o f rb c L
-Prim ers for PCR.
t Prim ers fo r sequencing.
^Primer fo r T-RFLP.
doi:10.1371 /journal.pone.0042024.t001
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K le p to p la s ty in Plakobranchus ocellatus
purified b y S a p /E x o l digestion. T h e sequencing reactions w ere
p erfo rm ed using a BigDye T e rm in a to r v3.1 Cycle S equencing K it
(Applied Biosystems, Foster City, CIA) w ith the prim ers used for
the P C R experim ents a n d two internal prim ers (T able 1). T h e
nucleotide sequences w ere d eterm in ed w ith a n ABI 3130x1
S equencer (Applied Biosystems). If the genetic distance betw een
two o r m ore rbcL sequences was less th a n 0.001 in/»-distance, those
sequence differences w ere considered as a P C R e rro r a n d the
m in o r different sequences w ere excluded from furth er analyses.
U sing the B lastn search a n d tree reconstruction o n partial
sequences [28], two ch im era sequences w ere d etected a n d
excluded from fu rth er processing.
Purified am plicons o f sea slug m tlO S rD N A w ere directly
sequenced w ith the prim ers used for the P C R experim ents
(T able 1). T h e sequencing was p erfo rm ed as described above.
[29] w ith the “— a u to ” option. A m biguously aligned sites w ere
rem oved autom atically using trim A l version 1.2 [30] w ith the “a u to m a te d l” option. T h e m ultiple sequence alignm ent finally
show ed 1254 positions. Phylogenetic analyses w ere p erform ed
w ith M E G A S [31] for the neighbor-joining (NJ) m ethod, a n d w ith
R A xM L version 7.2.8 [32] for the m axim um likelihood (ML)
m ethod. F or the N J m ethod, the m axim um com posite likelihood
m eth o d for nucleotide sequences [33] was em ployed. K akusan4
[34] was used to select the a p p ro p ria te m odel o f nucleotide
evolution for M L analysis, a n d the general tim e-reversible m odel
[35] in co rp o ratin g am ong-site rate v a riation a p p ro x im ated by a
discrete g am m a distribution w ith 4 categories (G T R + T ) was
chosen. T h e n o n p a ram etric b o o tstrap was used to exam ine the
robustness o f phylogenetic relationships (1000 pseudoreplicates for
N J a n d 300 for ML).
Sampling and genetic analysis o f algae
Terminal restriction fragm ent-length polym orphism
analysis o f rbcL from P. ocellatus
In o rd e r to e x p an d the reference d ataset o f rbcL sequences o f
potential source algae from the study region, species belonging to
the siphonous green algal o rd e r Bryopsidales w ere collected at
T enija, Bise (2 6 °4 2 'N 127°52'E ), Seragaki (2 6 °3 0 'N 127°52'E ),
a n d M a e d a (2 6 "2 6 'N 127"45'E ) in O kinaw a, at F aro de San
R afael (2 4 "1 8 'N 110"20'W ) in M exico, a n d at A suncion Island
(19"41'N 145"23'E ) in the M a ria n a Islands. N o perm ission was
req u ire d for sam ple collection. T h e sam ples w ere fixed in 99%
e thanol a n d kept a t —30"C until use. T h e species sam pled w ere
Rhipidosiphon lewmanomontiae, Rhipidosiphon sp., Caulerpa subserrata,
Halimeda borneensis, Chlorodesmis fastigiata, a n d Poropsis sp.
W e pressed v oucher specim ens from p a rt o f each sam ple a n d
deposited th em in the F ferbarium o f the C oastal B ran ch o f the
N a tu ral Ffistory M useum a n d Institute, C h ib a (C M N H ) (specim en
num bers C M N F F B A -6809-6816), J a p a n , or in the G h e n t
F ferbarium , Belgium (G.008 a n d FIV1774). T h e D N A was
extracted from the ethanol-fixed algal tissues w ith a D N easy
b lood a n d tissue kit (Qiagen), a n Isoplant kit (N ippon G ene,
T o y am a, Ja p a n ), o r a D N easy p lan t kit (Qiagen). T h en , algal rbcL
genes w ere am plified by P C R a n d directly sequenced w ith the
sam e prim ers used for P C R (Table 1) as described above.
W e p erfo rm ed term inal restriction fragm ent-length polym or­
phism (T-RFLP) analysis [36] to assess the diversity o f kleptoplasts
in the sea slugs a n d the seasonality o f their relative a b u n d an ce. T o
avoid the biases o f the am plification efficiency o f P C R due to the
h ig h /lo w m atch in g o f the prim ers, we new ly designed nested
consensus prim ers for T -R F L P based o n the rbcL sequences
o b tain e d (T able 1). T h e prim ers w ere designed to am plify the
fragm ent including a specific restriction site for distinguishing
different source algae o f the kleptoplasts. W e digested the rbcL
regions o b tain e d from P. ocellatus w ith various restriction enzym es
in silico using T R iF L e [37]. T a il (T herm o Fisher Scientific,
W alth am , MA) was found to b e the m ost suitable to identify the
m ultiple-source algal species o f kleptoplasts (T able 2).
T h e rbcL fragm ents w ere am plified from the D N A tem plates o f
P. ocellatus b y P C R w ith the fluoresceinated p rim e r trbcL -F a n d the
nonfluoresceinated p rim e r trb cL -R (T able 1). T h e rm a l cycling was
p erfo rm ed u n d e r the follow ing conditions: initial d é n atu ratio n at
96"C for 1 m in follow ed b y 35 cycles o f 20 s a t 96"C , 45 s at
57"C, a n d 1 m in a t 72"C. T h e com position o f the P C R m ixture
was the sam e as th a t for clone sequencing. A m plicons in the
reaction m ixtures (50 pi) w ere purified w ith a W izard SV G el a n d
P C R C lean -U p System (Prom ega, M adison, W I). T h e purified
am plicons w ere digested w ith 0.5 pi o f FastD igest T a il (Ferm entas,
Vilnius, L ithuania) a n d th en 0.5 pi o f 10 x FastD igest Buffer
(Ferm entas) in a total volum e o f 5 pi at 65 "Cl for 15 m in. T h e
Phylogenetic analyses o f kleptoplast rbcL
T h e rbcL sequences o b tain e d from P. ocellatus a n d algae w ere
aligned w ith those o f Bryopsidales a n d D asycladales species in the
D N A D a ta B ank o f J a p a n (DDBJ) using M A F F T version 6.818b
T a b l e 2 . In silico T-RF le n g th s o f rbcL se q u e n c e s.
