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Chlorophyll a synthesis by an animal using transferred algal nuclear genes. Symbiosis

Article in Symbiosis · December 2010 Impact Factor: 1.44 · DOI: 10.1007/s13199-009-0044-8

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Nicholas E Curtis Julie Schwartz

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Chlorophyll a synthesis by an animal using transferred algal nuclear genes

Sidney K. Pierce*, Nicholas E. Curtis, and Julie A. Schwartz

Department of Integrative Biology, SCA 110, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA, Email. [email protected]

(Received September 22, 2009; Accepted October 13, 2009)

Abstract Chlorophyll synthesis is an ongoing requirement for photosynthesis and a ubiquitous, diagnostic characteristic of plants and algae amongst eukaryotes. However, we have discovered that chlorophyll a (Chla) is synthesized in the symbiotic chloroplasts of the sea slug, Elysia chlorotica, for at least 6 months after the slugs have been deprived of the algal source of the plastids, Vaucheria litorea. In addition, using transcriptome analysis and PCR with genomic DNA, we found 4 expressed genes for nuclear-encoded enzymes of the Chla synthesis pathway that have been horizontally transferred from the alga to the genomic DNA of the sea slug. These findings demonstrate the first discovery of Chla production in an animal using transferred nuclear genes from its algal food.

Keywords: Horizontal gene transfer, chlorophyll synthesis, chloroplast symbiosis, kleptoplasty, Elysia chlorotica, Vaucheria litorea

1. Introduction chloroplast. In spite of this plant/animal dichotomy, we have discovered the long term ability to synthesize Chla in In plants and algae, the chlorophyll a (Chla) synthesis an animal. pathway, starting with 5-aminolevulinic acid (ALA), is A few years ago, we reported the first discovery of the contained within the chloroplast, and regulated by intricate transfer of functional, nuclear genes between multicellular interactions among the production of plastid- and nuclear- species (Pierce et al., 2007). The sacoglossan sea slug, encoded, cytoplasmically-processed enzymes used in the Elysia chlorotica, eats the chromophytic alga, Vaucheria reactions of the synthesis, the presence or absence of light, litorea. Certain cells that line the slug’s digestive as well as cofactor and substrate level kinetics. While diverticula are able to phagocytize undigested chloroplasts chlorophyll synthesis does not occur in animals, the which are maintained intracellularly, and continue to be synthesis of the identical porphyrin moieties of chlorophyll photosynthetically active for as long as the 10–11 month and cytochrome/hemoglobin proceeds from ALA to the life cycle of the slug (West et al., 1984). Although there is level of protoporphyrin IX along an identical synthesis no evidence of chloroplast division in the slug cytoplasm, pathway in both plants and animals, albeit in different synthesis of several chloroplast proteins occurs during the intracellular compartments using enzymes of identical endosymbiotic association, including proteins that are name, but distinctive sequence. At that point, the pathway encoded by algal nuclear genes (Pierce et al., 1996; Hanten directions diverge to either the chlorophylls or and Pierce, 2001). Our original discovery found expressed cytochrome/hemoglobin (see the review by Tanaka and genes for three of the algal light-harvesting complex Tanaka, 2007). Thus, while the porphyrin synthesis proteins, LHCV-1, LHCV-2 and FCP, in genomic DNA of enzymes in the initial portion of the pathway to Chla are the slug (Pierce et al., 2007). In addition, we located the present in the mitochondria or cytoplasm of animal cells, a same genes in the genomic DNA of unhatched veliger plant/algae-specific set of several, nuclear-encoded larvae, which lack symbiotic chloroplasts and do not feed enzymes is required to complete the synthesis inside the on V. litorea, confirming that the transferred algal genes were vertically transmitted in the slugs. Transferred gene sequences were also present in slug cDNA, indicating their *The author to whom correspondence should be sent. transcription (Pierce et al., 2007). 122 S.K. PIERCE ET AL.

