Sea Slug Elysia crispata

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1		Xochitl Guadalupe Vital	+ 2414 word(s)	2414	2021-11-12 07:53:33	\|
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This entry is adapted from the peer-reviewed paper 10.3390/ani11113157

Some species of sacoglossan sea slugs are able to steal chloroplasts from the algae they feed on and maintain them functional for several months, a process termed “kleptoplasty”. One of these photosynthetic slugs is Elysia crispata, found in coral reefs of the Gulf of Mexico. This sacoglossan inhabits different depths (0–25 m), being exposed to different food sources and contrasting light conditions.

Mollusca Heterobranchia kleptoplasty lipidomics phospholipids glycolipids

1. Introduction

Superorder Sacoglossa includes sea slug species that are able to acquire chloroplasts from the algae they feed upon and keep them functional inside their digestive gland cells, a process termed kleptoplasty ^[1]. Until recently, sacoglossan sea slugs were the only known metazoans displaying such a mechanism ^[2]. Van Steenkiste et al. ^[2] reported the first known case of other metazoans (flatworms) capable of maintaining functional algal chloroplasts for one week. The retention period of “stolen” functional chloroplasts (kleptoplasts) within sacoglossan sea slugs may vary from a few hours to several weeks or months, depending on sea slug species, its life stage and the chloroplast algal source ^[3]^[4]^[5]^[6]. Moreover, abiotic factors such as irradiance levels and temperature can also play a key role over the time frame during which kleptoplasts are retained ^[7]^[8]^[9]. For instance, Vieira et al. ^[7] found that retention of photosynthetic activity of chloroplasts in starved Elysia viridis under high-light conditions decreased exponentially and lasted only 6 to 15 days, compared to low-light conditions in which retention times were longer (15 to 57 days) ^[7].

Photosynthetic sea slugs are generally stenophagous, feeding on a specific genus or species of algae. For example, E. timida only consumes algal species of genus Acetabularia, and E. chlorotica feeds exclusively on genus Vaucheria ^[10]^[11]. However, one of the exceptions to this stenophagous pattern is E. crispata, which can consume up to 17 different species of macroalgae according to DNA analyses ^[12]. The main genera of macroalgae this sacoglossan is known to feed upon are Bryopsis, Penicillus and Halimeda ^[12]^[13], although occasionally other genera are also mentioned ^[6]^[14]^[15]. Although E. crispata can feed on several species, only chloroplasts from the macroalgae Bryopsis plumosa and Penicillus capitatus have been reported to be kept functional for more than 10 weeks ^[16]^[17], while those originating from Halimeda incrassata were kept functional for up to 49 days ^[6]. In the case of P. capitatus, kleptoplasts were still present in slug tissues with different degradation levels after 120 days of starvation ^[18]. In another study, after one week of starvation of E. crispata, chloroplasts from four algal species were detected by DNA analysis: P. capitatus, P. lamourouxii, H. incrassata and H. monile ^[13]. Christa et al. ^[6] also analyzed the DNA of chloroplasts from E. crispata under 7, 28, 35 and 49 days of starvation and found different unidentified algae belonging to Bryopsidales, as well as the consistent presence of H. incrassata in the 8 sea slug specimens surveyed. The rest of macroalgal species reported through DNA barcoding were identified in sea slugs analyzed immediately after collection, without a period of starvation to clean their gut. As such, if chloroplasts from those food sources can also be retained functional for a long-term in this sacoglossan remains to be studied.

Many of the known photosynthetic pigments are light harvesting compounds present in chloroplasts, whose main function is to absorb the light energy necessary to power photosynthesis ^[19]. Pigment composition is highly variable depending on the type of algae and acclimation to the light regime ^[20]. Some pigments (i.e., xanthophylls) have been related to photoprotection, and it has been hypothesized that they may play an important role in kleptoplasts retention by sea slugs ^[21]^[22]^[23]^[24]. Chlorophyll a and b have been reported to be synthesized in sea slugs’ cells by some authors ^[25]^[26], while others have found no evidence supporting this synthesis ^[27]. Even though it is not clear if pigments are broken down or synthesized in the animal cells, they are still present after several days of starvation in different species of Sacoglossa. The presence of these molecules can be related to long-term retention inside sea slug cells because they may alleviate light-induced oxidative stress ^[20]^[22]^[23]. As such, the characterization of photosynthetic pigments present in kleptoplasts provides a baseline to better understand photoregulation in sea slugs inhabiting coastal marine environments ^[28]^[29].

