Abstract
19 min readArticle Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Introduction Since the Hershey–Chase experiment (Hershey and Chase, 1952), which proved that DNA is the material transferred to bacteria in phage infections, horizontal gene transfer (HGT) has been considered essential for cross-species transformation (Arber, 2014). Although the prion hypothesis has rekindled the interest in proteins as an element of phenotype propagation (Crick, 1970; Wickner et al., 2015), HGT is still assumed to be the cause of transformation. For example, in a secondary plastid acquisition scenario in dinoflagellates, (1) a non-phototrophic eukaryote sequesters a unicellular archaeplastid; (2) the endogenous gene transfer to the non-phototrophic eukaryote leads to the shrinkage of the archaeplastidan nuclear DNA (nucDNA); and (3) the archaeplastidan nucleus disappears, and its plastid becomes a secondary plastid in the host (Reyes-Prieto et al., 2007). Chloroplast sequestration in sea slugs has attracted much attention due to the uniqueness of the algae-derived phenotype acquisition. Some species of sacoglossan sea slugs (Mollusca: Gastropoda: Heterobranchia) can photosynthesize using the chloroplasts of their algal food (Figure 1A and B; de Vries and Archibald, 2018; Kawaguti, 1965; Pierce and Curtis, 2012; Rumpho et al., 2011; Serôdio et al., 2014). These sacoglossans ingest species-specific algae and sequester the chloroplasts into their intestinal cells. This phenomenon is called kleptoplasty (Gilyarov, 1983; Pelletreau et al., 2011). The sequestered chloroplasts (named kleptoplasts) retain their electron microscopic structure (Fan et al., 2014; Kawaguti, 1965; Martin et al., 2015; Pelletreau et al., 2011; Trench, 1969) and photosynthetic activity (Cartaxana et al., 2017; Christa et al., 2014a; Cruz et al., 2015; Händeler et al., 2009; Taylor, 1968; Teugels et al., 2008; Wägele, 2001; Yamamoto et al., 2009). The retention period of photosynthesis differs among sacoglossan species (1 to >300 days; Figure 1B; Christa et al., 2015, Christa et al., 2014a, Christa et al., 2014b; Evertsen et al., 2007; Laetz and Wägele, 2017) and development stages and depends on the plastid 'donor' species (Curtis et al., 2007; Laetz and Wägele, 2017). Figure 1 Download asset Open asset Kleptoplasty in sea slugs. (A) Process of algal chloroplast retention by a sacoglossan sea slug (Pierce and Curtis, 2012). Sacoglossan sea slugs puncture the cell wall of food algae to suck out the protoplasm. The chloroplasts in the protoplasm are transported to the sea slug's intestinal tract, and the intestinal epithelial cells sequester chloroplasts by phagocytosis. The sequestered chloroplasts (kleptoplasts) maintain the photosynthetic activity in the cell for days to months. The sacoglossan cell contains no algal nuclei. Kleptoplast has never been found in germ cells of sea slug. (B) Phylogenetic distribution of kleptoplasty in the order Sacoglossa. Phylogenetic analysis showed that a common ancestor of Sacoglossa acquired non-functional chloroplast retention phenomena (without the maintenance of photosynthetic function), and multiple sacoglossan groups subsequently acquired the ability to maintain photosynthetic activity. Phylogenetic tree and kleptoplasty states are simplified from Christa et al., 2015. Christa et al., 2014b defined functional chloroplast retention for less than 2 weeks as 'short-term retention', and for more than 20 days as 'long-term retention'. Relationships within Heterobranchia are described according to Zapata et al., 2014. The red-colored taxa include the species used in the present study (P. ocellatus and E. marginata). (C–E) Photo images of PoB starved for 21 days. (C) Dorsal view. H, head; R, rhinophores; P, parapodia (lateral fleshy flat protrusions). Almost always, PoB folded parapodia to the back in nature. (D) The same individual of which parapodium was turned inside out (without dissection). The back of the sea slug and inside of the parapodia are green. This coloration is caused by the kleptoplasts in DG, which are visible through the epidermis. (E) Magnified view of the inner surface of parapodium. The diagonal green streaks are ridged projections on the inner surface of the parapodium. The cells containing kleptoplasts are visible as green spots. (F) Phylogeny of the P. cf. ocellatus species complex based on mitochondrial cox1 genes (ML tree from 568 nucleotide positions) from INSD and the whole mtDNA sequence. The sequence data for the phylogenetic analysis are listed in Figure 1—source data 1. Clade names in square brackets are based on Krug et al., 2013. Asterisks mark the genotypes from Krug et al., 2013. Study topics analyzed by previous researchers were described within the colored boxes