Furthermore, transfer of another algal nuclear gene on the sides of the aquarium. When found, they were gently between these species has recently been confirmed by removed and placed immediately into small culture dishes others (Rumpho et al., 2008). All of the above results were containing sterile artificial sea water containing rifampicin, produced using PCR-based experiments which were greatly where the embryos were maintained until they had impeded by the lack of slug and alga reference sequences in developed into veliger larvae (West et al., 1984), but had the public databases for both primer design and product not hatched. At the point DNA was extracted (see below), identification, as well as low sequence conservation of the veliger larvae, which do not contain symbiotic plastids, many of the genes of interest and contamination from the had not fed and were still inside their egg capsules. copious amounts of mucus produced by the highly mucogenic slugs. Genomic DNA purification In order to facilitate the hunt for additional transferred genes, we have recently turned our efforts to the production Genomic DNA was purified from pre-hatched and analysis of a transcriptome [expressed sequence tags E. chlorotica veliger larvae and V. litorea using the (EST)] library database from V. litorea (Schwartz et al., Nucleon® genomic DNA extraction kit, PhytoPure® (Tepnel submitted). Since EST’s are RNA-based, they are only a Life Sciences, Manchester, UK) following manufacturer’s representation of the genes being expressed at the moment instructions. the RNA is extracted. However, they provide the exact coding sequence of genes of interest, which greatly RNA isolation and mRNA purification facilitates not only identification, but also primer design and comparison with sequences in the slug. Using EST E. chlorotica: Total RNA was isolated from >2 month analysis, subsequently confirmed by PCR, we have already starved slugs as follows. Slugs were homogenized in located several additional transferred genes in the slug Trizol® Reagent (Invitrogen, Carlsbad, CA) and the genomic DNA (lhcv-3, lhcv-4, prk) (Schwartz et al., homogenate was centrifuged at 12,000 × g at 4oC to pellet submitted), as well as four genes in the Chla synthesis cellular debris. The supernatant was extracted with 1:6 (v/v) pathway. chloroform and centrifuged at 12,000 × g at 4oC. RNA was precipitated from the aqueous phase by adding 1:4 (v/v) isopropanol followed by 1:4 (v/v) 0.8M Na citrate/1.2 M 2. Materials and Methods NaCl solution and spun at 12,000 × g at 4oC. The RNA pellet was washed twice with 75% ethanol, air dried, Animals resuspended in diethylpyrocarbonate (DEPC)-treated water and quantified spectrophotometrically (260 nm). mRNA Specimens of Elysia chlorotica were collected in a salt was purified from total RNA using the Dynabeads® Oligo marsh on Martha’s Vineyard, MA. They were shipped to (dt)25 mRNA Purification Kit (Invitrogen) following Tampa, FL where they were kept in aquaria, without access manufacturer’s instructions and quantified spectrophoto- to algae, containing sterilized, aerated, artificial sea water metrically (260 nm). o (Instant Ocean, 1,000 mosm/kg H2O) at 10 C under a 14/10 V. litorea: Total RNA was isolated from the alga using hr light/dark cycle using fluorescent tubes (cool white). the Nucleon® genomic DNA extraction kit, PhytoPure® The slugs were starved for at least 2 months before use in following the manufacturer’s instructions, taking advantage the experiments. of the co-purification of RNA in that methodology. mRNA was purified as described above. Algae cDNA preparation The Vaucheria litorea used in the experiments came from a culture maintained in modified f/2 medium as E. chlorotica and V. litorea 1st and 2nd strand cDNAs described previously (Pierce et al., 1996). The original were synthesized from purified mRNA using the Mint inoculum for this culture came from the same salt marsh cDNA Synthesis Kit (Evrogen, Moscow, Russia) according that provided the slugs. At the time of the experiments the to manufacturer’s instructions. slugs and algae had not been in contact with each other for months and had undergone several biweekly changes of PCR and sequencing sterile media. Specific primers (Eurofins MWG/Operon, Huntsville, Veliger larvae AL) were designed from our EST sequences. Touchdown PCR reactions were done using 100 ng of genomic DNA or The aquaria containing the adult slugs were monitored cDNA, 12.5 pmol of each primer, 0.25 mM dNTP mix (ID daily for egg masses. Generally, the masses were deposited Labs, London, Ontario, Canada) and 1.25 units of CHLOROPHYLL SYNTHESIS BY AN ANIMAL 123

