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Identification of photosynthetic sacoglossans from Japan

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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.
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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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... In this study, we used two con-generic sacoglossan species, Elysia trisinuata and E. atroviridis, to study the effects of light and food on survival and weight retention. Both species are known to retain kleptoplasts in their digestive cells (Kawaguti and Yamasu 1965;Yamamoto et al. 2009). They are often sympatric and are among the most common sacoglossans in Japan (Trowbridge et al. 2008;Yamamoto et al. 2009). ...
... Both species are known to retain kleptoplasts in their digestive cells (Kawaguti and Yamasu 1965;Yamamoto et al. 2009). They are often sympatric and are among the most common sacoglossans in Japan (Trowbridge et al. 2008;Yamamoto et al. 2009). In addition, these two sacoglossans have some common algal food, but also are found on different species of potential algal food. ...
... E. trisinuata shows short-term chloroplast retention (<4 days; Yamamoto et al. 2013), whereas the retention period appears to be longer in E. atroviridis (at least 7 days; Klochkova et al. 2010). as the duration of kleptoplast retention may differ according to conditions such as algal species they use, the duration was also studied in both species using pulse-amplitude-modulated (PaM) fluorometry (evertsen et al. 2007;Händeler et al. 2009;Yamamoto et al. 2009). ...
Article
Sacoglossans use chloroplasts taken from algal food for photosynthesis (kleptoplasty), but the adaptive significance of this phenomenon remains unclear. Two con-generic sacoglossans (Elysia trisinuata and E. atroviridis) were collected in 2009–2011 from Shirahama (33.69°N, 135.34°E) and Mukaishima (34.37°N, 133.22°E), Japan, respectively. They were individually maintained for 16 days under four experimental conditions (combination of light/dark and with/without food), and their survival rate and relative (=final/initial) weights were measured. Both light and food had positive effects on the survival in E. trisinuata, whereas no positive effects of light or food on survival were detected in E. atroviridis. Both light and food had positive effects on relative weights in both species, but light had smaller effects than food. A significant interaction term between light and food was detected in E. trisinuata (but not in E. atroviridis) in that only the presence of both resulted in weight gains. This result suggests that E. trisinuata can obtain sufficient additional energy from photosynthesis for sustaining growth when fresh chloroplasts are continuously supplied from algal food. In addition, fluorescence yield measurements showed that unfed individuals of both E. trisinuata and E. atroviridis lost photosynthetic activity soon (
... Asterisk indicates molecular identification of food (overall 33 species). Food sources were either obtained in this study or taken out of literature [3,4,18,25,30,[34][35][36][37][80][81][82][83]. Classification of retention ability was done according to literature data [3,18,47,49,[80][81][82][83][84]. ...
... Food sources were either obtained in this study or taken out of literature [3,4,18,25,30,[34][35][36][37][80][81][82][83]. Classification of retention ability was done according to literature data [3,18,47,49,[80][81][82][83][84]. LtR = Long-term-retention, StR = short-term-retention, NR = no retention. ...
... DNA-Barcoding results of the present study were combined with results taken from literature to compare food sources of different retention forms (LtR, StR, NR). Literature data on food sources and retention form were taken from [3,4,18,25,30,[34][35][36][37][80][81][82][83][84] (Additional files 1 and 5). The retention ability of the majority of the species investigated here was measured by means of a PAM. ...
