Journal of Experimental Botany, Vol. 64, No. 13, pp. 3999–4009, 2013
doi:10.1093/jxb/ert197 Advance Access publication 11 July, 2013
Review paper
Crawling leaves: photosynthesis in sacoglossan sea slugs
Sónia Cruz1,*, Ricardo Calado1, João Serôdio1 and Paulo Cartaxana2
1 2 Departamento de Biologia and CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
Centro de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal
* To whom correspondence should be addressed. E-mail: [email protected]
Received 2 January 2013; Revised 3 June 2013; Accepted 4 June 2013
Abstract
Some species of sacoglossan sea slugs can maintain functional chloroplasts from specific algal food sources in the
cells of their digestive diverticula. These ‘stolen’ chloroplasts (kleptoplasts) can survive in the absence of the plant
cell and continue to photosynthesize, in some cases for as long as one year. Within the Metazoa, this phenomenon
(kleptoplasty) seems to have only evolved among sacoglossan sea slugs. Known for over a century, the mechanisms
of interaction between the foreign organelle and its host animal cell are just now starting to be unravelled. In the study
of sacoglossan sea slugs as photosynthetic systems, it is important to understand their relationship with light. This
work reviews the state of knowledge on autotrophy as a nutritional source for sacoglossans and the strategies they
have developed to avoid excessive light, with emphasis to the behavioural and physiological mechanisms suggested
to be involved in the photoprotection of kleptoplasts. A special focus is given to the advantages and drawbacks of
using pulse amplitude modulated fluorometry in photobiological studies addressing sacoglossan sea slugs. Finally,
the classification of photosynthetic sacoglossan sea slugs according to their ability to retain functional kleptoplasts
and the importance of laboratory culturing of these organisms are briefly discussed.
Key words: Endosymbiosis, kleptoplasty, PAM fluorometry, photobiology, photoprotection, photosynthesis, sacoglossa,
symbiosis.
Introduction
The incorporation and maintenance of functional chloroplasts into animal tissues (kleptoplasty or kleptoplastidy)
constitutes perhaps one of the most puzzling photosynthetic
systems. Captured plastids, derived from the ingestion of
algal tissue, are often referred in the literature as kleptoplasts
or kleptoplastids. Within the Metazoa, only sacoglossan
sea slugs (Heterobranchia = Opisthobranchia) are known
to display this type of association. These organisms have a
wide geographical distribution, being present in the majority of shallow tropical and temperate marine environments.
The most well-studied species of sacoglossans displaying the
ability to retain functional chloroplasts in their animal cells
include: Elysia viridis, which can be found, for instance, in
the coast of Scandinavia (e.g. Evertsen and Johnsen, 2009),
British Isles (e.g. Trowbridge, 2000; Trench et al. 1973) and
Iberian Peninsula (e.g. Vieira et al. 2009); Elysia timida
occurring in the Mediterranean coast (e.g. Jesus et al., 2010;
Giménez-Casalduero et al., 2011; Wägele et al., 2011); and
Elysia clarki (e.g. Curtis et al., 2006) and Elysia chlorotica
(e.g. Pierce et al., 2007; Rumpho et al., 2008; Pelletreau et al.,
2012) that occur in the southeast coast of North America.
As kleptoplasts do not constitute an organism per se, the
term kleptoplasty does not fit in the definition of symbiosis
(sensu stricto), i.e. ‘the living together of differently named
organisms’ (Raven et al., 2009). Nevertheless, this phenomenon is commonly reported as chloroplast symbioses or
endosymbioses. In sacoglossan sea slugs, kleptoplasts are
harboured intracellularly in cells of the digestive diverticula,
enabling their animal host to survive photo-autotrophically
for periods ranging from days to up to 1 year after being
‘stolen’ from algal cells (e.g. Clark et al., 1990; Evertsen
et al., 2007; Händeler et al., 2009). Due to these remarkable
© The Author [2013]. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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4000 | Cruz et al.
features, sacoglossan sea slugs have been frequently termed
‘leaves that crawl’ (Trench, 1975) or ‘solar-powered sea slugs’
(expression first used by Bill Rudman at the Sea Slug Forum,
www.seaslugforum.net, last accessed 19 June 2013).
Early studies using E. viridis demonstrated the capacity of
kleptoplasts to perform CO2 fixation and the importance of
photosynthesis as a carbon source for sacoglossan sea slugs
(e.g. Kawaguti and Yamasu, 1965; Trench et al., 1973; Hinde
and Smith, 1975). The incorporation of photosynthetically
fixed 14C was demonstrated in several low-molecular-weight
metabolites (e.g. glucose, galactose, or amino acids) through
the use of labelled bicarbonate (Trench et al., 1973; Trench
and Ohlhorst, 1976). Following these earlier studies with
E. viridis, photosynthesis and carbon incorporation into
newly synthesized molecules were also demonstrated for other
sacoglossan species (e.g. Clark et al., 1990). Main aspects of
photosynthetic sacoglossan sea slug nutrition are reviewed in
the present work, with emphasis on algal food sources and
the nutritional relevance of autotrophy.
