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Chlorophyllasynthesisbyananimalusingtransferredalgalnucleargenes.Symbiosis

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SYMBIOSIS (2009) 49, 121–131 ©Springer Science+Business Media B.V. 2009 ISSN 0334-5114

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. pierce@cas.usf.edu (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 In plants and algae, the chlorophyll a (Chla) synthesis

pathway, starting with 5-aminolevulinic acid (ALA), is contained within the chloroplast, and regulated by intricate interactions among the production of plastid- and nuclear-encoded, cytoplasmically-processed enzymes used in the reactions of the synthesis, the presence or absence of light, as well as cofactor and substrate level kinetics. While chlorophyll synthesis does not occur in animals, the synthesis of the identical porphyrin moieties of chlorophyll and cytochrome/hemoglobin proceeds from ALA to the level of protoporphyrin IX along an identical synthesis pathway in both plants and animals, albeit in different intracellular compartments using enzymes of identical name, but distinctive sequence. At that point, the pathway directions diverge to either the chlorophylls or cytochrome/hemoglobin (see the review by Tanaka and Tanaka, 2007). Thus, while the porphyrin synthesis enzymes in the initial portion of the pathway to Chla are present in the mitochondria or cytoplasm of animal cells, a plant/algae-specific set of several, nuclear-encoded enzymes is required to complete the synthesis inside the

*The author to whom correspondence should be sent.

chloroplast. In spite of this plant/animal dichotomy, we have discovered the long term ability to synthesize Chla in an animal.

A few years ago, we reported the first discovery of the transfer of functional, nuclear genes between multicellular species (Pierce et al., 2007). The sacoglossan sea slug, Elysia chlorotica, eats the chromophytic alga, Vaucheria litorea. Certain cells that line the slug’s digestive diverticula are able to phagocytize undigested chloroplasts which are maintained intracellularly, and continue to be photosynthetically active for as long as the 10–11 month life cycle of the slug (West et al., 1984). Although there is no evidence of chloroplast division in the slug cytoplasm, synthesis of several chloroplast proteins occurs during the endosymbiotic association, including proteins that are encoded by algal nuclear genes (Pierce et al., 1996; Hanten and Pierce, 2001). Our original discovery found expressed genes for three of the algal light-harvesting complex proteins, LHCV-1, LHCV-2 and FCP, in genomic DNA of the slug (Pierce et al., 2007). In addition, we located the same genes in the genomic DNA of unhatched veliger larvae, which lack symbiotic chloroplasts and do not feed 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 transcription (Pierce et al., 2007).

DOI 10.1007/s13199-009-0044-8

122 S.K. PIERCE ET AL.

Furthermore, transfer of another algal nuclear gene between these species has recently been confirmed by others (Rumpho et al., 2008). All of the above results were produced using PCR-based experiments which were greatly impeded by the lack of slug and alga reference sequences in the public databases for both primer design and product identification, as well as low sequence conservation of many of the genes of interest and contamination from the copious amounts of mucus produced by the highly mucogenic slugs.

In order to facilitate the hunt for additional transferred genes, we have recently turned our efforts to the production and analysis of a transcriptome [expressed sequence tags (EST)] library database from V. litorea (Schwartz et al., submitted). Since EST’s are RNA-based, they are only a representation of the genes being expressed at the moment the RNA is extracted. However, they provide the exact coding sequence of genes of interest, which greatly facilitates not only identification, but also primer design and comparison with sequences in the slug. Using EST analysis, subsequently confirmed by PCR, we have already located several additional transferred genes in the slug genomic DNA (lhcv-3, lhcv-4, prk) (Schwartz et al., submitted), as well as four genes in the Chla synthesis pathway.

2. Materials and Methods

Animals Specimens of Elysia chlorotica were collected in a salt

marsh on Martha’s Vineyard, MA. They were shipped to Tampa, FL where they were kept in aquaria, without access to algae, containing sterilized, aerated, artificial sea water (Instant Ocean, 1,000 mosm/kg H2O) at 10oC under a 14/10 hr light/dark cycle using fluorescent tubes (cool white). The slugs were starved for at least 2 months before use in the experiments.