IDProof™ DNA polymerase (ID Labs). Initial denaturation still in the 14C-ALA containing media as above. Following was done at 95oC for 2 min, followed by 20 cycles of the incubation, the slugs or filaments were removed from denaturing at 95oC (30 s) and annealing (30 s) where the their media, washed in medium without isotope and Chla annealing temperature was reduced 1oC every other cycle, was extracted immediately. To test the effect of light on then a 72oC extension period. This was followed by an Chla synthesis, the experiment was done the same way additional 20 cycles of denaturing at 95oC for 30 s, then 30 except that the 18 hr incubation under lights was replaced s at the lowest annealing temperature obtained in the with 18 hrs in the dark. touchdown and 72oC extension. Generally, the annealing temperatures started 5oC below the melting temperature of Chlorophyll a extraction the primers. PCR products were separated on 1% agarose gels containing 0.2 µg/ml ethidium bromide and visualized Chla was extracted by homogenizing the slugs and algal by UV illumination. DNA bands were excised from agarose filaments in cold, HPLC grade acetone (Pinckney et al., gels, purified using the QIAquick® Gel Extraction Kit 1996). Samples were kept cold and exposure to light (Qiagen, Valencia, CA) and cloned using the TOPO® TA minimized throughout the extraction procedure. Samples Cloning® Kit (Invitrogen) following manufacturer’s were kept at -20oC in the dark until chromatography. instructions. Clones were PCR amplified using M13 forward and reverse primers, purified and sequenced Chlorophyll a purification and scintillation counting (Eurofins MWG/Operon) in forward and reverse directions. Sequences were analyzed using the tblastx algorithm In order to determine the amount of 14C incorporation searching the GenBank nr database and then aligned using into Chla during the incubation period, Chla was purified the ClustalW2 sequence alignment program. Identified gene using HPLC and radioactivity determined. Chromato- sequences used in this paper were uploaded to GenBank graphy (System Gold, Beckman Coulter, Fullerton, CA) and the acquisition numbers are indicated below. was done using two C18 columns (Microsorb 100-3, 100 × A critical concern in these experiments is to be sure that 4.6 mm, 3 mm, Varian, Lakeforest, CA and Vydac 201TP, algal DNA is not somehow contaminating the slug extracts. 150 × 4.6 mm, 5 mm, Vydac, Hespira, CA) connected in Therefore, we always perform several controls in our series using a mobile phase starting with 80% MeOH: 20% experiments. All the PCR reagents were tested in the NH4CH3COOH (0.5 M, pH 7.2) changing to 80% absence of template with negative results. Furthermore, we MeOH/20% acetone, according to the protocol described tested the extracted slug DNA for the presence of non- elsewhere (Pinckney et al., 1998). The second column was o chloroplast targeted, V. litorea nuclear genes, either internal heated to 40 C. NH4CH3COOH was added to the samples transcribed spacer region (ITS) (see Pierce et al., 2007) or as an ion pair and injections were kept to 50 µl in order to spermidine synthase (SPDS), which was chosen from the not overload the analytical-sized columns. All chemicals V. litorea EST sequence data. Primers for both these genes were HPLC grade. Detection was done at 438 nm. This always produced products with both V. litorea DNA and protocol is designed to separate not only a wide array of cDNA extracts using our PCR protocols, but NEVER with photopigments, but also several Chla precursors and slug genomic DNA, cDNA or larval DNA. By now, we derivatives (Pinckney et al., 1998). The eluant was directed have performed our procedures on dozens of extracts from to a fraction collector set to collect at various intervals several batches of slugs at several times of year, in different following the sample injection. In order to obtain a lab rooms, in different buildings. In addition to these lab sufficient amount of material, several chromatographic runs hygiene controls, the starved slugs had stopped producing of each extract were necessary. The corresponding fractions digestive wastes for weeks before use in the experiments, so from each run were pooled, dried under a stream of N2, the gut contents were not included in the experiments. Lastly, residue dissolved in acetone, added to scintillation cocktail as mentioned above, the veliger larvae had not hatched (Scintisafe 30%, Fisher Scientific, Fair Lawn, NJ) and the from the egg masses, so they had not fed, do not have radioactivity determined with a scintillation counter symbiotic chloroplasts and have never touched sea water. (LS6000IC, Beckman Coulter, Fullerton, CA). Chla causes a large quenching effect (Nayar et al., 2003), so counting Chlorophyll a production efficiency was determined by spiking samples with a known amount of cpm and correcting the results to dpm At the end of a 8 hr dark period, slugs or algal filaments using the measured counting efficiency for each sample. were exposed to 15 µCi or 7.5 µCi (respectively) 14C-ALA (14C-4, 55 µCi/mmol, American Radiolabeled Chemical, St. Confirmation of radioactivity in Chla Louis, MO) in their respective media for 2 hr in the dark in a constant temperature (25oC) agitator. At the end of the As well as TLC, we used the well-documented two hours, the slugs or filaments were placed under intense (Llewellyn et al., 1990; Nayar et al., 2003) acid conversion illumination (2–75 watt Halogen flood lamps) for 18 hrs, of radiolabelled Chla to phaeophytin followed by HPLC 124 S.K. PIERCE ET AL. separation and fraction collecting to demonstrate the under lights in sea water containing 14C-labeled ALA and molecular location of 14C. In separate experiments, slugs or comparing the pattern of radiolabel distribution to that of algal filaments were labeled with 14C-ALA as described similarly treated V. litorea filaments, using HPLC and above. After the incubation period, Chla was extracted from fraction collection. In the algal filaments, 14C co-migrated the slugs or algae, the extract run in 50 µl aliquots through with several peaks on the HPLC chromatogram, some the HPLC protocol already described and the Chla peaks colored and some not, including Chla (Fig. 1). collected from the eluant and pooled, all as described Qualitatively, the chromatogram and distribution of above. Although the amount of Chla and the associated radioactivity in the V. litorea extracts is very typical of radioactivity in the extracts were reasonably robust, our those from measurements of Chla synthesis in organisms as analytical HPLC with its analytical-sized columns and 50 phylogenetically diverse as phytoplankton (Riper et al., µl sample loop required the collection of several dozen 1979) and barley (Wamsley and Adamson, 1994), for but Chla peaks. The pooled peaks were dried under nitrogen two examples of many. Although we were unable to load and dissolved in a small amount of acetone. HCl (3.6 M) comparable amounts of 14C-ALA into the slugs, was added and incubated for 5 min at room temperature, undoubtedly due to external mucus, the HPLC chromato- which removes the Mg2+ from Chla, thereby converting it to gram and distribution of radioactivity in the corresponding phaeophytin (Llewellyn et al., 1990; Nayar et al., 2003). fractions collected from the slug extracts were very similar The acid was then neutralized with 3.6 M NH4OH, the to the results from the alga (Fig. 2). Due to the several sample was spiked with non-radioactive Chla and hours-long incubation period in 14C-ALA, radioactivity rechromatographed on the HPLC. As before, several dozen appears in a variety of peaks along the chromatogram, some 50 µl injections were done, the eluant in the region of the of which are probably Chla precursors and Chla derivatives chromatogram where the Chla spike and the phaeophytin (Pinckney et al., 1996) as well as a robust number of counts peak eluted were collected, pooled and the radioactivity in in the Chla peak fraction. The almost complete lack of the pooled peaks determined by scintillation counting, radioactivity in the Chla peak when the experiment was corrected for background, quench and converted to dpm. done in the dark (Fig. 3), the conversion of both the Chla peak and its radioactivity to phaeophytin following acid treatment of the HPLC purified Chla (Fig. 4) and the co- 3. Results migration of radioactivity with the Chla band following thin layer chromatography (TLC) of the HPLC fraction (data not The genes in the Chla synthesis pathway of V. litorea, shown) all confirm that the radiolabel was a component of which we have identified in slug cDNA, and/or pre-hatched Chla rather than a co-elutant. We did not have authentic veliger larva genomic DNA (see Table 3, for example), are standard compounds to attempt to identify the other uroD (uroporphyrinogen decarboxylase), ChlD (ChlD radioactive peaks on the chromatogram, but as with the subunit of magnesium chelatase), ChlH (ChlH subunit of alga, the elution times of some, compared with other studies magnesium chelatase) and ChlG (chlorophyll synthase) (see above), suggests that they might be Chla precursors (Tables 1–4). UroD is a porphyrin synthesis enzyme and derivatives, as well as chlorophyll c. common to both plants and animals, catalyzing the same reaction, albeit with different sequences and different reaction sites (chloroplast vs. cytoplasm, respectively) 4. Discussion (Reith, 1995; Obornik and Green, 2005; Tanaka and Tanaka, 2007; Eberhard et al., 2008). We have found an Altogether, the results clearly demonstrate that uroD sequence in slug cDNA that matches a consensus E. chlorotica expresses several V. litorea nuclear genes that uroD sequence in the V. litorea EST almost exactly (9 code for enzymes in the Chla synthesis pathway. In bases different in a 1026 base transcript) (Table 1). The addition, Chla synthesis occurs in the slug cells and other 3 enzymes (Chl-D, -H and -G) are in the unique continues for months after the animal has been separated pathway that leads to Chla synthesis from protoporphyrin from its food alga. This not only expands on our earlier IX, including the terminal reaction catalyzed by chlorophyll discovery of horizontally transferred genes between synthase. The sequences of these 3 genes in the slug cDNA multicellular species (Pierce et al., 2007) but also is the first match the sequence from the alga almost exactly (Tables demonstration of the transfer of an entire biosynthetic 2–4). pathway between eukaryotic species. We did not yet find Together, the expression of the genes for the Chla genes for all the Chla synthesis pathway enzymes in our synthesis enzymes in the slug DNA, as well as the many- ongoing analysis of the 4 million bases in the V. litorea months long unabated photosynthesis by the endosymbiotic transcriptome library. Indeed, there is some possibility that chloroplasts, a process that requires regular Chla synthesis those genes may not be in the transcriptome or may not in plants, suggest that Chla synthesis is occurring in the have been transferred to E. chlorotica, indicating that slug cell. We tested this possibility by incubating slugs continued long-term Chla synthesis in the slug results CHLOROPHYLL SYNTHESIS BY AN ANIMAL 125