Lipids and fatty acids (FAs) are a major source of metabolic energy, the main constituents of biological membranes, and play a relevant role in cell signaling ^[30]. In mollusks, these molecules are important for gametogenesis and can be used as an energy source during periods of food shortage ^[31]. The FA profile in mollusks and marine invertebrates depends on environmental and biological factors, such as diet, temperature or reproductive cycle ^[32]. Few works have related lipids to kleptoplasty in sacoglossan sea slugs. Pelletreau et al. ^[33] proposed that lipids synthesized by kleptoplasts inside E. chlorotica were crucial for the establishment and stabilization of kleptoplasty; moreover, Trench et al. ^[34] studied the incorporation of CO₂ into glycolipids from the chloroplasts of Codium fragile to E. viridis. More recently, Rey et al. ^[35]^[36] analyzed the lipidome of two sea slug species, E. viridis and Placida dendritica feeding upon the same macroalgae, C. tomentosum, but with contrasting kleptoplasty performance. These studies revealed that the lipidome of the chloroplasts sequestered by E. viridis, which can perform long-term retention of functional chloroplasts, displays no major shifts and that the lipidome of this sea slug was unaffected by the absence of food for one week ^[35]. However, in P. dendritica, whose retention of chloroplasts is non functional, the lipidome varied significantly in this short period of time ^[36]. Therefore, information on composition and abundance of different lipid classes and FAs can help elucidate the importance and interaction of chloroplasts and animal cells.

Elysia crispata, the largest and most abundant species of Sacoglossa in the Caribbean and Gulf of Mexico, is one of the few species that can retain functional kleptoplasts for up to four months ^[1]^[6]^[13]. This sea slug can reach up to 15 cm in size ^[37], and inhabits mangrove areas and coral reefs from shallow water (<1 m) up to 25 m depth ^[38]. Different habitats at different depths provide diverse sources of macroalgal food, as well as contrasting environmental conditions, such as irradiance ^[39]. Distinct light conditions that change with depth can affect pigment composition and photosynthesis performance, and consequently, they may affect kleptoplasty as evidenced in experiments developed by Vieira et al. ^[7].

2. Sample Collection

Specimens of Elysia crispata (Figure 1) from the same population were collected in three different sampling sites, less than 400 m apart from each other and with an approximate area of 300 m², at the Verde coral reef (Sistema Arrecifal Veracruzano) in Veracruz, Southern Gulf of Mexico (19°12′8.80″ N, 96°4′17.20″ W) in a single day of September 2019. No macroalgae were spotted nearby, only turf algae were seen on top of hard substrate. Collecting was limited to the minimal sample size needed and was conducted under a permit issued by SAGARPA (PPF/DGOPA-082/19). Fourteen organisms measuring at least 40 mm in size (total length) were collected on hard substratum at 0–4 m depth (n = 7) and 8–12 m depth (n = 7). Their morphological characteristics matched the “crispata” ecotype described by Krug et al. ^[40]. Animals were transported to the laboratory, where they were maintained in a recirculating filtered seawater system (temperature: 26 °C, salinity: 35, light-dark photoperiod 12:12 h, with an irradiance of 40 µmol photons m⁻² s⁻¹). Animals were maintained for a month under starvation to ensure that sea slugs emptied their guts and digested short-term retained chloroplasts. Individuals were then flash frozen in liquid nitrogen and freeze-dried for biochemical analysis. The whole biomass of each sea slug was individually homogenized and used for extractions.

Figure 1. Specimen of Elysia crispata collected from the coral reef Verde (Sistema Arrecifal Veracruzano) in Veracruz, Southern Gulf of Mexico. Scale bar = 10 mm.

3. Photosynthetic Pigment Analysis

Photosynthetic pigments were extracted using methods detailed in Cruz et al. ^[29]. Briefly, samples were extracted in 95% cold buffered methanol with 2% ammonium acetate. Samples were ground with a plastic rod and sonicated for 30 s and vortexed. Samples were then transferred to −20 °C for 20 min in the dark. Extracts were filtered through 0.2 μm PTFE membrane filters and immediately injected into a Prominence-i LC 2030C High-performance liquid chromatography (HPLC) system (Shimadzu, Japan) with a photodiode array detector. Chromatographic separation was carried out using a Supelcosil C18 column (250 mm length; 4.6 mm diameter; 5 μm particles; Sigma-Aldrich, Burlington, MA, USA) for reverse phase chromatography and a 35 min elution program ^[41]. Photosynthetic pigments were identified from retention times and absorbance spectra, and concentrations calculated from the signals in the photodiode array detector. Pigment calibration was done using pure crystalline standards from DHI (Hørsolm, Denmark).

4. Lipid Analysis

Total lipids were extracted following a modification of the Bligh and Dyer method ^[42]. Briefly, animal tissues were homogenized, mixed with 800 µL of methanol and 400 µL of dichloromethane, vortexed and sonicated for 1 min, then, incubated on ice for 30 min on a rocking platform shaker and centrifuged at 2000 rpm for 10 min at room temperature. The organic phase was collected, and the biomass residue was re-extracted.

Ultrapure water was added (800 µL) and centrifuged at 2000 rpm for 10 min to recover the organic phase. An additional volume of 800 µL of dichloromethane was added to the aqueous phase, centrifuged at 2000 rpm for 10 min and the organic phase was recovered. Organic phases were dried under a nitrogen stream and preserved at –20 °C for further analysis. Total lipid extract weight was estimated by gravimetry.