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Sacoglossan sea slugs are well known for their unique ability among metazoans to incorporate functional chloroplasts (kleptoplasty) in digestive glandular cells, enabling the slugs to use these as energy source when starved for weeks and months. However, members assigned to the shelled Oxynoacea and Limapontioidea (often with dorsal processes) are in general not able to keep the incorporated chloroplasts functional. Since obviously no algal genes are present within three (out of six known) species with chloroplast retention of several months, other factors enabling functional kleptoplasty have to be considered. Certainly, the origin of the chloroplasts is important, however, food source of most of the about 300 described species is not known so far. Therefore, a deduction of specific algal food source as a factor to perform functional kleptoplasty was still missing. We investigated the food sources of 26 sacoglossan species, freshly collected from the field, by applying the chloroplast marker genes tufA and rbcL and compared our results with literature data of species known for their retention capability. For the majority of the investigated species, especially for the genus Thuridilla, we were able to identify food sources for the first time. Furthermore, published data based on feeding observations were confirmed and enlarged by the molecular methods. We also found that certain chloroplasts are most likely essential for establishing functional kleptoplasty. Applying DNA-Barcoding appeared to be very efficient and allowed a detailed insight into sacoglossan food sources. We favor rbcL for future analyses, but tufA might be used additionally in ambiguous cases. We narrowed down the algal species that seem to be essential for long-term-functional photosynthesis: Halimeda, Caulerpa, Penicillus, Avrainvillea, Acetabularia and Vaucheria. None of these were found in Thuridilla, the only plakobranchoidean genus without long-term retention forms. The chloroplast type, however, does not solely determine functional kleptoplasty; members of no-retention genera, such as Cylindrobulla or Volvatella, feed on the same algae as e.g., the long-term-retention forms Plakobranchus ocellatus or Elysia crispata, respectively. Evolutionary benefits of functional kleptoplasty are still questionable, since a polyphagous life style would render slugs more independent of specific food sources and their abundance.
... Then, the PAM probe (5 mm diameter) was set 8 mm above the cover glass, and the maximum quantum yield of Photosystem (PS) II (=Fν/Fm) was measured (Schreiber et al. 1995;Evertsen et al. 2007;Vieira et al. 2009). In the study of sacoglossans, Fν/Fm values >0.5 are generally regarded as indicating photosynthetic competence (Evertsen et al. 2007;Händeler et al. 2009;Yamamoto et al. 2009;Akimoto et al. 2014). Subsequently, the samples were darkadapted again for at least 30 min, and the photosynthesis versus irradiance curves were obtained by subjecting each sample to various light intensities (0,24,38,55,81,122,183,262,367 μmol photons m −2 s −1 in this order at 1-min intervals) to estimate the relative electron transport rates (rETR) (Vieira et al. 2009). ...
... showed negative phototaxis under our experimental conditions. The lack of kleptoplasty observed in these species needs further investigation, as Yamamoto et al. (2009) suggested that they are kleptoplastic. It is possible that they can retain chloroplasts only for a very short term (e.g. ...
Article
Several sacoglossan sea slugs utilise chloroplasts ingested from algae for photosynthesis (kleptoplasty), a unique trophic strategy unknown in other animals. Its adaptive significance, especially the behavioural adaptations involved in this phenomenon, has not been fully explored. To address this issue, the effects of kleptoplasty on phototaxis were investigated, both across and within species, using sacoglossans collected along Japanese coasts in 2012 and 2013. First, the presence of phototaxis and preferred light intensity was studied in five sacoglossans with various photosynthetic capabilities using an I-maze with a light gradient (4–330 μmol photons m−2 s−1). Each individual was allowed to move for 30 min to choose the optimal light intensity. Elysia hamatanii, E. trisinuata, and Plakobranchus ocellatus, all with high photosynthetic activity (i.e. kleptoplastic), showed positive phototaxis. Among them, E. hamatanii preferred the highest light intensity followed by P. ocellatus and E. trisinuata, and the order corresponded with the shallowness of their habitats. Conversely, Stiliger ornatus and Placida sp., with virtually no photosynthetic activity (non-kleptoplastic), showed neutral and negative phototaxis, respectively. Next, the phototaxis of E. hamatanii individuals with (fed) and without (starved) functional chloroplasts was compared to examine the effects of the presence of kleptoplasts on phototaxis within a species. Both fed and starved individuals showed positive phototaxis, but the preferred light intensity of starved individuals was lower than that of fed individuals. These results suggest that sacoglossans with functional chloroplasts exhibit positive phototaxis towards a preferred light intensity which may benefit photosynthesis efficiency.
... The ability to retain functional plastids varies among sacoglossan species, and some taxa digest the plastids instead of maintaining them, like taxa of the Oxynoacea (Händeler et al. 2009). While functional kleptoplasty is only known from the genus Costasiella in the Limapontioidea (Clark et al. 1981;Christa et al. 2014c), it is fairly common in the Plakobranchoidea (Clark et al. 1981;Händeler et al. 2009;Johnson 2010;Klochkova et al. 2010;Wägele and Martin 2013;Yamamoto et al. 2009). ...