C lade in Figure 1
P redicted T-RF length in silico (bp)
T-R FLP-category§
A b b re via tio n (clade)
Clade A (Proposis spp.)*
306
Proposis spp ./Halimeda borneensis
Prsp/Habo (A/D)
Clade B (Rhipidosiphon lewmanomontiae)
331
Rhipidosiphon lewmanomontiae
Rhle (B)
Clade C (Rhipidosiphon spp.)
366
Rhipidosiphon spp.
Rhsp (C)
Clade D (Halimeda borneensis)*
306
Proposis spp ./Halimeda borneensis
Prsp/Habo (A/D)
Clade E (Halimedineae spp. 1)*
138
Halimedineae spp. 1/Rhipiliaceae spp.
Hasp 1/Risp (E/G)
Clade F (Caulerpella spp.)*
191/291
Caulerpella spp.
Casp (F)
Clade G (Rhipiliaceae spp.)*
138
Halimedineae spp. 1/Rhipiliaceae spp.
Hasp 1/Risp (E/G)
Clade H (Halimedineae spp. 2)
260
Halimedineae spp. 2
Hasp2 (H)
'T-RF lengths of Proposis spp. and Halimeda borneensis were the same and indistinguishable.
b"-RF lengths o f Halimedineae spp. 1 and Rhipiliaceae spp. w ere identical and indistinguishable.
5Caulerpella spp. was com posed of tw o subtypes having tw o distinct T-RFs (191 and 291 bp, respectively).
§See Figures 3 and 4.
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dark) rhythm s. A fter incubation for 156 days (about 5 m onths), 3
individuals w ere ran d o m ly selected a n d dissected. T h e ir p a ra p o ­
dial tissues w ere also frozen a n d kept in liquid n itrogen until
analysis.
T h e n itrogen isotope analysis o f am ino acids was p erform ed
according to the m eth o d o f C hikaraishi et al. [25]. P a rt o f the
frozen p arap o d ial tissue o f each P. ocellatus individual was
hydrolyzed in 12N HC1 a t 100"C. T h e hydrolysate was w ashed
w ith H -hexane/dichlorom ethane (6:5, v/v ) to rem ove any hydrophobic constituents. A fter derivatization w ith thionyl c h lo rid e /2p ro p a n o l (1:4, v/v ) a n d subsequently w ith pivaloyl c h lo rid e /
dich lo ro m eth an e (1:4, v /v ), the derivatives o f the am ino acids w ere
extracted w ith n -h e x an e /d ic h lo ro m eth a n e (6:5, v /v ). T h e nitrogen
isotopic com position o f each am ino acid was d e term in ed by gas
ch ro m a to g ra p h y /c o m b u stio n /iso to p e ratio m ass spectrom etry
using a D elta plus X P isotope ratio mass spectrom eter (T herm o
F innigan M A T , B rem en, G erm any) coupled to a n 6890N gas
c h ro m a to g ra p h (Agilent T echnologies, M assy, France) via com ­
bustion a n d red u ctio n furnaces. N itrogen isotopic com positions
w ere expressed in 8 -notation against atm ospheric N 2 (air). T h e
trophic position o f the organism was calculated from the nitrogen
isotopic ratio in glutam ic acid a n d phenylalanine (T Po^/p^e value)
w ith the form ida T P Glu/Phc = (815N Glu- 8 15N Phc- 3 .4 ) / 7 .6 + l [24],
W e also d e term in ed the T P Glu/Phc value o f a giant clam ,
Tridacna crocea, h a rb o rin g a sym biotic dinoflagellate, zooxanthellae
(Symbiodinium spp.) [41], a n d o f an identified kleptoplast source
alga, R . lewmanomontiae. T h re e individuals o f T. crocea w ere
collected off Sesokojim a, O kinaw a (2 6 "3 8 'N 127"52'E ) o n 19
N ovem ber 2011. A fter 3 days o f incubation w ithout feeding a n d
light for tran sp o rt to the laboratory, the m antle tissues containing
zooxanthellae a n d a d d u cto r m uscles w ithout zooxanthellae w ere
dissected from each individual. T h e a d d u cto r m uscles w ere
analyzed using the sam e m eth o d as for the tissue o f P. ocellatus.
T o isolate zooxanthellae, m antles o f each individual w ere cut into
pieces, hom ogenized w ith a polytron-type h om ogenizer (T-10
basic hom ogenizer, IK A , Staufen, G erm any) w ith 20 m l o f
artificial seaw ater, a n d centrifuged at 750 Xg for 3 m in [42]. A fter
d é can tatio n o f the supernatant, the pellet was suspended w ith
20 m l o f artificial seaw ater. T h e suspension was filtered th ro u g h a
20-pm m esh nylon cloth to rem ove host tissue debris a n d w ashed
two tim es w ith 20 m l o f artificial seaw ater. Pelleted zooxanthellae
w ere used for the am ino acid n itrogen isotopic analysis. A thallus
o f R . lewmanomontiae was collected at the sam e site w ith P. ocellatus
digestion p ro d u c t was m ixed w ith 9 pi o f H i-D i form am ide
(Applied Biosystems) a n d 0.5 pi o f G S 1200 (Liz) (Applied
Biosystems) a n d th en d e n a tu re d b y heating at 95"C for 2 m in.
T h e m ixture was im m ediately chilled o n ice a n d th en electrophoresed w ith an ABI 3730x1 S equencer (Applied Biosystems).
T -R F L P profiles w ere analyzed using G e n eM ap p e r ver. 3.7
(Applied Biosystems). T h e internal size standards in electropho­
resis gave a n indication o f the length o f the term in al restriction
fragm ents (T-RFs) obtained. B ased o n the different lengths (base
pairs [bp]) o f T -R F s, the source algae o f the kleptoplasts w ere
distinguished. R elative am o u n ts o f the respective rbcL sequences
w ere estim ated from the heights o f T -R F peaks in T -R F L P
electropherogram s [36,38].
C h ro m ato g ram s w ith high-quality value (Q V > 7 5 ) w ere selected
for furth er analyses. F ragm ents sm aller th a n 100 b p a n d larger
th a n 1200 b p w ere considered noise a n d excluded from further
analyses. E ac h T -R F was identified b y m atch in g to the in silico
digestion lengths o f rbcL sequences from kleptoplasts (T able 2).