Maintenance of photosynthetic activity in the absence
of an algal nucleus is particularly interesting considering
the fact that the chloroplast genome is expected to encode
only a small fraction of the proteins considered necessary
for photosynthesis (Eberhard et al., 2008). Several hypotheses have been put forward to explain long-term functional
kleptoplasty. The most fascinating and controversial hypothesis is that of horizontal gene transfer (HGT) from the algal
nucleus to the sea slug, with nucleus-encoded transcription
factors or regulatory molecules being retargeted to the kleptoplasts. The discussion around HGT in sacoglossan sea
slugs and in E. chlorotica in particular, persists in the scientific
community. A number of recent studies have addressed and
reviewed plastid uptake and evolution within the Sacoglossa,
the biochemical interaction between kleptoplasts and their
animal host cells, and the molecular basis of plastid retention; overall, these studies aimed to explain how long-term
photosynthetic activity is retained in kleptoplasts (Rumpho
et al., 2007, 2011; Pierce et al., 2009, 2012; Johnson, 2011;
Pelletreau et al., 2011; Wägele et al., 2011; Pierce and Curtis,
2012).
Less attention has been paid to the effects of light exposure
on the photosynthetic activity of kleptoplasts in sacoglossan
sea slugs. This is particularly relevant as exposure to excessive
light has been shown to reduce functional kleptoplast longevity (Vieira et al., 2009; Klochkova et al., 2013). Algae and
plants have photoprotection mechanisms aiming to reduce
light absorption or dissipate the excess of energy absorbed
(Horton et al., 1996; Niyogi, 1999). However, little is known
concerning the maintenance of physiological photoprotective potential of isolated chloroplasts after being incorporated in the animal cells of sacoglossan sea slugs. In addition,
sea slugs may display photoprotective behaviour moving
away from high light (Gallop et al., 1980; Weaver and Clark,
1981) or folding their dorsal body flaps (parapodia, Fig. 1) to
reduce light absorption (Rahat and Monselise, 1979; Schmitt
and Wägelle, 2011).
Roughly one decade ago, pulse amplitude modulated
(PAM) chlorophyll (Chl) a fluorometry was used for the
first time to study photosynthesis in sea slugs (Wägele and
Johnsen, 2001). This real-time methodology is non-intrusive
and, as such, it opened a new window to study photosynthesis
in kleptoplasts in vivo. This approach allowed the screening
of photosynthetic activity and the study of kleptoplast longevity in a broad range of sacoglossan species (e.g. Evertsen
et al., 2007; Händeler et al., 2009).This review focuses on
the photobiology of sacoglossan sea slugs, emphasising the
Fig. 1. Sacoglossan sea slugs. (A) Large and small specimens of Elysia timida feeding on Acetabularia acetabulum (courtesy of Bruno
Jesus). (B) Elysia viridis feeding on Codium tomentosum. Lateral body flaps (parapodia) are present in both E. timida and E. viridis.
Bars, 1 mm.
Photosynthesis in sacoglossa | 4001
advantages and drawbacks of using PAM fluorometry in the
study of photosynthesis and photoprotection. In addition,
criteria employed to rate the level of functional kleptoplast
longevity are critically discussed, as well as the importance of
laboratory culture techniques for experimentation on photosynthetic sacoglossan sea slugs.
Nutrition: food sources and autotrophy
Photosynthetic sacoglossan sea slugs use their radular teeth
to penetrate the cell wall of algal filaments, suck and digest
the cellular content, and incorporate stolen algal chloroplasts
into tubular cells of their digestive diverticula. Early studies
have mostly relied on the observation of crawling activity of
sea slugs on macroalgae, along with different types of feeding
experiments, to determine the source of retained kleptoplasts
(e.g. Clark and Busacca, 1978; Jensen, 1980). However, the
presence of a slug on a given macroalgae, other than its food
source, can occur as a consequence of trial-and-error foraging behaviour on its search for food or simply as a need to
seek shelter when exposed to water turbulence (Gallop et al.,
1980). Simplistic approaches to determine the algal source
of kleptoplasts commonly yielded contradictory results.
For example, Caulerpa was described as the primary food
source of the sea slug Elysia crispata (Jensen, 1980), while
Clark and Busacca (1978) reported that the same species did
not consume Caulerpa but instead fed on macroalgae of the
genera Halimeda, Penicillus, Bryopsis, and Batophora. Hawes
(1979) used organelle ultrastructure characteristics identified by electron microscopy to determine the origin of kleptoplasts in E. viridis. While Codium fragile was identified as
the algal source for symbiotic plastids in this sea slug species, the authors also reported the existence of non-Codium
chloroplasts, suggested to be derived from Ulva. Therefore, it
is important to highlight that earlier published reports lacking strong experimental evidence on the algal food sources
of sacoglossan sea slugs should be analysed with caution,
as they may not accurately report the true algal origin of
kleptoplasts.
The occasional misidentification of the algal species used
as food by particular sacoglossan sea slugs in earlier studies
was later overcome through the use of more reliable biochemical and molecular approaches. As an example, by using the
chloroplast-encoded gene rbcL in combination with microscopic techniques, Curtis et al. (2006) showed that E. clarki
sequestered chloroplasts from four different species of algae
of genera Penicillus and Halimeda. In addition, it was also evidenced that newly metamorphosed E. clarki juveniles feed on
and sequester chloroplasts from algal species different from
those utilized by adult specimens (Curtis et al., 2007). The
sacoglossan sea slug Plakobranchus ocellatus was shown to
feed on a wide variety of ulvophyceaen chlorophytes, including members of the genera Halimeda, Caulerpa, Udotea, and
Acetabularia, as revealed by sequencing of tufA and rbcL
(Wägele et al. 2011; Christa et al., 2013).
