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Encocytobiosis Cell Res. (2009) 19, 112-119 112
Identification of photosynthetic sacoglossans from Japan
Yoshiharu Y. Yamamoto1,2, Yoichi Yusa3, Shoko Yamamoto3, Yayoi Hirano4, Yoshiaki
Hirano4, Taizo Motomura5, Takanori Tanemura1, Junichi Obokata1,6,*
1Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan; 2Faculty of Applied Biological
Sciences, Gifu University, Yanagido 1-1, Gifu City, 501-1193, Japan; 3Department of Biological Sciences,
Faculty of Science, Nara Women’s University, Kitauoyanishi, Nara, Nara 630-8506, Japan; 4Department of
Biology, Faculty of Science, Chiba University, Yayoi-cho, Inage-ku, Chiba, Chiba 263-8522, Japan; 5Muroran
Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Muroran 051-0003;
6Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8552, Japan.
* Corresponding author: obokata@gene.nagoya-u.ac.jp, obokata@kpu.ac.jp
Abstract
Some sacoglossan molluscs, including several
species of Elysiidae, are known to incorporate
algal chloroplasts, and the incorporated chlo-
roplasts are functional for days to months,
depending on species. This incorporation and
maintenance of foreign chloroplasts are known
as kleptoplasty. In this article, we surveyed
photosynthetic activity of several sacoglos-
sans collected in Japan, by analysis of in vivo
chlorophyll fluorescence. Our survey found 8
new species that have active chlorophylls,
judged by the parameter Fv/Fm, an indicative
of the functionality of photosystem II. Identified
sacoglossans with active chlorophylls belong
to the Bosella, Costasiella, Elysia, Julia,
Placida, Stiliger, and Thuridilla genera. Our
results strongly suggest that these species
possess kleptoplasts. Possession of chloro-
plasts was confirmed for one of the identified
sacoglossans, Elysia trisinuata, by transmis-
sion electron microscopy.
Introduction
Some sacoglossans are known to have ability
to photosynthesize using chloroplasts present
in their cells. These chloroplasts are not inher-
ited from the parents, but “stolen” from their
food algae, such as Codium or Caulerpa. Ac-
quired algal chloroplasts are maintained in
cells of digestive glands of sacoglossans for
days to months, depending on species. This
incorporation and maintenance is called as
kleptoplasty (TRENCH and OHLHORST 1976;
RUMPHO et al. 2007).
After the first report of kleptoplasty by Elysia
astroviridis (KAWAGUTI and YAMASU 1965),
several species have been shown to have
kleptoplasts by transmission electron micro-
scopic (TEM) analysis (TRENCH et al. 1969).
Functionality of kleptoplasts was demon-
strated by light-driven CO2-incorporation using
radioisotope (14C) (HINDE and SMITH 1974),
light-dependent O2-emission detected by
oxygen electrode (RUMPHO et al. 2000), and in
vivo chlorophyll fluorescence analysis for de-
tection of functional chlorophylls (EVERTSEN et
al. 2007). Now, kleptoplasts have been found
in more than 20 species of sacoglossans that
belong to genera Alderia, Bosellia, Caliphylla,
Elysia, Hermaea, Limapontia, Mourgona,
Oxynoe, Plakobranchus, Tridachia, and
Thuridilla (EVERTSEN et al. 2007).
In this report, we screened for Japanese
sacoglossans containing functional chloro-
plasts, judged by possession of active chlo-
rophylls that can be detected by in vivo chlo-
rophyll fluorescence analysis. We first report
possession of active chlorophylls in eight spe-
cies, including ones belonging to Costasiella,
Julia, Placida, and Stiliger genera. Our results,
together with previous studies in this field,
Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan 113
suggest that kleptoplasty occur in a wide range
of sacoglossans.
Materials and Methods
Sea slugs were collected in 2007 and 2008 at
Kominato (Kamogawa, Chiba, Japan), Misaki
(Miura, Kanagawa, Japan), Shirahama (Ni-
shimuro, Wakayama, Japan), Sesoko (Motobu,
Okinawa (Ryukyu), Japan), and Zanpa (Yomi-
tani-son, Okinawa, Japan). Collected sea
slugs were sent to Nagoya University and
subjected to in vivo chlorophyll fluorescence
analysis using FluorCam (Photon Systems
Instruments, Brno, Czech Republic) based on
the pump and probe system with a CCD cam-
era detector (NEDBAL and WHITMARSH 2004).