Algae

The Vaucheria litorea used in the experiments came

from a culture maintained in modified f/2 medium as described previously (Pierce et al., 1996). The original inoculum for this culture came from the same salt marsh that provided the slugs. At the time of the experiments the slugs and algae had not been in contact with each other for months and had undergone several biweekly changes of sterile media.

Veliger larvae

The aquaria containing the adult slugs were monitored

daily for egg masses. Generally, the masses were deposited

on the sides of the aquarium. When found, they were gently removed and placed immediately into small culture dishes containing sterile artificial sea water containing rifampicin, where the embryos were maintained until they had developed into veliger larvae (West et al., 1984), but had not hatched. At the point DNA was extracted (see below), the veliger larvae, which do not contain symbiotic plastids, had not fed and were still inside their egg capsules.

Genomic DNA purification

Genomic DNA was purified from pre-hatched

E. chlorotica veliger larvae and V. litorea using the Nucleon® genomic DNA extraction kit, PhytoPure® (Tepnel Life Sciences, Manchester, UK) following manufacturer’s instructions.

RNA isolation and mRNA purification

E. chlorotica: Total RNA was isolated from >2 month

starved slugs as follows. Slugs were homogenized in Trizol® Reagent (Invitrogen, Carlsbad, CA) and the homogenate was centrifuged at 12,000 × g at 4oC to pellet cellular debris. The supernatant was extracted with 1:6 (v/v) 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 NaCl solution and spun at 12,000 × g at 4oC. The RNA pellet was washed twice with 75% ethanol, air dried, resuspended in diethylpyrocarbonate (DEPC)-treated water and quantified spectrophotometrically (260 nm). mRNA was purified from total RNA using the Dynabeads® Oligo (dt)25 mRNA Purification Kit (Invitrogen) following manufacturer’s instructions and quantified spectrophoto-metrically (260 nm).

V. litorea: Total RNA was isolated from the alga using the Nucleon® genomic DNA extraction kit, PhytoPure® following the manufacturer’s instructions, taking advantage of the co-purification of RNA in that methodology. mRNA was purified as described above.

cDNA preparation

E. chlorotica and V. litorea 1st and 2nd strand cDNAs

were synthesized from purified mRNA using the Mint cDNA Synthesis Kit (Evrogen, Moscow, Russia) according to manufacturer’s instructions.

PCR and sequencing

Specific primers (Eurofins MWG/Operon, Huntsville,

AL) were designed from our EST sequences. Touchdown PCR reactions were done using 100 ng of genomic DNA or cDNA, 12.5 pmol of each primer, 0.25 mM dNTP mix (ID Labs, London, Ontario, Canada) and 1.25 units of

CHLOROPHYLL SYNTHESIS BY AN ANIMAL 123

IDProof™ DNA polymerase (ID Labs). Initial denaturation was done at 95oC for 2 min, followed by 20 cycles of denaturing at 95oC (30 s) and annealing (30 s) where the annealing temperature was reduced 1oC every other cycle, then a 72oC extension period. This was followed by an additional 20 cycles of denaturing at 95oC for 30 s, then 30 s at the lowest annealing temperature obtained in the touchdown and 72oC extension. Generally, the annealing temperatures started 5oC below the melting temperature of the primers. PCR products were separated on 1% agarose gels containing 0.2 µg/ml ethidium bromide and visualized by UV illumination. DNA bands were excised from agarose gels, purified using the QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA) and cloned using the TOPO® TA Cloning® Kit (Invitrogen) following manufacturer’s instructions. Clones were PCR amplified using M13 forward and reverse primers, purified and sequenced (Eurofins MWG/Operon) in forward and reverse directions. Sequences were analyzed using the tblastx algorithm searching the GenBank nr database and then aligned using the ClustalW2 sequence alignment program. Identified gene sequences used in this paper were uploaded to GenBank and the acquisition numbers are indicated below.