T h e relative a b u n d an c e was calculated from relative peak heights
using the form ula R A (%) = L1T.RF/F [ tot£li*100 (where R A is
relative ab u n d an c e, F I t - r f is the p eak height o f a specific T -R F ,
a n d Hjotai is the total o f all T -R F p e ak heights in each
chrom atogram ). T h e statistical significance o f the differences in
rbcL a b u n d an c e b etw een seasonally collected sam ples was assessed
w ith the p e rm u te d B ru n n er-M u n zel test [39] im plem en ted in the
“ law stat” package for “ R ” [40],
Am ino acid nitrogen isotopic analysis
T o estim ate the trophic position o f the sea slugs u n d e r natu ral
conditions a n d d u rin g starvation, w e used am ino acid nitrogen
isotopic analysis [24,25]. T h e individuals o f P. ocellatus for the
am ino acid n itrogen isotopic analysis w ere collected off T oguchi,
O kinaw a, J a p a n (2 6 "2 T N 127"44'E). N o perm ission was req u ired
for sam ple collection. A bout 50 individuals w ere collected o n 27
A pril 2010. F o r tran sp o rt to the lab o ra to ry in K an ag aw a, J a p a n ,
they w ere kept alive in artificial seaw ater (R hotom arine, R ei-Sea,
T okyo, Ja p a n ) a t ro o m tem p e ra tu re for 3 days w ithout lighting.
T h re e healthy individuals w ere ran d o m ly selected a n d dissected,
a n d p a rap o d ial tissues w ere frozen a n d kept in liquid n itrogen as
wild sam ples. T h e sam ples for starvation w ere collected o n 8 A pril
2008 (about 50 individuals) a n d in cu b ated in a n a q u ariu m filled
w ith artificial seaw ater not c o ntaining m acroalgae a t 24"C u n d e r
light (13 pinoi p hotons m -2 s- 1 ) w ith d a y -n ig h t (14-h lig h t/1 0 -h
T a b l e 3 . N u m b e r of c lo n e s o b ta in e d from k le p to p la s t rbcL clo n e se q u e n c e s.
C orresponding clades (in Figures 1 an d 2) o f clones
A
B
C
D
Po-2005-A (Apr.)
1
-
-
Po-2005-B (May)
18
-
1
Po-2005-C (June)
7
5
Po-2005-D (Aug.)
10
3
Po-2005-E (Aug.)
82
Po-2005-F (Sept.)
11
T o tal
E
F
G
H
-
7
-
-
8
16
-
2
-
-
-
21
ID of th e individual (month of collection)
Po-2005-G (Nov.)
13
Total num ber o f clones
142
^B ^B
^B ^B
^B
^B ^B
^B
^B ^B
4
4
3
-
-
7
-
4
1
-
8
-
-
4
1
13
2
-
24
5
2
90
-
-
15
5
10
6
9
20
19
-
24
209
Number of clones obtained in kleptoplast rbcL clone sequencing from each of 7 Plakobranchus ocellatus individuals. The source algae w ere identified from the
phylogenetic analysis (Figure 1).
doi:10.1371 /journal.pone.0042024.t003
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K le p to p la s ty ¡n Plakobranchus ocellatus
ocellatus kleptoplast AB619285
ocellatus kleptoplast AB619270
ocellatus kleptoplast AB619263
ocellatus kleptoplast AB619302
ocellatus kleptoplast AB619278
ocellatus kleptoplast AB619265
- P ocellatus kleptoplast AB619287
. P. ocellatus kleptoplast AB619269
.P . ocellatus kleptoplast ABS19288
. P. ocellatus kleptoplast AB619289
. P ocellatus kleptoplast AB619305
. P. ocellatus kleptoplast AB619286
. P ocellatus kleptoplast AB619271
-P . ocellatus kleptoplast AB619274
. P. ocellatus kleptoplast AB619313
- P ocellatus kleptoplast ABS19280
- P ocellatus kleptoplast AB619294
_ P. ocellatus kleptoplast AB619268
Clade A
■P. ocellatus kleptoplast AB619277
. P. ocellatus kleptoplast AB619281
(P roposis spp.)
, P oropsis sp. A B700349
1 P ocellatus kleptoplast AB619267
- P. ocellatus kleptoplast AB619284
. P ocellatus kleptoplast AB619311
- P . ocellatus kleptoplast AB619266
95/87 — P ocellatus kleptoplast AB619292
j - P ocellatus kleptoplast AB619276
NC/61
p - ocellatus kleptoplast AB619279
j - P ocellatus kleptoplast AB619300
9 é -P . ocellatus kleptoplast AB619308
NC/74
P ocellatus kleptoplast AB619297
97/99 r P ocellatus kleptoplast AB619282
97 )7 *— P. ocellatus kleptoplast AB619283
NC/87
P ocellatus kleptoplast AB619262
NC/63 100/88 P ocellatus kleptoplast AB619293
P. ocellatus kleptoplast AB619304
65/84
— P. ocellatus kleptoplast AB619306
L P. ocellatus kleptoplast AB619309
NC/66
R h ipidosiphon ja v e n s is F J432644
96/57p
ocellatus kleptoplast AB619298
P ocellatus kleptoplast AB619301
C lade B
100/76
Rhipidosiphon lew m anom ontiae AB700351
(R hipidosiphon
NC/73
P. ocellatus kleptoplast AB619290
100/73
lew
m anom ontiae)
j Rhipidosiphon lewm anom ontiae AB700350
98190^R hipidosiphon lew m anom ontiae HQ 871689
P.
.P .
P.
.P .
.P .
.P .
0.6
I
oí
CD
Q.
CD
03
CD
CO
*2
O
-O
«
CL
Q3_
CD
en
]
68 / 87/87
$0/100j— ch lo ro d e sm is fastigiata F J432639
C hlorodesm is fastigiata AB700352
94/81
Udotea conglutinata AY942168
Tí
R hipocephalus phoenix AY942172
Penicillus capitatus FJ432641
Udotea unistratea HQ 8716 92
U dotea spinulosa AY942160
Penicillus p yriform is AY942162
MC ;/66
.
100/100
97/1001- Penicillus dum etosus AY 942175
- Udotea g la u ce scen s F J432650
in n /a á ll R hipidosiphon sp. AB700348
79/511
ocellatus kleptoplast AB619299
C lade C (R hipidosiphon spp.)
93 98T p - ocellatus kleptoplast A B 619307
30,301 " ocellatus kleptoplast AB619295 ]
Udotea flabellum A Y 942166
F labellia pe tio la ta F J4 3 2 6 4 0
T ydem ania exp e dition is AY942161
rp
0.06
F ig u re 1. P h y lo g e n e tic tr e e b a s e d o n
rb c L se q u e n c e s . M axim um likelih o o d (ML) p h y lo g e n y o f t h e class U lv o p h y c e a e b a s e d o n 1254 n u c le o tid e
p o s itio n s o f th e c h lo ro p la s t-e n c o d e d rbcL g e n e . T h e p h y lo g ra m o f th e e n tire tre e o n th e u p p e r left Is c o lo re d to m a tc h th e Inset. C h la m y d o m o n a s
re in h a rd tii (class C h lo ro p h y c e a e ) w a s c h o s e n a s th e o u tg r o u p . R ed, rbcL s e q u e n c e s fro m P. o cellatus. Black, algal rbcL s e q u e n c e s . B o o tstra p s u p p o r t
(BS) v a lu e s > 5 0 % a re p ro v id e d a t th e n o d e s (n e lg h b o r-jo ln ln g /M L ).