for each cluster. Small black circles indicate nodes supported by a high bootstrap value (i.e., 80–100%). Thuridilla gracilis is an outgroup. Plakobranchus papua is a recently described species and previously identified as P. ocellatus (Meyers-Muñoz et al., 2016). Figure 1—source data 1 Sequence ID list used for phylogenetic analysis on Figure 1B. https://cdn.elifesciences.org/articles/60176/elife-60176-fig1-data1-v1.xlsx Download elife-60176-fig1-data1-v1.xlsx The absence of algal nuclei in sacoglossan cells makes kleptoplasty distinct from other symbioses and plastid acquisitions (de Vries and Archibald, 2018; Rauch et al., 2015). Electron microscopic studies have indicated that the sea slug maintains photosynthetic activity without algal nuclei (Hirose, 2005; Kawaguti, 1965; Laetz and Wägele, 2019; Martin et al., 2015; Pierce and Curtis, 2012). Because the algal nucleus, rather than the plastids, encodes most photosynthetic proteins, the mechanism to maintain photosynthetic proteins is especially intriguing, given that photosynthetic proteins have a high turnover rate (de Vries and Archibald, 2018; Pelletreau et al., 2011). Previous polymerase chain reaction (PCR)-based studies have suggested the HGT of algal nucleic photosynthetic genes (e.g., psbO) to the nucDNA of the sea slug, Elysia chlorotica (Pierce et al., 1996; Pierce et al., 2009; Pierce et al., 2007; Pierce et al., 2003; Rumpho et al., 2008; Schwartz et al., 2014). A genomic study of E. chlorotica (N50 = 824 bases) provided no reliable evidence of HGT but predicted that fragmented algal DNA and mRNAs contribute to its kleptoplasty (Bhattacharya et al., 2013). Schwartz et al., 2014 reported in situ hybridization-based evidence for HGT and argued that the previous E. chlorotica genome might overlook the algae-derived gene. Although an improved genome of E. chlorotica (N50 = 442 kb) was published recently, this study made no mention of the presence or absence of algae-derived genes (Cai et al., 2019). The genomic studies of sea slug HGT have been limited to E. chlorotica, and the studies have used multiple samples with different genetic backgrounds for genome assembling (Bhattacharya et al., 2013; Cai et al., 2019). The genetic diversity of sequencing data may have inhibited genome assembling. Although transcriptomic analyses of other sea slug species failed to detect HGT (Chan et al., 2018; Wägele et al., 2011), transcriptomic data were insufficient to ascertain genomic gene composition (de Vries et al., 2015; Rauch et al., 2015). Here, the genome sequences of another sacoglossan species, Plakobranchus ocellatus (Figure 1C–E), are presented to clarify whether HGT is the primary system underlying kleptoplasty. For more than 70 years, multiple research groups have studied P. ocellatus for its long-term (>3 months) ability to retain kleptoplasts (Christa et al., 2013; Evertsen et al., 2007; Greve et al., 2017; Kawaguti, 1941; Trench et al., 1970; Wade and Sherwood, 2017; Wägele et al., 2011). However, a recent phylogenetic analysis showed that P. ocellatus is a species complex (a set of closely related species; Figure 1F; Christa et al., 2014c; Krug et al., 2013; Maeda et al., 2012; Meyers-Muñoz et al., 2016; Yamamoto et al., 2013). Therefore, it is useful to revisit previous studies on P. ocellatus. This study first confirmed the photosynthetic activity and adaptive relevance of kleptoplasty to P. ocellatus type black (a species confirmed by Krug et al., 2013 via molecular phylogenetics; hereafter 'PoB'). The genome sequences of PoB (N50 = 1.45 Mb) and a related species, Elysia marginata (N50 = 225 kb), were then constructed. By improving the DNA extraction method, the genome sequences from a single sea slug individual in each species were successfully assembled. The comparative genomic and transcriptomic analyses of these species demonstrate the complete lack of photosynthetic genes in these sea slug genomes and supported an alternative hypothetical kleptoplasty mechanism. Results Does the kleptoplast photosynthesis prolong the life of PoB? To explore the photosynthetic activity of PoB, three photosynthetic indices were measured: photochemical efficiency of kleptoplast photosystem II (PSII), oxygen production rate after starvation for 1–3 months, and effect of illumination on PoB longevity. The Fv/Fm value, which reflects the maximum quantum yield of PSII, was 0.68–0.69 in the 'd38' PoB group (starved for 38 days) and 0.57–0.64 in the 'd109' group (starved for 109–110 days; Figure 2A). These values were only slightly lower than those of healthy Halimeda borneensis, a kleptoplast donor of PoB (Maeda et al., 2012), which showed Fv/Fm values of 0.73–0.76. The donor algae of PoB consisted of at least eight green algal species, and they