A critical concern in these experiments is to be sure that algal DNA is not somehow contaminating the slug extracts. Therefore, we always perform several controls in our experiments. All the PCR reagents were tested in the absence of template with negative results. Furthermore, we tested the extracted slug DNA for the presence of non-chloroplast targeted, V. litorea nuclear genes, either internal transcribed spacer region (ITS) (see Pierce et al., 2007) or spermidine synthase (SPDS), which was chosen from the V. litorea EST sequence data. Primers for both these genes always produced products with both V. litorea DNA and cDNA extracts using our PCR protocols, but NEVER with slug genomic DNA, cDNA or larval DNA. By now, we have performed our procedures on dozens of extracts from several batches of slugs at several times of year, in different lab rooms, in different buildings. In addition to these lab hygiene controls, the starved slugs had stopped producing digestive wastes for weeks before use in the experiments, so gut contents were not included in the experiments. Lastly, as mentioned above, the veliger larvae had not hatched from the egg masses, so they had not fed, do not have symbiotic chloroplasts and have never touched sea water.

Chlorophyll a production

At the end of a 8 hr dark period, slugs or algal filaments

were exposed to 15 µCi or 7.5 µCi (respectively) 14C-ALA (14C-4, 55 µCi/mmol, American Radiolabeled Chemical, St. Louis, MO) in their respective media for 2 hr in the dark in a constant temperature (25oC) agitator. At the end of the two hours, the slugs or filaments were placed under intense illumination (2–75 watt Halogen flood lamps) for 18 hrs,

still in the 14C-ALA containing media as above. Following the incubation, the slugs or filaments were removed from their media, washed in medium without isotope and Chla was extracted immediately. To test the effect of light on Chla synthesis, the experiment was done the same way except that the 18 hr incubation under lights was replaced with 18 hrs in the dark.

Chlorophyll a extraction

Chla was extracted by homogenizing the slugs and algal

filaments in cold, HPLC grade acetone (Pinckney et al., 1996). Samples were kept cold and exposure to light minimized throughout the extraction procedure. Samples were kept at -20oC in the dark until chromatography.

Chlorophyll a purification and scintillation counting

In order to determine the amount of 14C incorporation

into Chla during the incubation period, Chla was purified using HPLC and radioactivity determined. Chromato-graphy (System Gold, Beckman Coulter, Fullerton, CA) was done using two C18 columns (Microsorb 100-3, 100 × 4.6 mm, 3 mm, Varian, Lakeforest, CA and Vydac 201TP, 150 × 4.6 mm, 5 mm, Vydac, Hespira, CA) connected in series using a mobile phase starting with 80% MeOH: 20% NH4CH3COOH (0.5 M, pH 7.2) changing to 80% MeOH/20% acetone, according to the protocol described elsewhere (Pinckney et al., 1998). The second column was heated to 40oC. NH4CH3COOH was added to the samples as an ion pair and injections were kept to 50 µl in order to not overload the analytical-sized columns. All chemicals were HPLC grade. Detection was done at 438 nm. This protocol is designed to separate not only a wide array of photopigments, but also several Chla precursors and derivatives (Pinckney et al., 1998). The eluant was directed to a fraction collector set to collect at various intervals following the sample injection. In order to obtain a sufficient amount of material, several chromatographic runs of each extract were necessary. The corresponding fractions from each run were pooled, dried under a stream of N2, the residue dissolved in acetone, added to scintillation cocktail (Scintisafe 30%, Fisher Scientific, Fair Lawn, NJ) and the radioactivity determined with a scintillation counter (LS6000IC, Beckman Coulter, Fullerton, CA). Chla causes a large quenching effect (Nayar et al., 2003), so counting efficiency was determined by spiking samples with a known amount of cpm and correcting the results to dpm using the measured counting efficiency for each sample.

Confirmation of radioactivity in Chla

As well as TLC, we used the well-documented

(Llewellyn et al., 1990; Nayar et al., 2003) acid conversion of radiolabelled Chla to phaeophytin followed by HPLC

124 S.K. PIERCE ET AL.

separation and fraction collecting to demonstrate the molecular location of 14C. In separate experiments, slugs or algal filaments were labeled with 14C-ALA as described above. After the incubation period, Chla was extracted from the slugs or algae, the extract run in 50 µl aliquots through the HPLC protocol already described and the Chla peaks collected from the eluant and pooled, all as described above. Although the amount of Chla and the associated radioactivity in the extracts were reasonably robust, our analytical HPLC with its analytical-sized columns and 50 µl sample loop required the collection of several dozen Chla peaks. The pooled peaks were dried under nitrogen and dissolved in a small amount of acetone. HCl (3.6 M) was added and incubated for 5 min at room temperature, which removes the Mg2+ from Chla, thereby converting it to phaeophytin (Llewellyn et al., 1990; Nayar et al., 2003). The acid was then neutralized with 3.6 M NH4OH, the sample was spiked with non-radioactive Chla and rechromatographed on the HPLC. As before, several dozen 50 µl injections were done, the eluant in the region of the chromatogram where the Chla spike and the phaeophytin peak eluted were collected, pooled and the radioactivity in the pooled peaks determined by scintillation counting, corrected for background, quench and converted to dpm. 3. Results