BS < 5 0 % ; "NC", n o n c o n s e n s u s n o d e th a t w a s n o t o b s e r v e d In th e n e ig h b o rjo in in g tre e .
d o l:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 4 2 0 2 4 .g 0 0 1
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K le p to p la s ty ¡n Plakobranchus ocellatus
Pseudocodium sp. FJ607674
Pseudocodium sp. AM 909693
.........................................
100/81 1— rPseudocodium
devriesii AM 909689
100/100
100/100 í Pseudocodium okinawense AB301930
I Pseudocodium okinawense F J607675
100/100 Pseudocodium floridanum AM909692
76/92
Pseudocodium floridanum AY942178
90/98
Halim eda cryptica FJ624496
100/971
Halim eda pygm aea FJ624497
Halim eda m icronesica FJ624499
Halim eda fragilis FJ624498
98/59 Halim eda cylindracea FJ624510
100/95 ,H alimeda heterom orpha F J6 2 4 5 1 1
'53
Halim eda kanaloana FJ624512
76/Halim eda m acroloba F J624513
Halim eda m elanesica FJ624515
Halim eda borneensis F J624514
97/í
- P. ocellatus kleptoplast AB619264
C lade D
P. ocellatus kleptoplast AB619275
100/100
(.H alim eda
Halim eda borneensis AB700353
98| 99 P. ocellatus kleptoplast AB619273
] borneensis)
— Halim eda m onile F J624516
100/98
00/1OQJ ¡H a lim e d a incrassata FJ715719
6317m. Halim eda incrassata AY942167
100/991- Halim eda sim ulans FJ624517
)0/100------ Halim eda lacrim osa FJ624495
i ------ Halim eda gracilis FJ624491
100/991— Halim eda m agnidisca F J624530
np
i Halim eda discoidea AB038488
100/1 )0
„ Halim eda discoidea AB700354
NC/76
1.00/100 Halim eda lacunalis FJ624521
H;
5 7 71
/.’
Halim
eda tuna FJ624524
I
Halim eda copiosa FJ624508
inn/-i n n l i Halim eda opuntia AB700355
i n r v o i l - Halim eda opuntia AB038489
94/60 Halim eda m inim a FJ624504
— Siphonogram en abbreviata FJ432649
849
P. ocellatus kleptoplast AB619312 —
100/100
- P. ocellatus kleptoplast AB619296
C lade E
P ocellatus kleptoplast AB619310
(H alim edineae
P ocellatus kleptoplast AB619291
100/100
P ocellatus kleptoplast AB619314 ] spp.1)
-C aulerpella am bigua F J432638
100/100 -4 0 0 /100 r P. ocellatus kleptoplast AB619258
C lade F
L T I P. ocellatus kleptoplast AB619260
65/5 9 — p ocellatus kleptoplast AB619259 ] (C aulerpella
10 0 /1 0 0 1- Caulerpa okam urae A_B0_38_4_8_4
91/99
spp.)
Caulerpa lentillifera AB054009
-C a u le rp a taxifolia AB054Û16
100/100
Caulerpa sertularioides AB054014
100/100 |- Caulerpa prolifera AY942173
— Caulerpa webbiana AB054017
Caulerpa brachypus AB038483
97/100
Caulerpa subserrata AB700356
Caulerpa serrulata AB700357
59/64
Caulerpa scalpelliform is AB054013
— Caulerpa racem osa AB038485
i Caulerpa racem osa A B 054011
0.06
58/1 ¡ÿ- Caulerpa racem osa AB700358
-C a u le rp a num m ularia AB054010
1—Rhipiliopsis profunda FJ432647
C lade G
90/87
P. ocellatus kleptoplast AB619261
(R hipiliaceae spp,
P ocellatus kleptoplast AB619257
100/100
E
I
0.06
NC/68
I
CD
>5
o
3
CD
Q.
3 ’
CD
Q)
CD
"O
ÇP
CL
M
CD
CP
Í¿
90/98
f
]
10C 100 P. oce//afus_kleptoplast AB619256
----------- Pseudochlorodesm is sp. FJ432643
NC/61
100/100 i— Rhipilia nigrescens FJ432646
72/97
— Rhipilia tomentosa AY942164
R hipilia crassa FJ43JÄ45
100/100 jp . ocellatus kleptoplast A B619272 j c i a d e H (H alim edineae spp
51/■2 )
100/100
‘P ocellatus kleptoplast AB619303 ■
— Pseudochlorodesm is furcellata FJ432642
■Dichotom osiphon tuberosus A B038487
100/100
■Pedobesia ryukyuensis AB038482
3/66
100/100 ,
— Codium lucasii AB038481
Codium fragile M67453
100/100
62/58
■Bryopsis plum osa AB038480
Bryopsis hypnoides AY942169
O streobium quekettii FJ715720
Chlorocladus australasicus AY177750
100/100
98/99
------------ Bornetella sphaerica FJ535850
D asycladales
Acetabularia acetabulum FJ715714
— Chlam ydom onas reinhardtii NC 005353
88/81
100/100
F ig u re 2. P h y lo g e n e tic t r e e b a s e d o n rbcL s e q u e n c e s (c o n tin u e d ).
doi:10.1371/journal.pone.0042024.g002
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K le p to p la s ty in Plakobranchus ocellatus
Rhilidosiphon lewamanomontii
Relative fluorescence units fRFU)
100
6000-
150
Flagment length (bp)
200
250
300
350
400
350
400
350
400
500040003000
2000
1000
00
331 bp
Rhle(B)
Halimeda borneensis
Relative fluorescence units {RFU)
„100
1500]—
1400
150
200
250
300
1200
1000
800
600
400
200
Relative fluorescence units (RFU)
Q
305bp
Prsp/Habo(A/D)
Plakobranchus ocellatus
1500
1400
100
150
200
250
300
1200
1000
800
600
400
200
137bp
Hasp1/Rhsp(E/G)
291bp
Casp(F)
190bp
Casp(F)
331 bp
Rhle(B)
365bp
RhspiC)
305bp
Prsp/Habo(A/D)
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K le p to p la s ty in Plakobranchus ocellatus
F ig u re 3. E le c tro p h e ro g ra m s o f T-RFLP a n a ly sis. T-RFLP e le c tr o p h e r o g ra m s fro m (A) Rhipidosiphon lewmanomontiae, (B) Halimeda borneensis,
a n d (C) a s in g le in d iv id u al o f Plakobranchus ocellatus (s a m p le n o . P lo c 0 5 1 1 13A). F ra g m e n t le n g th s a n d c o rre s p o n d in g s o u rc e a lg a e a re s h o w n in th e
b o x u n d e r th e p e a k . O nly a s in g le p e a k w ith a le n g th id e n tic al to th a t p re d ic te d b y in silico T-RFLP w a s o b ta in e d fro m e a c h alg al in dividual (T able 2).