Table 1. Comparison of consensus nucleotide sequences of uroD (uropo rphyrinogen decarboxylase) located in the transcriptome data and cDNA of V. litorea (GU068606) with that found by PCR in E. chlorotica cDNA (GU068607). The two sequences are extremely similar in composition over the 1026 bases differing by 9 bases (bold). The slug se quences come from at least 3 separate mRNA extractions done on at least 3 different groups of slugs at different times of the year.

V. litorea cDNA AAAGATCCATTGTTGTTAAGGGCAGCA AGAGGAGAGGCTGTGGAAAGGGTTCCTGTTTGG E. chlorotica cDNA AAAGATCCATTGTTGTTAAGGGCAGCA AGAGGAGAGGCTGTGGAAAGGGTTCCTGTTTGG .... 60

V. litorea cDNA ATGATGAGACAGGCAGGCAGACACATG CAAGAATACAGAGACCTTGTCAAAAAGTACCCC E. chlorotica cDNA ATGATGAGACAGGCAGGCAGACACATGCAAGAATACAGAGACCTCATCAAAAAGTACCCC .... 120

V. litorea cDNA ACATTCAGAGAGAGATCGGAGATCCAC GAAGTGTCCACTGAAATTTCACTTCAGCCTTAC E. chlorotica cDNA ACATTCAGAGAGAGATCGGAGATCCAC GAAGTGTCCACTGAAATTTCACTTCAGCCTTAC .... 180

V. litorea cDNA AGGCGATATGGAACCGATGGAGTGATCTTATTTTCTGATATTCTGACTCCACTGCCTGGA

E. chlorotica cDNA AGGCGATACGGAACCGATGGAGTGATCTTATTTTCTGATATTCTGACTCCACTGCCTGGA .... 240

V. litorea cDNA ATGGGTGTCGATTTCAAAATTGAAGAG AAAACCGGGCCCAAATTGGTCCCAATGAGAACA E. chlorotica cDNA ATGGGTGTCGATTTCAAAATTGAAGAG AAAACCGGGCCCAAATTGGTCCCAATGAGAACG .... 300

V. litorea cDNA TGGGAAAGTGTCAATGCAATGCACACA ATTGATTCTGAAAAGGCATGTCCTTTTGTGGGG E. chlorotica cDNA TGGGAAAGTGTCAATGCAATGCACACA ATTGATTCTGAAAAGGCATGTCCTTTTGTGGGG .... 360

V. litorea cDNA CAAACTCTGAGAGATTTGAAAAAAGAG GTCGGATCAAATGCAACAGTCCTGGGTTTTGTG E. chlorotica cDNA CAAACTCTGAGAGATTTGAAAAAAGAGGTCGGATCAAATGCAACAGTCCTGGGTTTTGTG .... 420

V. litorea cDNA GGATGTCCGTACACACTTGCCACTTAC ATGGTTGAAGGAGGTTCAAGCAGAGAATATTTG E. chlorotica cDNA GGATGTCCGTACACACTTGCCACTTAC ATGGTCGAAGGAGGTTCAAGCAGAGAATATTTG .... 480

V. litorea cDNA GAAATTAAAAAGATGATGTTCACTGAG CCTGAGTTGTTGCATGCCATGCTGGCCAAAATT E. chlorotica cDNA GAAATTAAAAAGATGATGTTCACTGAG CCTGAGTTGTTGCATGCCATGCTGGCCAAAATT .... 540

V. litorea cDNA GCTGATTCAATAGGGGATTATGGGATT TATCAAATCGAAAGTGGTGCACAGGTGATTCAA E. chlorotica cDNA GCTGATTCAATAGGGGATTATGGGATTTATCAAATCGAAAGTGGTGCACAGGTGATTCAA .... 600

V. litorea cDNA GTCTTTGACTCTTGGGCAGGCCATCTC TCCCCCAAAGACTATGATGTTTTTGCGGCACCT E. chlorotica cDNA GTCTTTGACTCTTGGGCAGGCCATCTT TCCCCCAAAGACTATGATGTTTTTGCAGCACCT .... 660