The genes in the Chla synthesis pathway of V. litorea,

which we have identified in slug cDNA, and/or pre-hatched veliger larva genomic DNA (see Table 3, for example), are uroD (uroporphyrinogen decarboxylase), ChlD (ChlD subunit of magnesium chelatase), ChlH (ChlH subunit of magnesium chelatase) and ChlG (chlorophyll synthase) (Tables 1–4). UroD is a porphyrin synthesis enzyme common to both plants and animals, catalyzing the same reaction, albeit with different sequences and different reaction sites (chloroplast vs. cytoplasm, respectively) (Reith, 1995; Obornik and Green, 2005; Tanaka and Tanaka, 2007; Eberhard et al., 2008). We have found an uroD sequence in slug cDNA that matches a consensus uroD sequence in the V. litorea EST almost exactly (9 bases different in a 1026 base transcript) (Table 1). The other 3 enzymes (Chl-D, -H and -G) are in the unique pathway that leads to Chla synthesis from protoporphyrin IX, including the terminal reaction catalyzed by chlorophyll synthase. The sequences of these 3 genes in the slug cDNA match the sequence from the alga almost exactly (Tables 2–4).

Together, the expression of the genes for the Chla synthesis enzymes in the slug DNA, as well as the many-months long unabated photosynthesis by the endosymbiotic chloroplasts, a process that requires regular Chla synthesis in plants, suggest that Chla synthesis is occurring in the slug cell. We tested this possibility by incubating slugs

under lights in sea water containing 14C-labeled ALA and comparing the pattern of radiolabel distribution to that of similarly treated V. litorea filaments, using HPLC and fraction collection. In the algal filaments, 14C co-migrated with several peaks on the HPLC chromatogram, some colored and some not, including Chla (Fig. 1). Qualitatively, the chromatogram and distribution of radioactivity in the V. litorea extracts is very typical of those from measurements of Chla synthesis in organisms as phylogenetically diverse as phytoplankton (Riper et al., 1979) and barley (Wamsley and Adamson, 1994), for but two examples of many. Although we were unable to load comparable amounts of 14C-ALA into the slugs, undoubtedly due to external mucus, the HPLC chromato-gram and distribution of radioactivity in the corresponding fractions collected from the slug extracts were very similar to the results from the alga (Fig. 2). Due to the several hours-long incubation period in 14C-ALA, radioactivity appears in a variety of peaks along the chromatogram, some of which are probably Chla precursors and Chla derivatives (Pinckney et al., 1996) as well as a robust number of counts in the Chla peak fraction. The almost complete lack of radioactivity in the Chla peak when the experiment was 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-migration of radioactivity with the Chla band following thin layer chromatography (TLC) of the HPLC fraction (data not shown) all confirm that the radiolabel was a component of Chla rather than a co-elutant. We did not have authentic standard compounds to attempt to identify the other radioactive peaks on the chromatogram, but as with the alga, the elution times of some, compared with other studies (see above), suggests that they might be Chla precursors and derivatives, as well as chlorophyll c.