T h e e le c tr o p h e r o g ra m fro m th e s e a slu g (C) s h o w e d m u ltip le p e a k s c o rre s p o n d in g to th o s e o f in silico T-RFLP o f th e k le p to p la s t s o u rc e a lg a e (Table 2).
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 4 2 0 2 4 .g 0 0 3
single T -R F p e ak in each algal species, a n d its T -R F length was
identical w ith th a t p red icted in silico (H . borneensis = 306 b p a n d R.
lewmanomontiae = 331 bp) (Table 2, Figure 3A, B).
H igh-quality T -R F L P electropherogram s (quality value > 7 5 )
w ere obtain ed from 30 individuals o f P. ocellatus collected in 2005
a n d from 20 collected in 2007 (Figure 4A). O n e to five peaks w ere
found in each P. ocellatus individual (Figures 3C a n d 4A, T ab le SI).
E xcept for the peak corresponding to H alim edineae spp. 2 (clade H)
(260 bp), all p redicted restriction profiles w ere recovered. Forty-four
o u t o f 50 individuals o f P. ocellatus show ed m ultiple T -R F peaks (28
individuals in 2005 a n d 16 in 2007) (Figure 4A, T ab le SI).
A ssum ing th a t the genom e copy n u m b e r in the chloroplasts did
n o t change before a n d after sequestration, the relative a b u n d an c e
o f rbcLs was used to estim ate th a t o f the corresponding kleptoplasts.
As show n b y the boxplots in Figure 4B, the relative a b u n d an c e o f
kleptoplasts show ed m ark ed seasonal changes. Som e o f the
observed changes o c cu rre d over a relatively short tim e-span. F or
exam ple, in 2005, the a b u n d an c e o f H a s p l/R is p (clade E /G ) in
A ugust was significandy h igher th a n th a t in J u n e (p-value = 0.025)
(Figure 4B). Sim ilarly, in 2007, the percentage o f Caulerpella spp.
(clade F) significantly increased from J u ly to A ugust (pvalue = 0.010), while th a t o f R . lewmanomontiae (clade B) signifi­
cantly decreased in the sam e p erio d (p-value = 0.020) (Figure 4B).
In 2005, clear changes in a b u n d an c e w ere also observed for
Rhipidosiphon spp. (clade C), R . lewmanomontiae (clade B), a n d
H a s p l/R is p (clade E /G ) b u t these w ere n o t significant in the
p e rm u te d B ru n n er-M u n zel test (p-value = 0.097, 0.571, a n d 0.102,
respectively) (Figure 4B).
(off T oguchi, O kinaw a) o n 27 A pril 2010 a n d its S 15N was
analyzed as described above.
Results
Identification o f P. ocellatus kleptoplasts
T o confirm the genetic hom ogeneity o f P. ocellatus collected a n d
identify the source algae o f kleptoplasts, we sequenced th eir m tl6 S
rD N A a n d rbcL. T h e sequences o f m tl6 S rD N A fragm ents
[443 bp: D N A D a ta B ank o f J a p a n (DDBJ) accession nu m b er,
AB700359] w ere identical am o n g all exam ined P. ocellatus
individuals collected off T en ija, O kinaw a, J a p a n , suggesting th a t
all sam ples belonged to the sam e species.
By clone sequencing, we o b tain e d 209 rbcL sequences o f
kleptoplasts from 7 individuals o f P. ocellatus collected in 2005
(T able 3). A fter unifying highly sim ilar sequences (¡6-dista n c e< 0 .0 0 1 ), 59 sequences w ere used for subsequent analyses
(DD BJ accession num bers, A B 619256-A B 619314).
B lastn analyses show ed th a t the kleptoplast rbcL sequences w ere
the m ost sim ilar to those from green algae belonging to the
suborder H alim edineae (order Bryopsidales). In the phylogenetic
tree (Figures 1 a n d 2), the kleptoplast sequences did n o t form a
m onophyletic clade b u t w ere divided into eight clades w ithin the
H alim edineae (clades A - H in Figures 1 a n d 2). C lade B sequence
A B 619290 was identical to R . lewmanomontiae from T en ija
(AB700351). C lade D co rresp o n d ed to reference sequences o f H .
borneensis (FJ624514 a n d A B700353). T h e rem ain in g 6 clades o f
kleptoplasts (A, C, E, F, G , H) could n o t be identified a t the species
level a n d w ere assigned to h igher taxa. C lade A clustered w ith
Poropsis sp., clade C w ith Rhipidosiphon sp., clade F w ith the genus
Caulerpella, a n d clade G w ith the fam ily R hipiliaceae. Fam ilial
relationships w ere un clear for clades E a n d H , b u t they b o th
belong to the h igher tax o n H alim edineae. F or convenience, the
source algae w ith such im perfect identifications are indicated w ith
a h igher tax o n nam e+ spp. (e.g., R hipiliaceae spp.).
Based o n the rbcL sequences cloned from the 7 individuals, it
was already clear th a t individual sea slugs c o n ta in ed kleptoplasts
from m ultiple source species. As show n in T ab le 3, no single
individual h a d only one type o f kleptoplast. In m ost cases,
kleptoplasts from 3 o r 4 algal species w ere p re sen t in P. ocellatus
individuals.