Photosynthetic pigments have also been used for determining the origin of kleptoplasts. Different colour morphotypes
of E. timida were shown to feed exclusively on Acetabularia
acetabulum (Fig. 1A) on the basis of pigment profiles (Costa
et al., 2012). Using the same approach, Ventura et al. (2013)
showed that Thuridilla hopei fed not only on filamentous
green algae but also on brown algae. Through the pigment
profile of algal prey and sea slug host, it was possible to identify Codium tomentosum as the sole food source of E. viridis
(Fig. 1B) in three locations along the Portuguese Atlantic
coast (S Cruz, R Calado, J Serôdio, B Jesus, and P Cartaxana,
unpublished data). Most photosynthetic sacoglossan sea
slugs feed on siphonaceous green algae and sequester greenalgal (chlorophyte) plastids. This is not the case of E. chlorotica, the sacoglossan species displaying the longest retention
of functional kletoplasty known so far; its kleptoplasts are
red algal-derived plastids of secondary symbiotic origin from
the xanthophyte genus Vaucheria (Rumpho et al., 2011).
The importance of photosynthesis on the nutrition of photosynthetic sea slugs has been addressed by comparing animal
weight and survival rates of starved specimens (deprived of
exogenous food sources and unable to feed heterotrophically)
kept in the laboratory under dark (thus unable to acquire
photosynthates from their kleptoplasts) and light conditions.
A higher weight loss and a lower survival has been reported
for E. viridis kept in the dark (Hinde and Smith, 1975), as
well as for E. timida (Giménez-Casalduero and Muniain,
2008) and P. ocellatus (Yamamoto et al., 2013), thus demonstrating the importance of photosynthesis in the nutrition of
these species. Middlebrooks et al. (2012) observed an increase
of foraging effort and a decrease in photosynthetic activity
in 8- and 12-week-starved E. clarki. This behaviour was not
observed in 4-week-starved or satiated animals, in which the
photosynthetic activity of kleptoplasts remained high. The
most remarkable example in terms of dependency on photosynthetic fixed carbon is probably that of E. chlorotica, as this
species is capable of living autotrophically for its entire life
cycle upon incorporation of Vaucheria litorea chloroplasts.
Kleptoplast photosynthetic competence measured by net O2
evolution and incorporation of 14C recorded for E. chlorotica
starved for several months were comparable to those displayed by V. litorea macroalgal filaments (Green et al., 2000;
Rumpho et al., 2001).
Fewer studies have unequivocally demonstrated the importance of autotrophy on the nutritional state of natural populations of photosynthetic sacoglossan sea slugs. Using nitrogen
isotope analysis of amino acids, Maeda et al. (2012) showed
that digested algae played the major nutritional role in wild
P. ocellatus and that the nutritional benefits promoted by kleptoplast photosynthesis were only significant under starvation
in laboratory trials. Marín and Ros (1992) reported that the
importance of carbon fixation by kleptoplasts in E. timida
changed seasonally, being more significant during periods of
food shortage due to calcification of A. acetabulum. These
authors estimated that kleptoplasty contributed only 8% to
total organic carbon used by the animal host. Using stable
carbon isotope ratio data, Raven et al. (2001) determined
that total carbon derived from kleptoplastic photosynthesis
ranged between 16 and 60% in several Australian sacoglossans. The time frame during which algal chloroplasts remain
functional, as well as the nutritional relevance of autotrophy,
4002 | Cruz et al.
appears to be species specific. Then again, the nutritional benefits that kleptoplasty seems to confer are unquestionable, as
it may enhance slug survival and reproductive output through
periods of food scarcity.
Photobehaviour and photoprotection
In the study of sacoglossan sea slugs as photosynthetic systems, it is important to understand their relationship with
light. On one hand, these organisms seem to move towards
light, possibly to increase light harvesting at the kleptoplast
level. E. viridis showed a distinct tendency to move towards
light intensities of 12 W m–2 (approximately 60 μmol photons
m–2 s–1) to 24 W m–2 (approximately 120 μmol photons m–2
s–1), the approximate light interval at which maximum rates
of photosynthesis occurred in these sea slugs (Gallop et al.,
1980). Costasiella lilianae preferred light intensities up to
600 μmol photons m–2 s–1 over dark, while E. crispata and
Elysia tuca were attracted to light intensities up to 400 μmol
photons m–2 s–1 (Weaver and Clark, 1981). E. timida and
T. hopei preferred moderate natural light around 40 μmol
photons m–2 s–1 over darkness (Schmitt and Wägele, 2011).
However, light-driven behaviour in photosynthetic sacoglossan sea slugs may not be exclusively related to the presence of
kleptoplasts. For instance, in juveniles of E. timida, a distinct
preference for moderated light was present before the acquisition of kleptoplasts (Schmitt and Wägele, 2011). Phototaxis
is a widespread behaviour and factors such as predator avoidance, water turbulence, or the need to find food might also
play an important role on animal response to light.