Glycolipid (GL) quantification in total lipid extracts was performed using the orcinol colorimetric method ^[43]. Briefly, 200 µg of total lipid extract were transferred to a glass tube and 1 mL of orcinol solution (0.2% in 70% H₂SO₄) was added after dying dichloromethane under a nitrogen flow. Tubes were then incubated for 20 min at 80 °C. D-Glucose standards of 2-50 µg (standard solution of D-glucose 2.0 mg mL⁻¹) were used to prepare the calibration curve. At room temperature, the absorbance of standards and samples was measured at 505 nm using a microplate UV-Vis spectrophotometer. The conversion factor 100/35 (ca. 2.8) was used to estimate the total glycolipid content in total lipid extracts ^[44].

Phospholipid (PL) content from total lipid extract was quantified through the phosphorus assay, according to Bartlett and Lewis ^[45]. Lipid extracts were re-suspended in 300 µL of dichloromethane and 10 µL of each sample were transferred to a glass tube washed with 5% nitric acid. After drying under a nitrogen flow, 125 µL of perchloric acid (70%) was added and samples were incubated for 1 h at 180 °C in a heating block. A total of 825 µL of ultrapure water, 125 µL of NaMoO₄∙H₂O (2.5%), and 125 µL of ascorbic acid (10%) was added to each sample, with the mixture being homogenized in a vortex following each addition. Tubes were then incubated for 10 min at 100 °C in a water bath. Standards of 0.1-2 µg phosphate (standard solution of NaH₂PO₄∙2H₂O, 100 µg of phosphorus mL^-1) underwent the same treatment as samples, without the heating block step. At room temperature, absorbance of standards and samples was measured at 797 nm, using a microplate UV-Vis spectrophotometer. The conversion factor 775/31 (25) was used to estimate the total phospholipid content in total lipid extracts.

FA profile of E. crispata was analyzed by gas chromatography-mass spectrometry (GC-MS). FA methyl esters (FAME) were prepared using 30 µg of total lipid extract, 1 mL of the internal standard 19:0 (0.5 µg mL⁻¹ in n-hexane, CAS number 1731-94-8, Merck) and 200 µL of a methanolic solution of potassium hydroxide (2 M) ^[46]. After homogenization of this mixture, 2 mL of an aqueous solution of sodium chloride (10 mg mL⁻¹) were added. Sample was centrifuged at 2000 rpm for 5 min to separate the phases. The organic phase containing the FAME was transferred to a microtube and dried under a nitrogen stream. FAME were then dissolved in 40 µL of n-hexane, and 2 µL of this solution were injected on an Agilent Technologies 6890 N Network chromatograph equipped with a DB-FFAP column with 30 m length, an internal diameter of 0.32 mm, and a film thickness of 0.25 µm (J&W Scientific, Folsom, CA, USA). The GC was connected to an Agilent 5973 Network Mass Selective Detector operating with an electron impact mode at 70 eV and scanning the mass range m/z 50−550 in 1 s cycle in a full scan mode acquisition. The initial oven temperature was 80 °C, staying at this temperature for 3 min and increasing linearly to 160 °C at 25 °C min⁻¹, followed by linear increases to 210 °C at 2 °C min⁻¹ and 250 °C at 30 °C min⁻¹. Temperature was maintained at 250 °C for 10 min. The injector and detector temperatures were 220 °C and 250 °C, respectively. Helium was used as carrier gas at a flow rate of 1.4 mL min⁻¹. FAME present in the sample were identified by comparing their retention time and mass spectra with a commercial FAME standard mixture (Supelco 37 Component FAME Mix, ref. 47885-U, Sigma-Aldrich) and confirmed by comparison with the spectral library from ‘The Lipid Web’ ^[47]. FAME were quantified by using calibration curves of FAME standards acquired under the same instrumental conditions ^[48].

5. Conclusions

Authors provided a characterization of pigments, FA and lipid classes (PL and GL) from E. crispata sampled at different depths, which helps to fill a knowledge gap of one of the animal models most commonly employed to study kleptoplasty. To our knowledge, this is the first effort assessing differences in depth for a population of a sacoglossan sea slug, and the first work in the most western distribution recorded for this species.

The heterogeneity recorded in the profile of photosynthetic pigments and FA composition of E. crispata was not related with the habitat depth at the coral reef where they were sampled in Southern Gulf of Mexico. The total lipid, PL and GL contents found in this work were similar for specimens collected at shallow (0-4 m) and deeper (8-12 m) habitats. The conserved heterogeneity of their photosynthetic pigment profiles, as well as the high content of molecules exclusive of chloroplasts recorded on E. crispata, such as Chl a and GL after a month of food deprivation confirms that these sea slugs retain chloroplasts in good condition for long periods of time after stealing them from macroalgae.

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