V. litorea cDNA TACCAAAAGAAGGTTATCCAAAAAATCAAATCTTCTCATCCTGAAGTCCCCATCATCATT

E. chlorotica cDNA TACCAAAAGAAGGTTATCCAAAAAATCAAATCTTCTCATCCTGAAGTCCCCATCATCATT .... 720

V. litorea cDNA TACATAAACAAGAGTGGTGCACTTTTG GAAAGGATGAGTCAAAGTGGGGCAGATATCATC E. chlorotica cDNA TACATAAACAAGAGTGGTGCACTTTTG GAAAGGATGAGTCAAAGTGGGGCAGATATCATC .... 780

V. litorea cDNA AGCTTGGATTGGACAGTGACGATTGAA GAGGCTAGGAAAAGAATCGGCAACGATATTGGC E. chlorotica cDNA AGCTTGGATTGGACAGTGACGATTGAA GAGGCTAGGAAAAGAATAGGCAACGATATTGGC .... 840

V. litorea cDNA ATCCAGGGTAACCTTGATCCAGCCGCC TTGTTTGCACCAAATGAAGTTATCAAGGAAAGG E. chlorotica cDNA ATCCAGGGTAACCTTGATCCAGCTGCCTTGTTTGCACCAAATGAAGTTATCAAGGAAAGG .... 900

V. litorea cDNA ACTGAAGAAATTTTGAGGGCATGCGGA GGGAGAAACCATGTCATGAATTTGGGCCATGGA E. chlorotica cDNA ACTGAAGAAATTTTGAGGGCATGCGGA GGGAGAAACCATGTCATGAATTTGGGCCATGGA .... 960

V. litorea cDNA ATCGAAGCGACGACTTCAGAAGAAAAG GCTGAATTTTTCATCAATACCGTAAAAAACTTC E. chlorotica cDNA ATCGAAGCGACGACTTCAGAAGAAAAG GCTGAATTTTTCATCAATACCGTAAAAAACTTC .... 1020

V. litorea cDNA AGGTTC E. chlorotica cDNA AGGTTC ...... 1026

from some sort of long-term storage of massive amounts of pathway, as well as the terminal enzyme, Chl-G, indicate a enzyme in the symbiotic chloroplast. However, the good possibility that the genes for the intermediate steps presence and expression of Chl-D and –H in the slug, genes have been transferred as well. for the subunits of the initial enzyme in the Chla specific 126 S.K. PIERCE ET AL.

Table 2. Comparison of consensus sequences of Mg2+ chelatase subunit D (ChlD) located by PCR in V. litorea (GU068608) and E. chlorotica (GU068609) cDNAs using primer sequences based on the V. li torea transcriptome data. These gene fragments match exactly over the 975 bases. The E. chlorotica data come from 3 separate mRNA extr actions done on at least 3 different groups of slugs at different times of the year.

V. litorea cDNA AAAGAAATGAGCCTGAGCCGGAAAATCAGC CCGAAGATGACGAAGCCCCATCTGTACCCC E. chlorotica cDNA AAAGAAATGAGCCTGAGCCGGAAAATCAGC CCGAAGATGACGAAGCCCCATCTGTACCCC .... 60

V. litorea cDNA AAGAATTCATGTTTGGCATCGATTCAACGG TCATCGACCCTGAACTGTTGGATTTCGGAC E. chlorotica cDNA AAGAATTCATGTTTGGCATCGATTCAACGGTCATCGACCCTGAACTGTTGGATTTCGGAC .... 120

V. litorea cDNA GGAAGAACAATGCCGGCAGGTCTGGGAAGA GGGGAATGATCTTTAACATGGAAAGAGGGC E. chlorotica cDNA GGAAGAACAATGCCGGCAGGTCTGGGAAGA GGGGAATGATCTTTAACATGGAAAGAGGGC .... 180

V. litorea cDNA GGAAGAACAATGCCGGCAGGTCTGGGAAGAGGGGAATGATCTTTAACATGGAAAGAGGGC

E. chlorotica cDNA GATATATCAAGCCGATGCTTCCGAAAGGAAAAAAAGGGAAATTGGCGTTGGATGCGACGC .... 240

V. litorea cDNA TGAGATCAGCGGCGCCGTATCAATTGTCGA GAAGATCGCGCGCTCTCTCAAAGAACGACG E. chlorotica cDNA TGAGATCAGCGGCGCCGTATCAATTGTCGA GAAGATCGCGCGCTCTCTCAAAGAACGACG .... 300

V. litorea cDNA GGAATCCGACCAAAAGAACAGTCTTTGTCG AAAAGTCTGATCTGAGGGTCAAAAAGCTCG E. chlorotica cDNA GGAATCCGACCAAAAGAACAGTCTTTGTCG AAAAGTCTGATCTGAGGGTCAAAAAGCTCG .... 360

V. litorea cDNA CGCGGAAAGCCGGCTCACTTATCATTTTCT GCGTTGACGCGAGCGGGAGCATGGCGCTGA E. chlorotica cDNA CGCGGAAAGCCGGCTCACTTATCATTTTCTGCGTTGACGCGAGCGGGAGCATGGCGCTGA .... 420

V. litorea cDNA ACCGAATGAACGCCGCGAAAGGCGCAGCAA TGTCATTGCTGGGCGAAGCCTACAAAAGCA E. chlorotica cDNA ACCGAATGAACGCCGCGAAAGGCGCAGCAA TGTCATTGCTGGGCGAAGCCTACAAAAGCA .... 480

V. litorea cDNA GGGACAAAGTGTGCCTCATACCTTTCCAGG GGGAAAGGGCTGAAGTCCTCCTCCCACCTT E. chlorotica cDNA GGGACAAAGTGTGCCTCATACCTTTCCAGG GGGAAAGGGCTGAAGTCCTCCTCCCACCTT .... 540