On the other hand, sacoglossan sea slugs seem to avoid high
light intensities, possibly as a strategy to prevent premature
loss of kleptoplast photosynthetic function. When deprived
from their algal food source, Elysia nigrocapitata maintained
photosynthetically active kleptoplasts for 4 months in the
presence of low light (15 μmol photons m–2 s–1) (Klochkova
et al., 2010) but the longevity of functional kleptoplasts
was severely reduced under high light (200 μmol photons
m–2 s–1) (Klochkova et al., 2013). In a similar way, high light
(140 μmol photons m–2 s–1) reduced the longevity of photosynthetically active kleptoplasts in E. viridis when compared
to low light (30 μmol photons m–2 s–1) (Vieira et al., 2009).
In photosynthetic organisms, absorption of excessive light
(more light than that can be utilized in photosynthetic electron transport) can lead to increased production of oxidizing molecules and reactive oxygen species that can potentially
cause photooxidative damage and inhibit photosynthesis
(photoinhibition), mainly through the inactivation of photosystem II (PSII) reaction centre protein D1 (Edelman and
Mattoo, 2008). Photophobic behaviours reported for some
photosynthetic sea slugs have been assumed to be linked to
avoidance of exposure to potentially damaging irradiances
(e.g. Giménez-Casalduero and Muniain, 2008; Jesus et al.,
2010). E. crispata and E. tuca were shown to avoid light intensities above 400 μmol photons m–2 s–1 (Weaver and Clark,
1981), while in E. viridis photophobic response behaviours
were described at irradiances above 120–140 μmol photons
m–2 s–1 (Gallop et al., 1980; Vieira et al., 2009). P. ocellatus
was also observed to roll its body and lie on its side in bright
sunlight (Händeler et al., 2009).
Similar ‘behavioural photoprotection’ strategies have been
described in other organisms. Microalgae inhabiting intertidal mudflats and shallow coastal zones are thought to position themselves at a sediment depth where optimum light
conditions occur and avoid photoinhibitory irradiances (e.g.
Serôdio et al., 2006; Cartaxana et al., 2011). An analogous
behaviour has also been described for flatworms harbouring
endosymbiotic microalgae (e.g. Serôdio et al., 2011). These
adaptations would be functionally equivalent to chloroplast
avoidance movements (Kasahara et al., 2002) and leaf folding (Kao and Forseth, 1992) in plants, shown to provide effective photoprotection against photoinhibition.
An additional specialized photobehaviour has been
described in the sea slug E. timida. This species may use the
lateral body flaps (parapodia, Fig. 1A) to control exposure
of kleptoplasts to light (Rahat and Monselise, 1979; Schmitt
and Wägele, 2011). According to Rahat and Monselise (1979),
E. timida opened their parapodia wide (‘fully spread posture
resembling a flattened leaf’) under light intensities between
3 × 103 and 3 × 105 ergs cm–2 s–1 (approximately 14–1400 μmol
photons m–2 s–1) and closed them (‘contracted or arrow-like
form’) at lower or higher intensities. This behaviour was
observed over a 28 h period and was independent of the preincubation light regimes tested (e.g. continuous light, continuous dark, or 12/12 light/dark). This experimental evidence
indicates that the direct effect of light overrules a possible circadian cycle on parapodial position in E. timida (Rahat and
Monselise, 1979).
It is important to highlight that the presence of parapodia
has been recorded for all members of family Plakobranchidae
(e.g. genera Elysia, Thuridilla, and Plakobranchus), thus
including the majority of sea slugs known to display functional kleptoplasty (Händeler et al., 2009). The opening/closing of parapodia would therefore result in an efficient way
to control total body surface intercepting light. In this way,
it has been generally accepted that sea slugs can optimize
(and even prolong) the photosynthetic activity of functional
kleptoplasts using this specific photobehaviour (GiménezCasalduero and Muniain, 2006, 2008; Händeler et al., 2009;
Jesus et. al, 2010). Nonetheless, experimental evidence supporting the hypothesis that photobehavioural adaptations
in sacoglossan sea slugs can help optimize photosynthesis is
scarce. While Schmitt and Wägele (2011) reported that E. timida opened their parapodia when exposed to light, they found
no clear correlation between photosynthetically active radiation (PAR) intensities and the degree of parapodia opening.
It is critical to understand whether this photobehaviour
thus indeed confer an advantage to kleptoplasts: i.e. would
the opening of parapodia at lower light intensities (and the
reverse in excessive light) allow the host sea slug to maintain
optimal levels of photosynthetic activity in the kleptoplasts?
Finally, it would be interesting to find if this specialized
behaviour exists in other species of photosynthetic sacoglossan sea slugs other than E. timida. In the latter species, unlike
most photosynthetic sacoglossan sea slugs, the majority of
Photosynthesis in sacoglossa | 4003
kleptoplasts is distributed on the inner part of parapodia
(Fig. 1A). Therefore, the question remains whether this particular photobehaviour is unique for E. timida (given the
specificity of kleptoplasts distribution over the parapodia),
or if it is shared by most photosynthetic sacoglossan sea
slugs with parapodia (including those possessing kleptoplasts in the inner and outer sides of parapodia, for example
E. viridis, Fig. 1B).
In addition to behavioural adaptations, photosynthetic
organisms possess physiological mechanisms that help preventing excessive light absorption. These include adjustment
in light-harvesting antenna size, which can decrease light
absorption, while changes in the rate of electron transport
can optimize the use of light energy (reviewed by Horton
et al., 1996 and Niyogi, 1999). It is unknown if these photoacclimation processes occur in kleptoplasts of sacoglossan sea
slugs. In E. viridis specimens exposed to different light levels
while deprived from algal food source, kleptoplasts showed
no significant difference in the photoacclimation status determined by light-response curves (Vieira et al., 2009).