Fv/Fm (BAKER 2008) was measured per indi-
vidual using the camera system, and the av-
erage and standard deviation were calculated,
if possible. Assays were done within a week
after collection except for Costasiella cf. ku-
roshimae and Elysia ornata. These two spe-
cies were assayed in a month after collection.
Until assays, E. ornata, Placida sp., and Co-
stasiella cf. kuroshimae were kept together
with their food algae that are Codium, Codium,
and Avrainvillea, respectively. Because we
could not prepare food algae for the other sea
slugs, it was impossible to adjust days before
assay after start of starvation. This problem
caused some difficulty in strict comparison of
Fv/Fm among species.
For transmission electron microscopy (TEM),
samples were fixed with 3% glutaraldehyde in
0.1 M cacodylate buffer (pH 7.2) containing
3% NaCl for 2 h at 4˚C. In this fixative, the
samples were cut into small fragments. Then
they were postfixed with 1% OsO4 in 0.1 M
cacodylate buffer (pH 7.2) containing 3% NaCl
for 2 h at room temperature. They were dehy-
drated with a graded acetone series and em-
bedded in Spurr’s epoxy resin. Thin sections
were cut by a diamond knife on a Ultracut
(Reichert-Jung, Wien, Austria), mounted on
formvar-coated slot grids, stained with 4%
uranyl acetate and lead citrate (REYNOLDS
1963), and observed with a JEM-1011 electron
microscope (JEOL, Tokyo, Japan).
Results and Discussions
Sacoglossans collected at several shores in
Japan were subjected to in vivo chlorophyll
fluorescence analysis. Plakobranchus ocella-
tus and Pteraeolidia ianthina were used as
positive controls. P. ocellatus has kleptoplasts
(GREENE 1970b; HIROSE 2005), whose func-
tionality has been confirmed by detection of
light-dependent CO2-fixation (GREENE 1970a)
as well as Fv/Fm determined by in vivo chlo-
rophyll fluorescence analysis (EVERTSEN et al.
2007). P. ianthina is known to be a host of
endosymbiotic zooxanthella, rather than klep-
toplasts. P. ianthina has also been reported to
have photosynthetic activity due to the sym-
bionts, revealed by light-dependent carbon
fixation activity (HOEGH-GULDBERG and HINDE
1986).
As shown in Table 1, both P. ocellatus and P.
ianthina showed high Fv/Fm, as expected.
These results confirmed possession of active
chlorophylls in these organisms. It should be
noted that this analysis cannot distinguish
between kleptoplasts (P. ocellatus) and endo-
symbiotic algae (P. ianthina).
We collected 12 species in sacoglossa in
Japan, and subjected them to in vivo chloro-
phyll fluorescence analysis. Among them, 8
species showed measurable Fv/Fm values
(Table 1). These results indicate that the posi-
tive species, that belong to genera of Costa-
siella, Bosellia, Elysia, Thuridilla, Julia, Placida,
and Stiliger, possess active chlorophylls and
thus photosynthetically functional chloroplasts.
Considering that all these species belong to
sacoglossa, these results strongly suggest that
they have kleptoplasts. Photographs of posi-
tive species are shown in Figure 1.