V. litorea cDNA CAAGTTCAATAGCAATGGCAAAAAGCCGTT TGGAGACGATGCCGTGTGGAGGTGGGTCAC E. chlorotica cDNA CAAGTTCAATAGCAATGGCAAAAAGCCGTTTGGAGACGATGCCGTGTGGAGGTGGGTCAC .... 600

V. litorea cDNA CGCTCGCTCATGCAATCAACGTCGCTGTAC GGACAGGGATTAACGCCATCAAATCACAGG E. chlorotica cDNA CGCTCGCTCATGCAATCAACGTCGCTGTAC GGACAGGGATTAACGCCATCAAATCACAGG .... 660

V. litorea cDNA ACGTGGGAAAAGTGGTGATTGTGATGGTAAGCGATGGTCGAGCGAATGTGCCCCTCGCGG

E. chlorotica cDNA ACGTGGGAAAAGTGGTGATTGTGATGGTAAGCGATGGTCGAGCGAATGTGCCCCTCGCGG .... 720

V. litorea cDNA TCAGTAACGGCACGCAGCTCCCTGAAGATG AGAAGATGTCCAGGGAGGAGTTGAAGGAGG E. chlorotica cDNA TCAGTAACGGCACGCAGCTCCCTGAAGATG AGAAGATGTCCAGGGAGGAGTTGAAGGAGG .... 780

V. litorea cDNA AGGTGTTGAACACTGCGAAGGCGCTGAGGG AGTTGCCGGCCTTTAGCTTGGTGGTGTTGG E. chlorotica cDNA AGGTGTTGAACACTGCGAAGGCGCTGAGGG AGTTGCCGGCCTTTAGCTTGGTGGTGTTGG .... 840

V. litorea cDNA ACACTGAGAATAAGTTCGTGAGCACTGGCA TGGCGAAGGAGTTGGCTGCAGCGGCTGGTG E. chlorotica cDNA ACACTGAGAATAAGTTCGTGAGCACTGGCATGGCGAAGGAGTTGGCTGCAGCGGCTGGTG .... 900

V. litorea cDNA GGAGATATCATTATATTCCAAAAGCAACGG ATCAAGCGATGGCGAAGGTGGCCAGCGAAG E. chlorotica cDNA GGAGATATCATTATATTCCAAAAGCAACGG ATCAAGCGATGGCGAAGGTGGCCAGCGAAG .... 960

V. litorea cDNA CAATTTCAAGCATCA E. chlorotica cDNA CAATTTCAAGCATCA ...... 975

Each generation of E. chlorotica must take up plasts as long as V. litorea is available. However, under chloroplasts from the algal food. However, once ensconced field or lab starvation conditions, photosynthesis continues into the digestive cells, the symbiotic plastids encounter an for months at a level sufficient to sustain slug reproduction array of algal genes transmitted from the parent slugs, without the input of fresh plastids. The remarkable which are sufficient to keep photosynthesis operating. longevity of the chloroplast symbiosis in E. chlorotica During the life cycle, the slugs will feed and take up chloro- suggests that many more algal genes have been transferred CHLOROPHYLL SYNTHESIS BY AN ANIMAL 127

Table 3. Comparison of consensus sequences of ChlH (ChlH subunit of mag nesium chelatase) in cDNA (GU068610) and genomic DNA (GU068611) from V. litorea and also cDNA from E. chlorotica adults (GU 068612) and genomic DNA from pre-hatched E. chlorotica veliger larvae (GU068613). The sequences differ in 1 nucleotide between al gae and slug (bold). The sequence data were the same among at least 3 different DNA or mRNA extractions from at least 3 different groups of organisms at different times of the year.

V. litorea cDNA AGGCTTTGTATGCCAGAACCAAAC TCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA V. litorea genomic DNA AGGCTTTGTATGCCAGAACCAAAC TCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA E. chlorotica cDNA AGGCTTTGTATGCCAGAACCAAAC TCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA E. chlorotica larval genomic DNA AGGCTTTGTATGCCAGAACCAAAC TCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA .... 60

V. litorea cDNA GTGGGTACGAGGGCACAAGGGAGA TCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG V. litorea genomic DNA GTGGGTACGAGGGCACAAGGGAGA TCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG E. chlorotica cDNA GTGGGTACGAGGGCACAAGGGAGA TCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG E. chlorotica larval genomic DNA GTGGGTACGAGGGCACAAGGGAGATCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG .... 120

V. litorea cDNA CCACTGCAGGGGAGGTGGACAACT TTATCTACGAAGATGCGAACGATGTTTTCATCAAAG V. litorea genomic DNA CCACTGCAGGGGAGGTGGACAACT TTATCTACGAAGATGCGAACGATGTTTTCATCAAAG E. chlorotica cDNA CCACTGCAGGGGAGGTGGACAACT TTATCTACGAAGATGCGAACGATGTGTTCATCAAAG E. chlorotica larval genomic DNA CCACTGCAGGGGAGGTGGACAACT TTATCTACGAAGATGCGAACGATGTGTTCATCAAAG .... 180

V. litorea cDNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA

V. litorea genomic DNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA

E. chlorotica cDNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA

E. chlorotica larval genomic DNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA .... 240

V. litorea cDNA CCACTTTTCTGGAGGCCAATGGAA GGGGCTACTGGGACACCTCGGATGATAATATAGAAC V. litorea genomic DNA CCACTTTTCTGGAGGCCAATGGAA GGGGCTACTGGGACACCTCGGATGATAATATAGAAC E. chlorotica cDNA CCACTTTTCTGGAGGCCAATGGAA GGGGCTACTGGGACACCTCGGATGATAATATAGAAC E. chlorotica larval genomic DNA CCACTTTTCTGGAGGCCAATGGAA GGGGCTACTGGGACACCTCGGATGATAATATAGAAC .... 300