are closely related to H. borneensis (Maeda et al., 2012). Although it is not clear whether the Fv/Fm is the same for all donor algae, Fv/Fm is almost identical (~0.83) in healthy terrestrial plants regardless of species (Maxwell and Johnson, 2000). Moreover, the values of H. borneensis were similar to those of other green algae (e.g., Chlamydomonas reinhardtii, Fv/Fm = 0.66–0.75; Bonente et al., 2012). Hence, we assumed that Fv/Fm values are similar among the donor species. The Fv/Fm value suggested that PoB kleptoplasts retain a similar photochemical efficiency of PSII to that of the food algae for more than 3 months. Figure 2 with 1 supplement see all Download asset Open asset Photosynthetic activity of PoB. (A) Jitter plot of Fv/Fm values indicating the photochemical efficiency of PSII. Habo, H. borneensis; d38, starved PoB for 38 days; d109, starved for 109 to 110 days (12 hr light/12 hr dark cycle; the light phase illumination was 10 µmol photons m−2 s−1). The magenta line indicates the mean, and the black dot indicates each individual's raw value (n = 3 per group). (B) Dynamics of seawater oxygen concentration. To better demonstrate the PoB photosynthesis, a change of oxygen concentration every second was visualized. A PoB individual, one of the d38 samples (ID1), was put into the measurement chamber, and the light conditions were changed in tens of minutes. The measurements in other individuals are visualized in Figure 2—figure supplement 1, along with the diagrams of the measuring equipment. Each point represents the oxygen concentration value per second. Gray signifies a dark period, and yellow means an illuminated period (50 µmol photons m−2 s−1). Temp, water temperature. Although the values fluctuated by the noise due to ambient light and other factors, the oxygen concentration decreased in the dark conditions and increased in the light conditions. When the illumination condition was changed, the changing pattern kept the previous pattern for a few minutes. This discrepancy may reflect the distance between the organism and oxygen sensor and the time for adaptation to brightness by the kleptoplast. (C) Jitter plots of PoB oxygen consumption and generation. D, dark conditions; L, light conditions; G, gross rate of light-dependent oxygen generation (L minus D). (D) Jitter plots of PoB longevity (n = 5 per group). D, Continuous dark; L/D, 12 hr light/12 hr dark cycle. The p from Welch's two-sample t-test was used. A source file of Fv/Fm jitter plot, time course of oxygen concentration, and longevity analysis are available in Figure 2—source data 1–3, respectively. Figure 2—source data 1 Summarized data of Fv/Fm and oxygen generation activity analysis of P. ocellatus used for Figure 2. https://cdn.elifesciences.org/articles/60176/elife-60176-fig2-data1-v1.xlsx Download elife-60176-fig2-data1-v1.xlsx Figure 2—source data 2 Raw data of oxygen concentration dynamics. This zip archive contains raw data of oxygen concentration dynamics. The measurement data for each time series are stored in separate files, with the experiment ID (ID1–6) and types of the experiment (mock or P. ocellatus sample) indicated in the name. https://cdn.elifesciences.org/articles/60176/elife-60176-fig2-data2-v1.zip Download elife-60176-fig2-data2-v1.zip Figure 2—source data 3 Summary of the longevity of analyzed P. ocellatus individuals used for Figure 2D. https://cdn.elifesciences.org/articles/60176/elife-60176-fig2-data3-v1.xlsx Download elife-60176-fig2-data3-v1.xlsx Based on the measurement of oxygen concentrations in seawater, starved PoB individuals ('d38' and 'd109') displayed gross photosynthetic oxygen production (Figure 2B and C). The mock examination without the PoB sample indicated no light-dependent increase in oxygen concentrations, i.e., no detectable microalgal photosynthesis in seawater (Figure 2—figure supplement 1). The results demonstrated that PoB kleptoplasts retain photosynthetic activity for more than 3 months, consistent with previous studies on P. cf. ocellatus (Christa et al., 2014c; Evertsen et al., 2007). The longevity of starved PoB specimens was then measured under different light conditions. The mean longevity was 156 days under continuous darkness and 195 days under a 12 hr light/12 hr dark cycle (p=0.022; Figure 2D), indicating that light exposure significantly prolongs PoB longevity. This observation was consistent with that of Yamamoto et al., 2013 that the survival rate of PoB after 21 days under starvation is light dependent. Although a study using P. cf. ocellatus reported that photosynthesis had no positive effect on the survival rate (Christa et al., 2014c), our results indicated that this finding does not apply to PoB. Given the three photosynthetic indices' data, we concluded that