... Former investigations based on CO 2 fixation experiments, for example, indicate potentially functional kleptoplasts in Hermaea bifida, although evidences are not conclusive (Kremer and Schmitz 1976;Taylor 1971). Higher F v /F m values in the Japanese oxynoacean Julia zebra Kawaguchi, 1981 measured by Yamamoto et al. (2009) certainly need to be re-investigated since no further information on the time frame and conditions of the slugs after collection were given. ...
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Sacoglossa is a rather small taxon of marine slugs with about 300 described species, yet it is quite fascinating scientists for decades. This is mainly because of the ability of certain species to incorporate photosynthetically active plas-tids of their algae prey, a phenomenon known as functional kleptoplasty. With the stolen plastids, these slugs endure weeks (short-term retention) or months (long-term retention) of starvation, though contribution of the plastids to the surviv-al and factors enhancing plastid longevity are unknown. Likewise, contrasting hypotheses on evolution of functional kleptoplasty exist and the phylogenetic relationship of Sacoglossa taxa is still under debate. We analyzed the phylo-genetic relationship of 105 sacoglossan species to address the question of the origin of functional kleptoplasty. Based on our phylogenetic analysis and the ancestral character state recon-struction, we conclude that functional short-term retention most likely originated two times and long-term retention at least five times. Previous suggestions that functional long-term kleptoplasty is established with specific plastids are supported by our food analyses in Elysia clarki that finally harbors only plastids of certain algae species over a prolonged starvation period.
... Händeler et al. (2009) published measurements of nearly 30 species, including many genera never studied before. Yamamoto et al. (2009) focussed on Japanese sacoglossans, while Klochkova et al. (2010) focussed on sacoglossans in Korean waters. ...
Chapter
In this chapter, we summarize our knowledge on photosynthesis properties in the enigmatic gastropod group Sacoglossa. Members of this group are able to sequester chloroplasts from their food algae and store them for weeks and months in order to use them in a similar way as plants do. Only four to five sacoglossan species are able to perform photosynthesis for months, others are less effective or are not able at all. The processes involved are not clear, but we show by this chapter that many factors contribute to the developing of a photosynthetic seaslug. These include extrinsic (environment, origin and properties of the nutrition and the plastids) and intrinsic factors (behaviour, physiological and anatomical properties). Maintenance of plastids is not enhanced by a horizontal gene transfer (HGT) from the algal genome into the slug genome, as was hypothesized for many years. We outline here the questions that now have to be asked and the research that has to be done to understand the factors that actually contribute to this unique metazoan phenomenon, which is not understood at all.
... Mourgona (Evertsen & Johnson, 2009), have until now not been confirmed through in situ PAM measurements. The latter method, although commonly used for studying photosynthetic ability of Sacoglossa (reviewed by Cruz et al., 2013), has only been applied to only about 40 sacoglossan species, representing roughly 10% of those known (Ha¨ndeler et al., 2009;Yamamoto et al., 2009;Klochkova et al., 2010Klochkova et al., , 2013. Of these, only 11 limapontioidean species have been examined regarding their ability to incorporate functional plastids. ...
Article
The evolution and origin of functional kleptoplasty (sequestration and retention of functional plastids) within the Sacoglossa is still controversial. While some authors have suggested that it is a synapomorphy of the parapodia-bearing Plakobranchoidea, others have suggested an earlier origin at the base of the more inclusive clade Plakobranchacea. The latter is supported by the presence of kleptoplasts in Costasiella ocellifera, a ceras-bearing member of Limapontioidea, in which they remain functional for several weeks and fix CO2. However, the phylogenetic relationships of Costasiella, especially with regard to the Plakobranchoidea, have not been satisfactorily demonstrated, and the photosynthetic ability and the importance of photosynthesis within the genus remain poorly studied. In this study we analyse the phylogenetic position, photosynthetic activity and importance of photosynthesis for survival during starvation of five Costasiella species, but focusing on C. ocellifera. We demonstrate that Costasiella is a basal member of the Limapontioidea, however a final conclusion on the origin of functional kleptoplasty within Sacoglossa is still not possible. Three Costasiella species maintain functional chloroplasts (of which C. ocellifera shows long-term retention, and both C. kuroshimae and C. sp. 1 short-term retention) and together form a monophyletic group, feeding mainly on Avrainvillea. The two nonphotosynthetic species, C. nonatoi and C. sp. 2, represent the sister clade and feed on algae other than Avrainvillea. Intriguingly, C. ocellifera survived under nonphotosynthetic conditions for a minimum of 38 d, demonstrating that photosynthates may not be essential in order to survive starvation. These findings support our previous suggestion that during starvation kleptoplasts primarily represent a sort of larder, whose function might benefit from ongoing photosynthesis.