V. litorea cDNA TGTTGCAGGATCTGTACCAAGAGG TGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT V. litorea genomic DNA TGTTGCAGGATCTGTACCAAGAGG TGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT E. chlorotica cDNA TGTTGCAGGATCTGTACCAAGAGG TGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT E. chlorotica larval genomic DNA TGTTGCAGGATCTGTACCAAGAGG TGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT ...... 357

than we have uncovered so far-perhaps even pieces of, or is likely similar amongst the slug species (but see below), even entire, algal chromosomes are involved. Nevertheless, the differences in chloroplast source and longevity suggest these results clearly show that the successful transfer of that the transferred gene array is different between slug functional nuclear genes between multicellular species not species. This specificity of transferred gene arrays within only can occur, but also can involve many genes which are sea slug species suggests, in turn, that gene movements expressed producing alien proteins that reach cellular have occurred many times across species and in different targets and are capable of function. amounts. Second, on a broader scale, our results here and These results seem important at several levels. First, the previously (Pierce et al, 2007) clearly show that completely phenomenon of chloroplast symbiosis has been of interest unrelated organisms can transfer genes between them, for almost half a century. Although many species of elysiid integrate the transfers into the host genome and, not only sea slugs are capable of intracellular sequestration of express the genes, but also successfully use the gene chloroplasts, some protists do it as well (Gast et al., 2007; products. Thus, at least some multicellular species do not Johnson et al., 2007). The division of the symbiotic have to wait for a mutation to occur for an evolutionary chloroplasts has not been seen so far in any kleptoplastic change to take place. Much as in prokaryotes and protists, species, including E. chlorotica. Also, while E. chlorotica mechanism(s) for a successful swap of DNA between even may hold the longevity record for plastid maintenance, the distantly related species is both present and active in symbiotic organelles in other species persist from but a few metazoans. days [for example, Elysiella pusilla (Evertson et al., 2007)] Clearly, genome sequencing is necessary to determine to several months [E. clarki (Pierce et al., 2006)] and the entire scope of algal genes in the genome of involve a variety of algal taxa, often, unlike E. chlorotica, E. chlorotica. In addition to the pathway we have found, to with more than one species of alga per species of sea slug work efficiently, the chlorophyll biosynthesis requires (Curtis et al., 2006). While the mechanism of sequestration retrograde signaling by the chloroplasts to help regulate 128 S.K. PIERCE ET AL.

Table 4. Comparison of consensus sequences of the chlorophyll synthase gen e (ChlG), the terminal enzyme in the Chla synthesis pathway, in cDNA from V. litorea (GU068614) and E. chlorotica (GU068615). The sequences match 100% over the 800 base run. As with the preceding figures, these fragments were produced by PCR using primer s equences made from the V. litorea transcriptome data. The E. chlorotica data come from 3 separate mRNA extractions done on at least 3 different groups of slugs at different times of the year.

V. litorea cDNA TCACACCTGGAATCCATTCGCAGGGCCAGA TGCAGTTGATTTACAAGATGCTGGGATTGA E. chlorotica cDNA TCACACCTGGAATCCATTCGCAGGGCCAGA TGCAGTTGATTTACAAGATGCTGGGATTGA .... 60

V. litorea cDNA CTTGGCCAAAGCTCTGACTTGTATGATATT GGCTGGGCCCTTTCTAACTGGCTTTACCCA E. chlorotica cDNA CTTGGCCAAAGCTCTGACTTGTATGATATTGGCTGGGCCCTTTCTAACTGGCTTTACCCA .... 120

V. litorea cDNA AACCATCAACGATTGGTATGACCGAGATAT TGATGCGATCAATGAGCCATATCGACCCAT E. chlorotica cDNA AACCATCAACGATTGGTATGACCGAGATAT TGATGCGATCAATGAGCCATATCGACCCAT .... 180

V. litorea cDNA TCCTTCTGGAGCTATTTCTGAGGGTCAAGTGAAAGCGCAAATTGCCTTTCTTCTAGTTGG

E. chlorotica cDNA TCCTTCTGGAGCTATTTCTGAGGGTCAAGTGAAAGCGCAAATTGCCTTTCTTCTAGTTGG .... 240

V. litorea cDNA TGGATTGGCTTTGTCGTATGGTTTGGATCT ATGGGCAGGGCACCAAATGCCCACTGTTTT E. chlorotica cDNA TGGATTGGCTTTGTCGTATGGTTTGGATCT ATGGGCAGGGCACCAAATGCCCACTGTTTT .... 300

V. litorea cDNA TTTGTTGTCATTGTTTGGGACTTTCATTTC ATACATATACTCAGCCCCGCCACTGAAATT E. chlorotica cDNA TTTGTTGTCATTGTTTGGGACTTTCATTTC ATACATATACTCAGCCCCGCCACTGAAATT .... 360

V. litorea cDNA GAAACAGAATGGCTGGGCAGGTAATTTTGC CTTGGGCTCAAGCTACATTAGCTTGCCGTG E. chlorotica cDNA GAAACAGAATGGCTGGGCAGGTAATTTTGCCTTGGGCTCAAGCTACATTAGCTTGCCGTG .... 420

V. litorea cDNA GTGGTGTGGTCAGGCTATGTTTGGTGAGCT CAACTTGCAAGTTGTGGTCCTAACTTTGCT E. chlorotica cDNA GTGGTGTGGTCAGGCTATGTTTGGTGAGCT CAACTTGCAAGTTGTGGTCCTAACTTTGCT .... 480