Besides adjusting light absorption, algae and plants have
ways of dissipating excessive light energy that has already
been absorbed. These include alternative electron transport
pathways (reviewed by Niyogi, 1999 and Goss and Jakob,
2010) and thermal dissipation of excitation energy (reviewed
by Horton et al., 1996; Niyogi, 1999; Müller et al., 2001;
Szabó et al., 2005). The latter involves non-photochemical
processes that quench singlet-excited Chl, being therefore
collectively called non-photochemical quenching (NPQ). The
major fraction of NPQ depends on thylakoid ΔpH and is
called energy-dependent quenching (qE). These NPQ component forms within minutes of exposure to light and is the
fastest to relax in darkness. It is now broadly accepted that
this major component of NPQ involves changes in xanthophyll composition (e.g. reviewed by Demming-Adams, 2003).
The increase in the ΔpH in excessive light activates a de-epoxidation enzyme which converts a di-epoxy xanthophyll associated with the light-harvesting complexes, to the epoxy-free
pigment via the so-called xanthophyll cycle. Vascular plants
and green and brown algae possess the violaxanthin cycle
(violaxanthin, antheraxanthin, and zeaxanthin) while the diadinoxanthin cycle (diadinoxanthin and diatoxanthin) is present in the algal classes Bacillariophyceae, Xanthophyceae,
Haptophyceae, and Dinophyceae (e.g. reviewed by Goss and
Jakob, 2010). In addition to NPQ and alternative electron
transport mechanisms, numerous antioxidant molecules and
scavenging enzymes are present to deal with the inevitable
generation of reactive molecules, especially reactive oxygen
species (reviewed by Niyogi, 1999 and Murata et al., 2007).
In photosynthetic sacoglossan sea slugs, it is largely unknown
if photoprotection mechanisms occurring in the chloroplasts
of the algal food source are maintained in the isolated kleptoplasts. To date, only one study has shown a functional xanthophyll cycle within kleptoplasts of a sacoglossan sea slug,
E. timida, with a strong linear relationship with NPQ measurements (Jesus et al., 2010).
Despite all these photoprotective defences, damage to the
photosynthetic machinery still occurs. Repair of damaged
PSII involves disassembly, proteolysis, and introduction of
newly synthesized proteins, primarily protein D1 (reviewed
by Chow 1994 and Murata et al., 2007). In plants, the repair
of components of the photosynthetic apparatus, including
the de novo synthesis of D1, is known to require the translation of nuclear-encoded proteins (Edelman and Mattoo,
2008), which poses unique problems for kleptoplasts in
sacoglossan sea slugs deprived of its nuclear algal resources.
Although the reasons for premature loss of photosynthetic
activity in E. viridis exposed to high-light conditions were not
investigated (Vieira et al., 2009), the authors suggested that
it was related to the photoinactivation of PSII reaction centre protein D1. In E. chlorotica, inhibition of photosynthetic
activity at high light intensities (2250 μmol photons m–2 s–1)
was not consistently observed (West, 1979). In this species,
capacity for de novo synthesis of not only D1 protein but also
other key proteins such as the large subunit of RuBisCO, and
several light-harvesting complex polypeptides has been demonstrated (Mujer et al., 1996; Pierce et al., 1996; Green et al.,
2000; Hanten and Pierce, 2001). This could account for the
capacity of this species to maintain photosynthetic activity at
high irradiances. On the other hand, it is unknown if E. viridis is capable of synthesizing molecules such as D1 protein,
a factor that could explain the premature loss of photosynthetic activity observed in kleptoplasts exposed to high light.
Further research is needed to understand if photoprotection
mechanisms are generally maintained in kleptoplasts of different photosynthetic sacoglossan species. It would be interesting to investigate in a broad range of species if the existence
of photoprotection mechanisms in kleptoplasts confers an
advantage to the host by prolonging their photosynthetic
capacity in periods of food scarcity. Can the efficiency of photoprotection and repair mechanisms play a role in the longterm maintenance of photosynthetic activity in species known
to endure long periods of starvation in laboratory conditions?
Advantages and drawbacks of using PAM
fluorometry in sacoglossan sea slugs
Many experimental techniques are available today for the
study of in vivo photosynthesis. In recent years, the technique
of Chl a fluorescence has become ubiquitous in photobiology research (e.g. reviewed by Schreiber, 2004; Strasser et al.,
2004; Oxborough, 2004). In the study of functional chloroplasts in sacoglossan sea slugs, Chl a fluorescence measured by PAM fluorometry (e.g. Wägele and Johnsen, 2001;
Evertsen et al., 2007; Händeler et al., 2009; Jesus et al., 2010;
Wägele et al., 2011; Middlebrooks et al., 2012; Klochkova
et al., 2013) has been replacing to a large extent other methodologies such as O2 production (e.g. Rumpho et al., 2000;
Giménez-Casalduero and Muniain, 2006) and light-driven
CO2 incorporation using 14C incubation (e.g. Trench et al.,
1973; Hinde and Smith, 1975; Clark et al., 1990; Marín and
Ros, 1992). PAM fluorometry has a number of well-known
operational advantages, which help explaining its growing
popularity: (i) the ability to carry out truly non-destructive
measurements, allowing to follow the same specimens over
4004 | Cruz et al.