Article
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Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.
Chapter
Alga is an informal name that refers to a diverse group of photosynthetic eukaryotes that have a polyphyletic origin in the tree of life. Although genomics has provided powerful tools for understanding the evolution of algal photosynthesis many issues remain unresolved. These include explaining the intermingling of plastid-lacking taxa such as ciliates and oomycetes among plastid-containing groups of chromalveolates. Does this pattern reflect a single ancient endosymbiosis in the chromalveolate ancestor followed by independent plastid losses or multiple secondary endosymbioses? Here we review current knowledge about chromalveolate evolution and phylogeny with a focus on secondary and tertiary endosymbiosis and survey recent genome-wide analyses to assess the potentially broad and lasting impacts of plastid transfer on eukaryote evolution. We assess the evidence for ‘footprints’ of photosynthetic pasts that remain even when the plastid is lost. These data comprise remnant algal genes in the nucleus of plastid-lacking taxa that have putatively originated via intracellular gene transfer from the former endosymbiont. We also provide a survey of recent work done in the field of protein import (i.e., via translocons) into chromalveolate and other plastids derived from secondary endoysmbiosis. We contrast the similarities and differences between primary and secondary plastid protein import machineries and speculate on the key innovations that led to their establishment. And finally, we take a careful look at the remarkable case of sea slug (Elysia chlorotica) kleptoplasty and photosynthesis and review recent work aimed at explaining this phenomenon in different metazoa. In particular, we critically assess support for the hypothesis that sea slug photosynthesis is explained by massive horizontal gene transfer (HGT) from the genome of the captured alga.
Article
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Sometimes the elementary experiment can lead to the most surprising result. This was recently the case when we had to learn that so-called "photosynthetic slugs" survive just fine in the dark and with chemically inhibited photosynthesis. Sacoglossan sea slugs feed on large siphonaceous, often single-celled algae by ingesting their cytosolic content including the organelles. A few species of the sacoglossan clade fascinate researcher from many disciplines, as they can survive starvation periods of many months through the plastids they sequestered, but not immediately digested - a process known as kleptoplasty. Ever since the term "leaves that crawl" was coined in the 1970s, the course was set in regard to how the subject was studied, but the topics of how slugs survive starvation and what for instance mediates kleptoplast longevity have often been conflated. It was generally assumed that slugs become photoautotrophic upon plastid sequestration, but most recent results challenge that view and the predominant role of the kleptoplasts in sacoglossan sea slugs.
Article
The kleptoplastic sacoglossan Elysia chlorotica shares a requisite, intracellular symbiosis with the plastids (= chloroplasts) of the Xanthophyte alga Vaucheria litorea. Although wild specimens have been used to address a range of biological questions, no studies have thoroughly characterized animal development during the initial establishment of the symbiosis under controlled laboratory conditions. Laboratory culture conditions were modified and the time required for successful metamorphosis was reduced by 40 % relative to previous work. Plastids were not initially stable within the host; “permanent kleptoplasty” was obtained only after ≥7 days of feeding on V. litorea. Feeding for shorter time periods resulted in the loss of plastids and abnormal development; this phase was characterized as “transient kleptoplasty”. Individuals in the transient state exhibited a significantly greater decrease in length compared to animals with permanent kleptoplasts after the same starvation period. To test the effect of food availability after obtaining permanent kleptoplasty, animals were subjected to various dietary regimes followed by a recovery period of constant feeding. Thirty percent of animals survived prolonged starvation (>4 weeks) after only the initial week of feeding required to establish permanent kleptoplasty. All treatments showed rapid growth when re-exposed to Vaucheria. Thus, during initial development E. chlorotica experiences enhanced fitness when Vaucheria is available for consumption. However, the animal rapidly establishes permanent kleptoplasty, which bestows flexible food requirements and resistance to food limitation, a likely advantage for E. chlorotica in salt marsh environments where Vaucheria sp. abundance is sporadic.
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