Trophic position o f P. ocellatus
T o estim ate the tro p h ic position o f P. ocellatus, the S15N values o f
glutam ic acid (S15N g iu) a n d phenylalanine (S15Nphe) o f the freshly
collected a n d starved sam ples o f P. ocellatus w ere m easured, a n d
TPoiu/phe values w ere calculated. T h e m ean TPoiu/phe value o f 3
freshly collected P. ocellatus individuals was 1 .9 ± 0 .1 (m ean ± “ l a
for the T P values,” n = 3) (T able 4). O n the o th er h and, the m ean
TPoiu/phe values o f P. ocellatus th a t h a d b e en starved u n d e r light for
ab o u t 5 m onths (156 days) was 1 .3 ± 0 .1 (n = 3) (T able 4). T h e
TPoiu/phe value o f R . lewmanomontiae, w hich was a source alga o f
kleptoplasts in P. ocellatus, was 1.0 (n= 1). T h e m ea n T P o w p h e
value o f T. crocea a d d u cto r m uscle was 2 .0 ± 0 .1 (n = 3), a n d th a t o f
zooxanthellae isolated from T. crocea m a n d e was 0 .9 ± 0 .0 (n = 3).
Seasonality o f P. ocellatus kleptoplasts
T o exam ine w h eth er the com position o f kleptoplasts changed
over tim e, T -R F L P analysis o f rbcL w ith T a il digestion was
perform ed. H alim ed in eae spp. 1 (clade E) a n d R hipiliaceae spp.
(clade G) could n o t b e distinguished in this analysis because their
pred icted T -R F lengths w ere identical a t 138 b p (T able 2). T h ey
are h ereafter referred to as “ H a s p l/R is p (clade E /G ).” T h e T -R F
o f Poropsis spp. (clade A) could n o t be differentiated from th a t o f H .
borneensis (clade D) (T -R F length = 306 bp, T ab le 2), a n d th ey are
referred to as “ P rs p /H a b o (clade A /D ).” T h e heights o f those T R F peaks w ere re g ard e d as the sum o f the respective two algal
source clades (T able 2).
T o establish the applicability o f in silico T -R F predictions in
actual T -R F L P analysis, we c o m p a red the p red icted T -R F
fragm ent lengths w ith those o f PC R -am plified rbcL fragm ents
from two algae, H . borneensis a n d R . lewmanomontiae. W e o b tain e d a
PLoS ONE I w w w .p lo s o n e .o rg
D iscussion
By com bining different m ethods, o u r results offer several new
insights into the ecology o f functional kleptoplasty in P. ocellatus.
Because u n in cu b a ted w ild individuals w ere studied, the results
shed light o n the n a tu ra l state o f this sea slug species.
M ultiple algal sources o f kleptoplasts in P. ocellatus
T h e phylogenetic analyses o f the chloroplast-encoded rbcL gene
identified a t least 8 algal sources o f kleptoplasts in P. ocellatus
(Figure 1). T h is n u m b e r exceeds the estim ates from previous
lab o ra to ry feeding experim ents o n P. ocellatus [18,20] O u r d a ta
dem onstrate th a t P. ocellatus has one o f the b ro a d est food algal
spectra in the Sacoglossa, including a t least 8 species in 5 different
algal g enera [7]. T h e m ultiplicity o f kleptoplasts in individual sea
8
J u ly 2 01 2 I V o lu m e 7 | Issue 7 | e 4 2 0 2 4
K le p to p la s ty in Plakobranchus ocellatus
2005
100
2007
TUI
8 75
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CS
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§ 50
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CD
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a)
a:
H asp1/R hsp
(Clade E/G)
1
â 25
o
10, 28, 23,
Apr. Apr. May
21,
Jun.
3,
July
7,
Aug.
16, 13,
S e p t Nov.
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May
15,
July
15,
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S am pling date
B
2005
2007
100
Rhsp
Maximum
‘ U pper quartlle
50
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Apr Apr. May Jun. July Aug.
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Nov.
May
July
Aug.
Sept.
Sam pling date
F ig u re 4. T im e s e rie s o f rbcL s e q u e n c e c o m p o s itio n in Plakobranchus ocellatus. (A) R elative a b u n d a n c e o f re s p e c tiv e rbcL in in d iv id u a ls o f
Plakobranchus ocellatus c o lle c te d m o n th ly in 2 0 0 5 a n d 2 0 0 7 . S o u rc e a lg a e o f k le p to p la s ts a re s h o w n b y a b b re v ia tio n s o f th e c la d e s (T able 2). (B)
T u k ey 's b o x p lo ts o f t h e re la tiv e a b u n d a n c e o f rbcL s e q u e n c e s . D ata p o in ts o f fe w e r th a n th re e in d iv id u á is o f P. ocellatus a re s h o w n a s d ia m o n d s . T he
PLoS ONE I w w w .p lo s o n e .o rg
9
J u ly 2 01 2 I V o lu m e 7 | Issue 7 | e 4 2 0 2 4
K le p to p la s ty in Plakobranchus ocellatus
brackets with
indicate a significant difference of the percentage value in the perm uted Brunner-Munzel test. The value beside the symbol
indicates p-value.
doi:10.1371/journal.pone.0042024.g004
slug specim ens also confirm s th a t individuals have m ultiple food
sources. T his m ultiplicity o f kleptoplasts was first re p o rte d in a
sacoglossan, Elysia clarki (2 -4 species) [9], a n d also recently
suggested in P. ocellatus [17]. T hese findings strongly suggest that,
in the n a tu ra l setting, the sea slugs feed o n several species a n d
re ta in th eir chloroplasts in digestive gland cells.
A com m on conclusion o f our w ork a n d previous feeding studies on
P. ocellatus [7,18,20] is that their food/source algae are all siphonous
green algae o f the order Bryopsidales. T h e genus Caulerpella, w hich
was identified as a food source in a previous study [20], was shown to
be a m ajor contributor to the kleptoplast pool. O n the other hand, we
did not detect 3 algal species that w ere eaten by P. ocellatus in a
laboratory setting in previous studies: C. hildebrandtii (Udoteaceae);
Bryopsis sp.; a n d R . javensis [18,20], Such discordance m ay be because
the algae in question are not preferred by P. ocellatus in their natural
habitats, or because the algae are ingested b u t their chloroplasts are
not retained. F or some o f the algal species involved, misidentifications
could also lead to the m ism atch. For exam ple, Rhipidosiphon is a genus
o f dim inutive, fan-shaped algae in w hich there are several
m orphologically similar but genetically distinct species (Verbruggen,
unpublished results), a n d kleptoplast clade C m ay originate from
algae th at closely resem ble R . javensis. Similarly, it is possible that in
the previous studies the source algae belonging to clade A (genus
Poropsis) could have been m isidentified as C. hildebrandtii. Because these
taxa are anatom ically very similar', a n d because the genus Poropsis is
little know n a n d not com m only included in taxonom ic reference
works, it m ay have b een regarded as C. hildebrandtii in previous studies.