the increase in PoB survival days was due to photosynthesis. A previous study using the short-term (retention period of 4–8 days) kleptoplastic sea slug, Elysia atroviridis, indicated no positive effect of light illumination on their survival rate (Akimoto et al., 2014). These results supported that light exposure does not affect sacoglossan longevity in the absence of kleptoplasts. Although plastid-free PoB may directly indicate that kleptoplasty extends longevity, there is no way to remove kleptoplasts from PoB, except during long-term starvation. PoB feeds on nothing but algae and retains the kleptoplast for 3–5 months. Although future research methods may allow for more experimental analyses of kleptoplast functions, the currently most straightforward idea is that kleptoplast photosynthesis increases the starvation resistance of PoB. Do kleptoplasts encode more photosynthetic genes than general plastid? To reveal the proteins synthesized from kleptoplast DNA (kpDNA), we sequenced the whole kpDNA from PoB and compared the sequences with algal plastid and nuclear genes. Illumina sequencing provided two types of circular kpDNA and one whole mitochondrial DNA (mtDNA) (Figure 3A; Supplementary file 1; Figure 3—figure supplements 1–3). The mtDNA sequence was almost identical to the previously sequenced P. cf. ocellatus mtDNA (Figure 3—figure supplement 3B; Greve et al., 2017). The sequenced kpDNAs corresponded to those of the predominant kleptoplast donors of PoB (Maeda et al., 2012), i.e., Rhipidosiphon lewmanomontiae (AP014542; hereafter 'kRhip') and Poropsis spp. (AP014543; hereafter 'kPoro'; Figure 3B; Figure 3—figure supplement 4). Figure 3 with 10 supplements see all Download asset Open asset Gene composition of PoB kpDNAs. (A) Gene map of two kpDNAs from PoB. Gene positions are described in circles colored according to the gene's functional category (see keys in the box). Genes on the outside and inside of each circle are transcribed in the clockwise and anticlockwise directions, respectively (for detailed maps, see Figure 3—figure supplements 1 and 2). (B) Phylogenetic positions of sequenced kleptoplasts among green algal plastids. The original ML tree (Figure 3—figure supplement 4) was created based on rbcL genes (457 positions) and converted to the tree. Red indicates sequenced kpDNA or cpDNA in the present study. Underlines indicate algal species used in RNA-Seq sequencing. (C) An UpSet plot of plastid gene composition. Species abbreviations are defined in Figure 3—source data 1. The horizontal bar chart indicates the gene numbers in each species. The vertical bar chart indicates the number of genes conserved among the species. Intersect connectors indicate the species composition in a given number of genes (vertical bar chart). Connections corresponding to no gene were omitted. Connectors were colored according to the gene's conservation level in Bryopsidales: Core gene, conserved among all analyzed Bryopsidales species; Dispensable gene, retained more than two Bryopsidales species; Rare gene, determined from a single or no Bryopsidales species. Gray shading indicates non-Viridiplantae algae, and magenta shading indicates PoB kleptoplasts. Cyme (C. merolae) and Vali (V. litorea) had more than 100 genes that Bryopsidales does not have (e.g., left two vertical bars). (D) Box-plots of tblastn results. The vertical axis shows the database searched (kPoro and kRhip, PoB kpDNAs; nCale, nucDNA of C. lentillifera). Each dot represents the tblastn result (query is the A614 dataset). Red dots show the result using the chlD gene (encoding magnesium-chelatase subunit ChlD) as the query sequence; this sequence is similar to the kleptoplast-encoded chlL gene. The right pie chart shows the proportion of queries with hits (E-value < 0.0001). (E) Heat map of tblastn results of representative photosynthetic nuclear genes (a subset of data in D). The source species of the query sequences are described on the top. The source files of plastid gene composition and tblastn analysis are available in Figure 3—source data 1 and 2. Figure 3—source data 1 Plastid gene composition. This zip archive contains source files of plastid gene composition and of orthologous analysis. https://cdn.elifesciences.org/articles/60176/elife-60176-fig3-data1-v1.zip Download elife-60176-fig3-data1-v1.zip Figure 3—source data 2 Tblastn analysis of kleptoplast/chloroplast DNA. This zip archive contains source and result files of tblastn analysis of kleptoplast/chloroplast DNA. https://cdn.elifesciences.org/articles/60176/elife-60176-fig3-data2-v1.zip Download elife-60176-fig3-data2-v1.zip To determine whether kpDNA gene repertoires were similar to green algal chloroplast DNAs (cpDNAs), H. borneensis cpDNA was sequenced, and 17 whole