One of the positive species, Elysia trisinuata,
was subjected to electron microscopic analysis
(Figure 2). As expected, cells of digestive
glands contained chloroplasts (Ct in the Figure
2B), judged by their ultrastructure. As shown in
Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan
114
Table 1: Sacoglossans with active chlorophylls identified in this study
Species Sampling place Sampling date Number of
individuals Size (mm) Fv/Fm
SACOGLOSSA
Costasiellidae
Costasiella cf. ku-
roshimae Zanpa 2008 Dec. 5 1-3 0.661 ± 0.0395
Elysiidae
Bosellia sp. Sesoko 2008 Apr. 4 7-11 0.716 ± 0.052
Elysia ornata Shirahama 2007 Sep. 3 20-40 0.815 ± 0.0201
Ibid. Misaki 2007 Oct. 5 20-40 0.844 ± 0.0190
Elysia trisinuata Misaki 2007 Aug. 6 15-30 0.853 ± 0.042
Thuridilla vatae Sesoko 2008 Jun. 1 14 0.725
Juliidae
Julia zebra Sesoko 2008 Apr. 1 2 0.616
Limapontiidae
Placida sp. (sensu
(BABA 1986) Sesoko 2008 Apr. 9 4 - 8 0.475 ± 0.075
Stiliger ornatus Shirahama 2008 Oct. 3 7-10 0.595 ± 0.0795
Plakobranchidae
Plakobranchus ocel-
latus Sesoko 2008 Jun. 1 22 0.811
NUDIBRANCHIA
Pteraeolidia ianthina1Kominato 2008 Oct. 4 20-50 0.831 ± 0.023
Kominato: Kamogawa, Chiba; Misaki: Miura, Kanagawa; Shirahama: Nishimuro, Wakayama; Sesoko: Motobu,
Okinawa; Zanpa: Zanpamisaki, Yomitani-son, Okinawa. 1 Known to retain Symbiodinium by endosymbiosis
(BURGHARDT et al. 2008).
the figure, some chloroplasts possess starch
grains (white ovals, Figure 2B), suggesting
functionality of these kleptoplasts.
As shown in Table 1, Fv/Fm varies from 0.85
(Elysia trisinuata) to 0.48 (Placida sp.) ac-
cording to species, and variation within a spe-
cies was much smaller than difference among
species. As for Elysia ornata, we analyzed two
batches, one from Shirahama collected in
September and the other from Misaki caught in
October. Although these two groups were
caught at different places in different seasons,
again they showed small difference in Fv/Fm
(0.815 ± 0.0201 and 0.844 ± 0.0190, respec-
tively).
Fv/Fm is a parameter that reflects maximum
efficiency of photosystem II, and thus it indi-
cates intactness of photosystem II (BAKER
2008). Injury of photosystem II, that can be
caused by environmental stresses, results in
reduction of Fv/Fm (BAKER 2008). Although
degree of injury against environmental
stresses can vary among individuals, we found
that there is little variation of Fv/Fm among the
analyzed individuals of the same species.
In the case of kleptoplasts, decrease of
Fv/Fm was observed during starvation, corre-
sponding to the loss of kleptoplasts (EVERTSEN
et al. 2007). Periods until loss of kleptoplasts
vary from days to months, depending on spe-
Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan 115
Thin bar: 0.5 mm, thick bar: 10 mm
Figure 1: Sacoglossans with functional chlorophylls
Thin and thick scale bars indicate 0.5 mm and 10 mm, respectively. A: Bosellia sp., B: Elysia trisinuata, C:
Elysia ornata, D: Julia zebra, E: Stiliger ornatus, F: Costasiella cf. kuroshimae, G: Thuridilla vatae, H: Placida
sp. (sensu BABA, 1986)
cies. Sacoglossans at a higher level of klepto-
plasty maintain chloroplasts for a longer period
(CLARK et al. 1990). The reduction of Fv/Fm
during starvation is related to the loss of klep-
toplasts, but it is not due to the reduction of the
chloroplast number, because Fv/Fm should
not be affected by amount of chloroplasts or
chlorophylls in an individual (BAKER 2008).
starvation is not clear. Theoretically, the reduc-
tion can be caused by either increase of in-
jured photosytem II as mentioned above, or
saturation of the electron transport system
after photosystem II in the dark (BAKER 2008).
Anyway, this reduction causes variations of
Fv/Fm within a species. In spite of the above
tendency, observed Fv/Fm was quite similar
Reason for the reduction of Fv/Fm during within a species (Table 1). This would be in
Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan
116
Figure 2: Transmission electron micrographs of Elysi sinuata
A. Section of digestive glands. B. part of a cell in dig e glands. Ct: chloroplast. Electron-dense black cir-
a tri
e tiv in chloroplasts are starch grains.
atch of assays.
risinuata, and Plakobranchus
ia
s:
possession of kleptoplasts or of endosymbiotic
As
s
s
cles in chloroplasts are plastoglobules, and electron-sparse ovals
art due to the same starvation period for a there are two possible cytological situation
p
bAmong the sacogrossans shown in the table,
Elysia ornata, E. t
nthobaptus showed the highest Fv/Fm val-
ues. These species can survive for the longest
periods (more than one month) in the list dur-
ing starvation (GREENE 1970b) (data not
shown). On the other hand, Placida sp. shows
the lowest Fv/Fm value among the positive list,
and this species survived for the shortest pe-
riod during starvation among the positive spe-
cies, that is, less than a week (data not shown).