O u r kleptoplast survey also revealed several new food algae o f P.
ocellatus. A m ong th em are R . lewmanomontiae (clade B), PI. borneensis
(clade D), R hipiliaceae spp. (clade G), a n d potentially Rhipidosiphon
spp. (clade B). In ad dition to these taxa, we found kleptoplasts o f two
lineages th a t could n o t be readily assigned to a family (Flaliniedineae
spp. 1 a n d spp. 2). T h e m ost closely related rbcL sequences o f these
two clades are Pseudochlorodesmis species (Figure 1). T h e dim inutive
siphon o f Pseudochlorodesmis, if b ra n ch e d a t all, does so only a few
tim es [43]. Because the algae rarely exceed a few m illim eters in
length, it should not com e as a surprise th a t they m ay have b een
overlooked in previous studies. T h e observed ulvophyceaen
m acroalgae in sam pling sites are listed in T ab le S3.
A n interesting observation is th a t P. ocellatus do not seem to
discrim inate am o n g food sources based on size. A m ong the sources
o f kleptoplasts are some larg er seaweeds such as PI. borneensis (10 cm)
a n d R . lewmanomontiae (3 cm) as well as several m illim eter-scale taxa
(.Pseudochlorodesmis, Caulerpella). O n e com m onality betw een these
algae is th a t they all belong to suborder Flaliniedineae o f the ord er
Bryopsidales (Figure 1) [44], w hich is consistent w ith the hypothesis
on the relationships betw een the ra d u la r tooth shape a n d food algal
cell wall com ponents [45]. T h e ra d u la r to o th o f P. ocellatus has a
triangular shape suitable for p u n c tu rin g a hole in the halim edineaen
cell wall, w hich is com posed o f xylan [45,46], after w hich the
cytoplasm can b e ingested.
Changing annual kleptoplast com position
T -R F L P analysis show ed th a t the kleptoplast rbcL com position
o f the sea slugs ch an g ed considerably from m o n th to m onth.
Because kleptoplasts have n o t b e en observed to divide [2,17], these
changes m ust result from the replacem ent o f kleptoplasts w ith
newly obtain ed chloroplasts by feeding, different spans o f longevity
am o n g kleptoplasts derived from various source algae, a n d /o r
differential kleptoplast D N A replication (m ultim erization) rates
[47,48] due to those source algae.
D ifferent spans o f longevity a n d change in chloroplast genom e
m ultim erizations o f the kleptoplasts w ould leave a specific im print
on the kleptoplast com position over tim e, i.e., the recovered
kleptoplast com position w ould rem ain the sam e b u t th eir relative
ab u n d an c e should in cre ase/d e cre ase gradually over tim e. O u r
results do not suggest such a p a tte rn b u t ra th e r suggest th a t large,
ap p aren tly stochastic changes occur in kleptoplast com position.
A lthough we can n o t entirely exclude the possibility th a t P. ocellatus
individuals im m igrated into the pop u latio n at the collection sites,
previous studies suggested th a t P. ocellatus b re ed in a restricted
season in spring a n d rarely m igrate as adults [18,49], T herefore,
the hypothesis th a t kleptoplasts are replaced w ith newly obtain ed
chloroplasts from ingested algae appears to b e the m ost plausible
e x planation for o u r results. A lthough the seasonal change in the
Table 4. S15N values of amino acids and estim ated trophic positions of Plakobranchus ocellatus, th e alga Rhipidosiphon
lew m anom ontiae, and the giant clam Tridacna crocea.
Specim en
N um ber o f specim ens
A veraged §1SN v a lu e § (SD)
G lu ta m ic acid
P h enylalan ine
T rop hic po sitio n #
Plakobranchus ocellatus
Freshly collected* (Apr. 2010)
3
17.4 (0.9)
7.3 (1.3)
1.9 (0.08)
Starved* (Apr.-Nov. 2008)
3
20.0 (0.5)
14.4 (0.9)
1.3 (0.07)
1
12.3
8.8
1.0
Rhipidosiphon lewmanomontiae (Apr. 2010)
Tridacna crocea (Nov. 2011)
Adductor muscle
3
16.1 (0.6)
5.5 (0.1)
2.0 (0.07)
Zooxanthellae*
3
8.2 (0.4)
5.3 (0.1)
0.9 (0.04)
^Collected off Toguchi, Okinawa, Japan.
^Starved for 156 days (5 months) in a laboratory aquarium after collection off Toguchi.
5Symbiodinium spp. isolated from th e mantle of T. crocea.
§% o, relative to air.
# Trophic position (TPG,U/Phe) = (815NG,u- 8 15Nphe-3.4)/7.6+1.
doi:10.1371 /journal.pone.0042024.t004
PLoS ONE I w w w .p lo s o n e .o rg
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K le p to p la s ty in Plakobranchus ocellatus
algal co m m unity m ay affect the kleptoplast com positions in P.
ocellatus, this p o in t rem ains to be studied in future.
synthetic activity lost is necessary to sustain the re q u ire d level o f
photosynthetic activity.
A lthough P. ocellatus is capable o f retain in g kleptoplasts for up to
10 m onths, o u r results indicate th a t the nu tritio n al con trib u tio n o f
kleptoplasts m ay b e insufficient even in a n a tu ra l h a b ita t w here
food algae are plentiful. In o th er sacoglossan species such as Elysia
timida, E . viridis, a n d Thuridilla carlsoni, the n utritional c o ntribution
o f functional kleptoplasts m ay be even less, because the
photosynthetic activity o f th eir kleptoplasts is less a n d re tention
periods are shorter th a n in P. ocellatus [10]. T h e ecological role o f
kleptoplasts u n d e r n a tu ra l conditions w h en food is a b u n d a n t
rem ains unclear. F unctional kleptoplasty m ay reduce the algal
feeding a n d the energy cost to ingest th eir calcified thallus [6]. O u r
p re sen t study show ed th a t the nu tritio n al yield from kleptoplasts
does n o t exceed th a t from food digestion b u t suggested th a t they
provide a n energy source (e.g., sugars) a n d essential nutrients (e.g.,
am ino acids) u n d e r starved conditions. T h e T P Giu/phe value o f 1.3
d u rin g starvation suggests th a t the con trib u tio n o f kleptoplasts to
am ino acid synthesis is m u ch greater th a n th a t o f autophagic
digestion o f reserved am ino acids a n d proteins, w hich w ould
increase the T P Giu/phe value. It has b e en p ro p o sed th a t the
function o f kleptoplasts m ay be to co m p lem en t nutrients u n d e r
starved conditions [6]. O u r p re sen t results sup p o rt this hypothesis.