cpDNA sequences were obtained from public databases (Figure 3C; Figure 3—figure supplements 5 and 6). PoB kpDNAs contained all the 59 conserved chloroplastic genes in Bryopsidales algae (e.g., psbA and rpoA), although they lacked four to five of the dispensable genes (i.e., petL, psb30, rpl32, rpl12, and ccs1; Figure 3C). To test whether kpDNAs contained no additional photosynthetic genes, a of photosynthetic genes was then used the which were from the algal transcriptomic data and public algal genomic data files 2 and A tblastn using A614 obtained no reliable hits (E-value < the kpDNA except for the chlD gene, which the chlL gene (Figure A positive an algal nucDNA database et al., found reliable for of the queries (Figure suggesting that the has high Hence, it was concluded that kpDNAs lack multiple photosynthetic genes (e.g., psbO) as general green algal there transferred algal genes to the PoB nucleic To determine whether the PoB nucleic genome contains algae-derived genes (i.e., evidence of we sequenced the nuclear genome of PoB. A for sequences in gene genomic and were The genome contained of the genome = 1.45 gene Supplementary file Figure supplements 1–3). analysis using the showed high of the eukaryote conserved gene indicating that the gene was complete to HGT file 4). The gene were in INSD and with of PoB gene found no evidence of algae-derived the database found PoB gene with hits to or eukaryotic of the hits encoded as or Supplementary file was the most source of algae of the sequences and was the second most source However, the of using multiple results analysis with et al., the algal of the genes file A using the A614 which contains sequences of the gene donor (e.g., transcriptomic data of H. determined no positive HGT evidence file analysis for all PoB gene genes were predicted to from species other than These genes may be due to but no genes were found from predicted that of the PoB genes from and its The genes to taxa as (e.g., the genes to other than (e.g., and genes from as transferred genes. However, these genes contained no photosynthetic gene. of the PoB genes as transferred genes encoded For the genes, no species. A the public database of the genes as genes genes were not with photosynthesis, except one gene to However, this to was not reliable it to be from on public Our via the database indicated the of the PoB gene to of a The of the analyses are in Supplementary file indicated that genes might have from or other but provided no evidence of algae-derived gene with Gene no PoB gene was as a gene although the same found to genes in the five algal species (Figure PoB genes to the of were found (Figure Supplementary file However, an with animal and algal genes not these algal (Figure supplement was considered that the sequence conservation the caused these in the The of these genes had no to photosynthesis file genes response structure mitochondrial inner and Figure with supplements see all Download asset Open asset for transferred algal genes in the PoB (A) of the number of genes to or The of or genes was compared among PoB, two mollusk species and five algal species (see abbreviations in Figure Figure 3—source data 1). A was visualized in the on the (B) plot of the the of the A614 gene set (query to the PoB The view with the tblastn Figure supplement The dot shows the source algae of each query sequence (see keys in the box). The horizontal axis shows the of the query to the sequences The vertical axis shows the of the sequences between the query and PoB of the and the by are for a query (i.e., a of the query sequence and a of (C) plot of results for the A614 photosynthetic genes, and genes, used as query sequences the database of from DNA of PoB. The shows the distribution of the number of with per the value of identical from the (D) plot of HGT indices for genes in PoB, the two mollusk species and and one algae species (C. Each dot represents a gene. A high or value means the of algal or respectively. the for HGT for and 100 for indicate PoB genes the (E) of the results of for in the PoB RNA-Seq head; The indicates the number of RNA-Seq as (see The show the RNA-Seq and analyzed The indicate the query the queries no corresponding were from the For queries using the the mean of the from each was The number of for each is given on the The source files of algal gene analysis using and are available in raw data Figure data 1. Figure data 1 gene from PoB This zip archive contains source files of algal gene from PoB genome and Download To that the gene not overlook a photosynthetic gene, the A614 the PoB and C. genome sequences was directly searched using tblastn and and the C. genome and hits were using the same only 1 and 2
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