Therefore, there seems to be a rough correla-
tion between Fv/Fm and survival period during
starvation.
As for molluscs with photosynthetic activity,
algae (VENN et al. 2008). We suggest that the
photosynthetic sacoglossans shown in Table 1
have kleptoplasts, because almost all of the
known photosynthetic sacoglossans possess
kleptoplasts and there is no report on endo-
symbiosis of algae in adult sacoglossans.
Sacoglossans with kleptoplasts, including
our results, are summarized in Table 2.
hown in this table, around 30 species are now
known to have kleptoplasts. The largest group
in the table is Elysiidae, and most of the fami-
lies in Sacoglossa are included in the table,
except for two families, Gascoignellidae and
Volvatellidae. This demonstrates widespread
distribution of kleptoplasty in Sacoglossa.
Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan 117
Ta le 2: List of sacoglossans reported to possess functional chloroplasts
pecies Method Reference
b
S
Caliphyllidae
Caliphylla mediterranea l. 1990)
ainea C (CLARK et al. 1990)
Cae m
l. 1990)
v/Fm his work
(= E. subornata)1 BUSACCA 1978)
. 2000)
pata)2
m 007)
et al. 2007)
osa
ta
BUSACCA 1978)
viridis al. 1973; HINDE and SMITH 1974)
rloni m
lata m
Ha l. 1990)
m
tiidae
esta l. 1990)
nsu (BABA 1986) v/Fm his work
ssa Chla74)
Oum BUSACCA 1978)
idae tus
)m 007), this work
TEM (HIROSE 2005)
14C (CLARK et a
Mourgona germ 14
ostasiellidae
Costasiella cf. kuroshim Fv/F This work
Elysiidae
Bosellia mimetica 14C (CLARK et a
Bosellia sp. F T
Elysia australis 14C (HINDE 1980)
Elysia cauze Chla(CLARK and
Elysia chlorotica O2(RUMPHO et al
Elysia crispata (= Tridachia cris Chla(CLARK and BUSACCA 1978)
Elysia hedgpethi 14C, Chl (GREENE 1970a)
Elysia ornata Fv/F This work
Elysia pusilla Fv/Fm (EVERTSEN et al. 2
Elysia sp. Fv/Fm (EVERTSEN
Elysia timida Fv/Fm (EVERTSEN et al. 2007)
Elysia toment Fv/Fm (EVERTSEN et al. 2007)
Elysia trisinua Fv/Fm This work
ibid. TEM This work
Elysia tuca Chla(CLARK and
Elysia 14C (TRENCH et
Thuridilla ca Fv/F (EVERTSEN et al. 2007)
Thuridilla lineo Fv/F (EVERTSEN et al. 2007)
Thuridilla vatae Fv/Fm This work
ermaeidae
Hermaea cruciat 14C (CLARK et a
Juliidae
Julia zebra Fv/F This work
Limapon
Alderia mod 14C (CLARK et a
Placida sp. (se F T
Limapontia depre 14C, (HINDE and SMITH 19
Stiliger ornatus Fv/Fm This work
xynoidae
Oxynoe antillabr Chla(CLARK and
Plakobranch
Plakobranchus ocella
3
(=P. ianthobaptus Chla(GREENE 1970a)
ibid. Fv/F (EVERTSEN et al. 2
ibid.
The shows sacoglossans containing funct loropla (CLARK et al.
199 hl: extraction and measurement of c hyll con II activity determined by in vivo
table ional ch sts (Level 4 and higher categories
0)). C hlorop tent, Fv/Fm: PS
chlorophyll fluorescence measurement (pulse amplitude modulation (PAM) or pump & probe methods), O2:
light-dependent O2 emission, 14C: light-dependent incorporation of NaH14CO3. aPossession of chlorophylls does
not directly mean functionality of chloroplasts, but stable maintenance of chlorophylls suggest it (EVERTSEN et al.
2007). References for synonymous species are: 1(CLARK 1984), 2(GOSLINER 1995), 3(JENSEN 1992).
Yamamoto et al. – Identification of photosynthetic sacoglossans from Japan
118
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