A lthough a d u lt P. ocellatus are th o u g h t to be n ourished b y the
photosynthetic activity o f functional kleptoplasty, the p re sen t study
suggests th a t the sea slug continually feeds o n siphonous green
m acroalgae in its n a tu ra l h a b ita t a n d th a t digested algae are the
m ajo r source o f n u trition, while kleptoplast photosynthesis plays a
very m in o r role. F u rth e r studies on the feeding behavior,
photosynthetic activity, a n d nu tritio n al con trib u tio n o f kleptoplasts
in P. ocellatus are necessary to u n d e rstan d the ecological role o f the
functional kleptoplasty o f this species.
Trophic position o f P. ocellatus
A ssum ing a n e rro r in the T P Giu/phe value (i.e., l a = 0.12) [24],
the values o f w ild P. ocellatus (m ean TPoiu/phe value = 1.9) w ere
identical to the trophic position o f algivorous organism s, i.e.,
p rim a ry consum ers (T able 4, S2). T hese results clearly indicate
that, u n d e r n a tu ra l circum stances, P. ocellatus individuals m ainly
o b tain am ino acids by digestion o f ingested algae. T his is in stark
co n trast to P. ocellatus individuals th a t h a d b e en starved for 5
m onths a n d h a d a T P o w p h e value o f 1.3, w hich is interm ediate
betw een values typical o f p h o to tro p h ic organism s (prim ary
producers) a n d algivores (prim ary consum ers).
T hese results suggest th a t kleptoplasts o f P. ocellatus could
pro d u ce am ino acids, for w hich the T P Glu/Phe value w ould be 1.0,
sim ilar to th a t o f p rim a ry producers. T h e p ro d u c tio n o f am ino
acids from inorganic n itrogen was also re p o rte d in kleptoplasts o f
E . viridis in a trac er ex p erim en t using 15N -inorganic n itrogen [14],
T eugels a n d cow orkers have p ro p o sed th a t kleptoplasts synthesize
glutam ic acid from am m o n iu m derived from seaw ater or m ade
from nitrate a n d /o r urea, a n d th a t o th er am ino acids are
synthesized from glutam ic acid [14], It is n o t clear w h eth er the
enzym es o f these pathw ays re m a in active in the kleptoplasts [17]
o r w h eth er th eir genes are expressed in the host anim al nucleus to
w hich they w ere transferred from the algal nu clear genom e [50].
W h en the a m o u n t o f am ino acids from the digestion o f algae is
m u ch larg er th a n th a t from kleptoplasts, the T P Giu/phe value
w ould becom e 2.0. A ccordingly, the observed T P Giu/phe values
(i.e., 1.3 ± 0 .1 ) for c ultured individuals im ply som e significant
con trib u tio n o f the kleptoplast-produced am ino acids to P. ocellatus
d u rin g starvation.
T h e g iant clam T. crocea harb o rs zooxanthellae, w hich
con trib u te significandy to the host n u trition, b u t it also feeds on
free-living plankters [51]. Previous studies show ed th a t the
zooxanthellae secrete a p hotosynthate, glycerol o r glucose, in the
m antle tissue [42,52,53] b u t the zooxanthellae are also digested in
the stom ach [54,55]. T hose previous results suggested th a t
essential am ino acids are derived from the digestion o f zoo x an ­
thellae a n d plankters. T h e T P Giu/phe value o f the g iant clam T.
crocea was 2.0, w hich is in ag ree m e n t w ith previous results [54,55]
show ing th a t T. crocea gain am ino acids by the digestion o f
zooxanthellae in sym biotic relationships as well as the digestion o f
free-living plankters. T h e d a ta together w ith those o f n a tu ra l a n d
starved individuals suggest th a t w ild P. ocellatus o b tain am ino acids
m ainly from hetero tro p h ic digestion o f algae as in the case o f T.
crocea, w hile starved individuals obtain significant am o u n ts o f
am ino acids from photosynthetic p ro d u c tio n in kleptoplasts.
Supporting Information
Figure SI
O v e r v ie w s o f P lan kobranchu s o cella tu s. (A)
D orsal views o f a freshly collected intact P. ocellatus individual. (B)
A n anesthetized individual w ith spread p a rap o d ia. T h e tissue
region in the re d square was dissected a n d used for D N A
extraction.
(TIF)
T able SI P e r c e n ta g e a b u n d a n ce o f rbcL s e q u e n c e s fr o m
s o u r c e a lg a e in P. ocellatus*.
(XLS)
T able S2
(XLS)
T able S3
(XLS)
Feeding o f P. ocellatus under natural conditions
A ck n ow led gm en ts
T h e p re sen t results indicate th a t P. ocellatus gain kleptoplasts
from m ultiple algal species, th a t each individual harb o rs a
heterogeneous p o p u latio n o f kleptoplasts from m ultiple algal
species, th a t the kleptoplast com position changes w ith tim e, a n d
th a t the sea slugs m ainly gain am ino acids by digesting ingested
algae in natu re, although kleptoplasts are p ro b ab ly capable o f
p ro d u c in g am ino acids. T hese com b in ed findings suggest th a t wild
P. ocellatus repeatedly feed o n siphonous green algae a n d acquire
new kleptoplasts w h en th ere is sufficient algal biom ass to feed the
P. ocellatus p opulation. T h e ecological role o f functional klepto­
plasty has b e en hypothesized to be a n u tritio n resource du rin g
periods o f food insecurity [6,15]. T h e photosynthetic activity o f
kleptoplasts in starved P. ocellatus is red u ced by a b o u t 10% p e r
m o n th [10]. R eplenishing kleptoplasts equivalent to the p h o to ­
PLoS ONE I w w w .p lo s o n e .o rg
W e thank Ms. Rie N akano, Dr. T ohru Iseto, Dr. Seiji Takegata, an d all
staff in E. H irose’s laboratory for their support in the field; D r. T o m Schils
for providing some o f the algal specimens; Dr. N aohiko O hkouchi and Ms.
Yoko Sasaki for their advice an d help on the nitrogen isotopic study; and
Dr. D hugal Lindsay for his advice on writing the manuscript.
Author C ontributions
Conceived and designed the experiments: E H T. M aruyam a. Perform ed
the experiments: T . M aeda M K K T H V TY SS Y T YC M T . Analyzed the
data: T. M aeda E H YC K T TY H V Y T M T T. M aruyam a. C ontributed
reagents/m aterials/analysis tools: T . M aeda E H H V K I J T YC K T TY
Y T. W rote the paper: T . M aeda E H YC K T TY H V Y T M T T.
M aruyam a.
11
J u ly 2 01 2 I V o lu m e 7 | Issue 7 | e 4 2 0 2 4
K le p to p la s ty in Plakobranchus ocellatus
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