time and reducing the need of extensive replication normally required by the large inter-individual variability often
observed; (ii) the possibility of obtaining real-time and rapid
measurements; and (iii) the capacity to detect and measure
very low signals, as in the case of small individuals or very
low Chl a content, due to the high specificity and sensitivity
for Chl a fluorescence. In this context, fluorescence imaging
systems (see Oxborough, 2004) are particularly promising to
unveil the spatial heterogeneity of the photosynthetic activity
of host animals; moreover, it can allow researchers to relate
photosynthetic activity with morphological structures, and
evaluate the effects of shift in body configuration (e.g. opening/closing of parapodia) on photophysiological parameters.
Chl a fluorescence provides information on the state of
PSII and how absorbed energy is being used. The flow of
electrons through PSII is indicative, under many conditions,
of the overall rate of photosynthesis (e.g. Genty et al., 1989;
Baker and Oxborough, 2004). While Chl a fluorescence is
easy to measure, its determination per se also exhibits certain limitations. In addition, under inadequate experimental
design, data retrieved can lead to erroneous interpretations.
Ideally, Chl a fluorescence should not be used alone but
rather combined with other techniques, such as gas exchange
methods, to obtain a full picture of the responses of photosynthetic organisms to their environment (Maxwell and
Johnson, 2000).
Regardless of the numerous advantages of PAM fluorometry, some drawbacks associated with the use of this technique in motile animals, as well as when comparing different
photosynthetic matrixes are often disregarded. For instance,
specific light-dependent behaviour (opening/closing of parapodia) can induce significant changes in the light level to
which kleptoplasts are exposed during the construction of
light-response curves that measure effective quantum yield
(φPSII) as a function of irradiance. Under each irradiance
level, the relative electron transport rate (rETR) is calculated by multiplying φPSII by the exposure irradiance (E). The
construction of rETR versus E curves allow the estimation
of a number of photosynthetic parameters, namely lightuse efficiency (α), maximum rETR (rETRm), and minimum
saturation irradiance (Ek) (see Table 1 for notation). As discussed in detail by Cruz et al. (2012), if the increased light
attenuation caused by the closing of parapodia is not considered, overestimated rETR values are likely to be recorded.
Another important aspect to be considered is the depth integration of the fluorescence signal when comparing different
photosynthetic matrixes. Detection of fluorescence emitted
from chloroplasts at deeper layers, thus exposed to a lower
PAR and thereby presenting higher φPSII, cause light curves
to saturate at higher irradiances or to show less photoinhibition (Serôdio, 2004). In this way, when comparing the
physiological response of sea slugs and macroalgae, depth
integration of the fluorescence signal will affect rETR values
differently in each organism. From our own experience, this
is likely to occur when comparing E. viridis and its algal food
source C. fragile, where the thickness of the algal sample is
much higher than that of the sea slug. The effect of depth
integration in rETR versus E curves was tested using intact
and sliced C. tomentosum and the results were significantly
affected by differences in light absorption by the sample (S.
Cruz, unpublished data).
A number of assumptions are considered when estimating overall photosynthetic capacity by calculating rETR (e.g.
amount of absorbed light and its distribution between the two
photosystems) and is therefore of paramount importance to
take this in consideration when using rETR to compare different samples (see Maxwell and Johnson, 2000). If sources
of error, as those described above for rETR versus E curves,
are not considered, comparisons among species or between
the motile sea slug and its respective algal food source (e.g.
Evertsen and Johnsen, 2009; Jesus et al., 2010) are likely to
be biased.
Relative ETR versus E curves based on short (<1 min) light
steps, commonly termed rapid light-response curves (RLC),
have been introduced as an alternative to more time-consuming steady-state light-response curves (SSLC) (Schreiber
et al., 1997; White and Critchley, 1999). While presenting several operational advantages, RLC are equally prone to errors
introduced by animal movement. Moreover, while SSLC are
only affected by the acclimation status formed as a response
Table 1. Notation for commonly used PAM fluorometry parameters as defined in the text
Notation
Parameter
Fo, Fm
Ft, Fm′
Minimum and maximum fluorescence emitted by a dark-adapted sample (arbitrary units)
Steady-state and maximum fluorescence emitted by a light-adapted sample (arbitrary units)
Fv, ΔF
Fv/Fm
Variable fluorescence (Fm – Fo and Fm′ – Ft, respectively) (dimensionless)
Maximum quantum yield of PSII of a dark-adapted sample (dimensionless)
φPSII
E
Spectrally averaged ambient PAR (400–700 nm (μmol photons m–2 s–1)
rETR
rETRm
α
Ek
SSLC
RLC
NPQ
Effective quantum yield of PSII (ΔF/Fm′) (dimensionless)
Relative electron transport rate (E × ϕPSII) (dimensionless)
Maximum relative electron transport rate of the rETR versus E curve (dimensionless)
Initial slope parameter of the rETR versus E curve
Minimum saturation irradiance estimated from the interception of rETRm and α
Steady-state light-response curves: rETR versus E curve
Rapid light-response curves: rETR versus E curve
Non-photochemical quenching of chlorophyll a fluorescence [(Fm–Fm′)/Fm′] (dimensionless)
Photosynthesis in sacoglossa | 4005
to long-term light exposure conditions (long-term photoacclimation), RLC are in addition affected by the particular
conditions of recent light history (short-term photoacclimation) (Cruz and Serôdio, 2008 and references therein).