V. litorea cDNA GTATTCTTGGGCAGGCCTTGGAATTGCAAT AGTAAATGACTTCAAATCAGTTGAGGGGGA E. chlorotica cDNA GTATTCTTGGGCAGGCCTTGGAATTGCAAT AGTAAATGACTTCAAATCAGTTGAGGGGGA .... 540

V. litorea cDNA TAGAGCCATGGGTTTACAGTCTCTTCCTGT GGCTTTTGGTATAGAAAAAGCCAAGTGGAT E. chlorotica cDNA TAGAGCCATGGGTTTACAGTCTCTTCCTGTGGCTTTTGGTATAGAAAAAGCCAAGTGGAT .... 600

V. litorea cDNA ATGTGTGAGTAGCATTGACATTACTCAATT GGGCATAGCCGCATGGCTATATTATATTGG E. chlorotica cDNA ATGTGTGAGTAGCATTGACATTACTCAATT GGGCATAGCCGCATGGCTATATTATATTGG .... 660

V. litorea cDNA AGAGCCTACCTATGCATTCGTTTTATTGGGCCTCATTCTTCCTCAGATATATGCACAATT

E. chlorotica cDNA AGAGCCTACCTATGCATTCGTTTTATTGGGCCTCATTCTTCCTCAGATATATGCACAATT .... 720

V. litorea cDNA TAAGTATTTTTTGCCGGATCCAGTTGAGAA TGATGTCAAATACCAAGGATTTGCTCAGCC E. chlorotica cDNA TAAGTATTTTTTGCCGGATCCAGTTGAGAA TGATGTCAAATACCAAGGATTTGCTCAGCC .... 780

V. litorea cDNA ATTTCTTGTATTTGGGATTT E. chlorotica cDNA ATTTCTTGTATTTGGGATTT ...... 800

nuclear gene expression (Green et al., 2000). It is not yet gastropods is largely accomplished using intracellular clear how refined this control system is in the symbiotic vacuoles (Owen, 1966). During ingestion, food material is plastids in the E. chlorotica digestive cells, but algal mechanically broken down into small pieces or sucked up, nuclear genes that code for the proteins that signal between in the case of the sacoglossan. It then passes into the the chloroplast and nucleus as well as proteins involved in digestive tubules where the epithelial cells phagocytize the targeting and trafficking are obvious candidates. This pieces into lysosomal vacuoles where digestion proceeds. especially long-lived chloroplast symbiosis in E. chlorotica The chloroplasts are engulfed from the lumen of the tubules presents a unique opportunity to study the evolution of by either the same phagocytic mechanism, or an analogous intracellular organelles as it is occurring. process, according to the few studies that have examined it Finally, from both theoretical and applied perspectives (McLean, 1976; Mondy and Pierce, 2003). Although some understanding the gene transfer mechanism may be of other reports have incorrectly stated that the symbiotic considerable significance. The uptake of the chloroplasts chloroplasts are naked in the cytoplasm of the digestive has only been examined in a few instances. However, the tubule cell (Graves et al., 1979; Rumpho et al., 2001) ancient literature makes clear that digestion in herbivorous they are actually surrounded by a tightly applied animal CHLOROPHYLL SYNTHESIS BY AN ANIMAL 129

Figure 1. Typical HPLC chromatogram of Chla extracted from V. litorea (upper chart) and separated according to the protocol described in the methods section. The Chla peak is labeled such at approximately 43.5 min. The lower chart represents the 14 Figure. 2. Typical HPLC chromatogram (upper chart) of Chla radioactivity ( C) in fractions collected from the HPLC column extracted from E. chlorotica and separated by the same protocol eluant also as described in the methods. The large peak in that produced the V. litorea results in Fig. 1. The peak at radioactivity at approximately 43.5 min coelutes exactly with the approximately 43.5 min corresponds to the elution time of both Chla peak in the upper chart. Although we did not identify them, standard Chla (Sigma Chemicals) as well as the Chla peak in the the smaller radioactive peaks just preceding the Chla location are V. litorea chromatogram. The lower chart represents the (14C) most likely intermediates in the Chla synthesis pathway (see radioactivity profile in fractions of eluant collected during the Pinckney et al., 1996). The large peak of radioactivity starting at HPLC run. The large peak of radioactivity at approximately 43.5 about 4 min, which was not detected on the HPLC chromatogram, min corresponds exactly with the elution of the Chla peak. The is right at the column void volume. large peak starting at about 4 min into the run is right at the column void volume. The identities of the other radioactive peaks are unknown. Those just preceding and following Chla are likely Chla precursors and degradation products (Llewellyn et al., 1990; Pinckney et al., 1996; Nayar et al., 2003). The broad peak from membrane (Mondy and Pierce, 2003; Curtis et al., 2006). 13–15 min is in the region where chlorophyll c and fucoxanthin elute in this HPLC protocol (Llewellyn et al., 1990). Instead of being digested, the plastids reside inside the vacuole for the duration of their association. There is some possibility that the algal genes, especially if they are transferred in the form of chromosomes or pieces of chromosomes, enter the host cell by a similar process. Alternatively, circumstantial evidence indicates that Acknowledgements endogenous retroviruses could be the transfer agent, at least in E. chlorotica (Pierce et al., 1999; Mondy and Pierce, We gratefully acknowledge the generous financial 2003) although the increasing number of transferred genes support of a private donor, who wishes to remain being found in this species may make viral transfer a less anonymous. The work reported here could not have been attractive hypothesis. done without that donor’s help. 130 S.K. PIERCE ET AL.

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