Another example of a photobiological parameter that can
be biased by sea slug movement is the characterization of
induction and relaxation of Chl a fluorescence kinetics. This
type of measurement is commonly used to study the operation of photoprotective processes and the occurrence of photoinhibition in plants and algae. The lowering of fluorescence
yield as a result of photoprotective or photoinhibitory processes is quantified by the NPQ of Chl a fluorescence based
on the variation of maximum fluorescence from dark-adapted
to light-adapted state (Fm and Fm′ respectively). A reference
level of Fm is needed and, as described in detail by Cruz et al.
(2012), movements of the measuring target will cause nonphysiological changes in the ground fluorescence signal (Fo or
Ft), therefore altering the reference level and compromising
the relation between Fm and Fm′ to be used in NPQ calculations (Table 1).
The problems raised by motility in photosynthetic organisms are not exclusive to the study of sea slugs. For example,
vertical migratory responses of microphytobenthos (benthic
microalgae inhabiting intertidal flats of estuaries and shallow coastal zones) to changes in irradiance can hamper the
photophysiological studies using PAM fluorometry on intact
biofilms (e.g. Cartaxana and Serôdio, 2008 and references
therein). In summary, motility might significantly affect specific photophysiological studies using PAM fluorometry and,
depending on the aims of the study, the drawbacks referred
above must be taken in consideration by researchers prior to
any experimentation and/or data interpretation.
Placing sea slugs in small vials (Evertsen et al., 2007;
Evertsen and Johnsen, 2009) or continuously adjusting the
animal position to the optical fibre (Jesus et al., 2010; Schmitt
and Wägele, 2011; Costa et al., 2012) does not impede animal
motility and does not solve the problems discussed above.
Alternative immobilization techniques have been used with
sacoglossan sea slugs, such as placing the animal in the well of
a concavity microscope slide, filled with seawater and covered
with a coverslip (Vieira et al., 2009; Serôdio et al., 2010; Costa
et al., 2012). Nevertheless, for some species and depending
on the light treatment, this technique does not fully impair
sea slug motility, even under such a limited space. Pelletreau
et al. (2012) compressed between glass plates specimens of
E. chlorotica embedded in agarose for visualization under a
microscope. The latter approach could be suitable for Chl
a fluorescence measurements. Cruz et al. (2012) have evaluated the use of anaesthetics on the photosynthetic activity of
kleptoplasts in E. viridis. Eugenol (clove oil) may be suitable
for Chl a fluorescence measurements requiring short periods
of immobilization only, as a prolonged exposure (120 min)
resulted in a decrease of RLC parameters. All immobilization
approaches described present advantages and disadvantages,
and researchers must evaluate if the error associated with
animal movement is preferable to that associated with the
method used for its immobilization and/or potential interference with photosynthesis.
Levels of kleptoplast longevity
The main Chl a fluorescence parameter used in the study of
photosynthetic activity in sea slugs has been the maximum
quantum yield of PSII (Fv/Fm). This parameter has been
increasingly used in the screening of photosynthetic activity
in a broad range of sacoglossan species (e.g. Händeler et al.,
2009; Yamamoto et al., 2013) and in the study of plastid longevity in host animal cells (e.g. Evertsen et al., 2007; Händeler
et al., 2009; Jesus et al., 2010; Serôdio et al., 2010). Evertsen
et al. (2007) employed PAM fluorometry in a variety of sacoglossan species, combined his results with previous classifications for ranking functional retention of kleptoplasts (Clark
et al., 1990) and proposed a new eight time-course-level scale.
A simplified ranking system employing a reduced number of
levels was later proposed by Händeler et al. (2009) (1, no functional retention; 2, short-term retention lasting about 1 week;
and 3, long-term retention for over a month) and by Pierce
and Curtis (2012) (1, non-functional; 2, photosynthesis less
than a month; or 3, photosynthesis for up to 3 months; and 4,
photosynthesis for a year or more). While both classifications
exclude specific periods of time, this approach seems reasonable from a functional perspective.
Nonetheless, it must be stressed that any classification based
on PAM fluorometry, or other methodologies, is compromised
by a series of environmental or maintenance factors. For example, seasonal differences in wild-collected specimens (Hinde
and Smith, 1975; Marín and Ros, 1992) are often overlooked.
Temperature has also been shown to significantly affect the
retention time of functional chloroplasts in E. viridis (Hinde
and Smith, 1972, 1975). The water volume and quality in which
individual sea slugs are maintained during starvation periods
can also affect their survival (e.g. Yamamoto et al., 2013). The
light regime under which sea slugs are starved is often disregarded in the scientific literature, although it has clearly been
shown that light conditions can strongly affect the longevity of kleptoplasts, as already discussed. Recently, Pelletreau
et al. (2012) demonstrated the importance of feeding history
on plastid stability and growth during the initial development
of E. chlorotica. A good example of how previous life history
(e.g. age, feeding, light exposure) of wild-collected samples can
strongly influence plastid longevity is that described by Evertsen
and Johnsen (2009). The authors reported that only 1 week after
in situ collection, starved E. viridis showed no functional chloroplasts present inside their digestive cells (φPSII=0). However, in
the same study, if individuals were able to feed in the laboratory
before exposed to a starvation, the photosynthetic activity was
maintained for at least 73 days. Different food sources may also
account for differences in the longevity of kleptoplasts within
the same species (Wägele et al., 2004). While some sacoglossan
sea slugs are very selective in their food choice (stenophagous),
others have the ability to retain kleptoplasts from a range of
species of macroalgae, as already discussed.
Finally, extrapolation of possible survival of slugs by analysing trend lines should be done with caution (Händeler
et al., 2009). While some authors assumed a linear decay of
chloroplast functionality (e.g. Evertsen et al., 2007; Evertsen
and Johnsen, 2009) based on Fv/Fm values, others have shown
4006 | Cruz et al.
a very different trend in the temporal shifts of Fv/Fm values in
starved animals (Vieira et al., 2009; Jesus et al. 2010).
Laboratory culturing
Culturing of photosynthetic sacoglossan sea slugs is of
utmost importance for several reasons. First, these organisms are needed at all stages of their life cycle to address specific scientific questions experimentally. As the association
between sea slugs and kleptoplasts is already established in
specimens collected from the wild, the early steps in development, uptake, and retention of plastids cannot be investigated
(Rumpho et al., 2011). Additionally, experiments performed
with animals cultured in the laboratory provide a significantly
better control in a number of issues that may bias experimental trials, such as age, fitness, previous food habits, and photoacclimation (Rumpho et al., 2007). Furthermore, determining
the specific conditions that favour the growth of sea slugs in
the laboratory can help researchers to better understand their
ecological traits. Finally, by establishing optimized culture
protocols the number of wild specimens needed for laboratorial experimentation can be significantly reduced; this
approach will certainly contribute to protect vulnerable species that are known to occur at low densities in their natural
habitats (Clark, 1994; Pelletreau et al., 2012).
Published reports on the culturing of photosynthetic
sacoglossan sea slugs in the laboratory are scarce (e.g. West,
1979; West et al., 1984; Trowbridge, 2000; Curtis et al., 2007;
Rumpho et al., 2008; Pelletreau et al., 2012; Dionísio et al.,
2013). Trowbridge (2000) cultured the planktotrophic veligers
of E. viridis for about 30 days until metamorphosis using a
diet of Rhodomonas baltica. Macroalgae induced E. viridis
competent larvae to metamorphose, with effective feeding on
C. fragile and chloroplast acquisition being recorded 2–3 days
post settlement (Trowbridge, 2000). Similarly, the metamorphosis of E. chlorotica larvae provided with Isochrysis galbana
was only triggered in the presence of V. litorea (Rumpho et al.,
2011). However, earlier studies on E. chlorotica reported that
different populations showed either direct development (no
free-living larval stage) or free-swimming veligers (West, 1979;
West et al., 1984). In laboratory culture, the population with
direct development metamorphosed in the egg capsule without substrate, while planktonic veligers produced by the other
population required the alga Vaucheria sp. to trigger metamorphosis. Lecithotrophic larval development and metamorphosis in the absence of macroalgal cues were described for
E. timida (Marín and Ros, 1993) and E. clarki (Curtis et al.,
2007). Pelletreau et al. (2012) successfully bred repeated generations of E. chlorotica in the laboratory from small-sized egg
masses, with metamorphosis occurring around 18 days posthatching. Contrary to data recorded for E. viridis, immediate
feeding of E. chlorotica juveniles with macroalgae was crucial
to achieve normal development (Trowbridge, 2000; Pelletreau
et al., 2012). Pelletreau et al. (2012) reported a minimum
period of 7 days for permanent kleptoplasty to be established
in E. chlorotica, but a robust ability to withstand starvation
at a very early stage of post-metamorphic development.
Furthermore, these authors were able to grow specimens for
over 2 years, contradicting previous reports of E. chlorotica
senescence after approximately 10 months of adult life; in the
field or in the laboratory, previous studies reported the occurrence on each year of adult E. chlorotica synchronous death
due to an infection by an endemic virus (Pierce et al., 1999).
Concluding remarks
Symbiosis has contributed greatly to generate biological
diversity and novel functions, including photosynthesis in animals. Photosynthetic sacoglossan sea slugs exploit the plastids
captured from specific algal prey: a rare opportunity to examine how genetic and biochemical components from two distantly related taxa have evolved to form a unique functional
photosynthetic animal. An overview of functional kleptoplasty
in sacoglossan sea slugs clearly shows that the photosynthetic
longevity of kleptoplasts is variable and species specific. Both
physiological mechanisms and behaviour traits are likely to
contribute to prolong the functional life of kleptoplasts and
more in depth photobiological studies are required. The
increasing use of PAM fluorometry in photobiological studies
in sacoglossan sea slugs is likely to play a key role towards a
better understanding of kleptoplasty; however, researchers
must be fully aware of the principles and assumptions that this
technique requires to produce reliable experimental results.
Acknowledgements
SC was supported by Fundação para a Ciência e a Tecnolo­
gia (FCT, Portugal) with the postdoctoral grant SFRH/BPD/
74531/2010 and by the Seventh Framework Program (FP7)
Marie Curie Career Integration Grant (CIG) PCIG11-GA2012-322349. The authors wish to thank two anonymous
reviewers for critical comments on the manuscript.
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