4. Discussion Altogether, the results clearly demonstrate that

E. chlorotica expresses several V. litorea nuclear genes that code for enzymes in the Chla synthesis pathway. In addition, Chla synthesis occurs in the slug cells and continues for months after the animal has been separated from its food alga. This not only expands on our earlier discovery of horizontally transferred genes between multicellular species (Pierce et al., 2007) but also is the first demonstration of the transfer of an entire biosynthetic pathway between eukaryotic species. We did not yet find genes for all the Chla synthesis pathway enzymes in our ongoing analysis of the 4 million bases in the V. litorea transcriptome library. Indeed, there is some possibility that those genes may not be in the transcriptome or may not have been transferred to E. chlorotica, indicating that continued long-term Chla synthesis in the slug results

CHLOROPHYLL SYNTHESIS BY AN ANIMAL 125

Table 1. Comparison of consensus nucleotide sequences of uroD (uroporphyrinogen 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 sequences 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 AAAGATCCATTGTTGTTAAGGGCAGCAAGAGGAGAGGCTGTGGAAAGGGTTCCTGTTTGG E. chlorotica cDNA AAAGATCCATTGTTGTTAAGGGCAGCAAGAGGAGAGGCTGTGGAAAGGGTTCCTGTTTGG .... 60

V. litorea cDNA ATGATGAGACAGGCAGGCAGACACATGCAAGAATACAGAGACCTTGTCAAAAAGTACCCC E. chlorotica cDNA ATGATGAGACAGGCAGGCAGACACATGCAAGAATACAGAGACCTCATCAAAAAGTACCCC .... 120

V. litorea cDNA ACATTCAGAGAGAGATCGGAGATCCACGAAGTGTCCACTGAAATTTCACTTCAGCCTTAC E. chlorotica cDNA ACATTCAGAGAGAGATCGGAGATCCACGAAGTGTCCACTGAAATTTCACTTCAGCCTTAC .... 180

V. litorea cDNA AGGCGATATGGAACCGATGGAGTGATCTTATTTTCTGATATTCTGACTCCACTGCCTGGA E. chlorotica cDNA AGGCGATACGGAACCGATGGAGTGATCTTATTTTCTGATATTCTGACTCCACTGCCTGGA .... 240

V. litorea cDNA ATGGGTGTCGATTTCAAAATTGAAGAGAAAACCGGGCCCAAATTGGTCCCAATGAGAACA E. chlorotica cDNA ATGGGTGTCGATTTCAAAATTGAAGAGAAAACCGGGCCCAAATTGGTCCCAATGAGAACG .... 300

V. litorea cDNA TGGGAAAGTGTCAATGCAATGCACACAATTGATTCTGAAAAGGCATGTCCTTTTGTGGGG E. chlorotica cDNA TGGGAAAGTGTCAATGCAATGCACACAATTGATTCTGAAAAGGCATGTCCTTTTGTGGGG .... 360

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

V. litorea cDNA GGATGTCCGTACACACTTGCCACTTACATGGTTGAAGGAGGTTCAAGCAGAGAATATTTG E. chlorotica cDNA GGATGTCCGTACACACTTGCCACTTACATGGTCGAAGGAGGTTCAAGCAGAGAATATTTG .... 480

V. litorea cDNA GAAATTAAAAAGATGATGTTCACTGAGCCTGAGTTGTTGCATGCCATGCTGGCCAAAATT E. chlorotica cDNA GAAATTAAAAAGATGATGTTCACTGAGCCTGAGTTGTTGCATGCCATGCTGGCCAAAATT .... 540

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

V. litorea cDNA GTCTTTGACTCTTGGGCAGGCCATCTCTCCCCCAAAGACTATGATGTTTTTGCGGCACCT E. chlorotica cDNA GTCTTTGACTCTTGGGCAGGCCATCTTTCCCCCAAAGACTATGATGTTTTTGCAGCACCT .... 660

V. litorea cDNA TACCAAAAGAAGGTTATCCAAAAAATCAAATCTTCTCATCCTGAAGTCCCCATCATCATT E. chlorotica cDNA TACCAAAAGAAGGTTATCCAAAAAATCAAATCTTCTCATCCTGAAGTCCCCATCATCATT .... 720

V. litorea cDNA TACATAAACAAGAGTGGTGCACTTTTGGAAAGGATGAGTCAAAGTGGGGCAGATATCATC E. chlorotica cDNA TACATAAACAAGAGTGGTGCACTTTTGGAAAGGATGAGTCAAAGTGGGGCAGATATCATC .... 780

V. litorea cDNA AGCTTGGATTGGACAGTGACGATTGAAGAGGCTAGGAAAAGAATCGGCAACGATATTGGC E. chlorotica cDNA AGCTTGGATTGGACAGTGACGATTGAAGAGGCTAGGAAAAGAATAGGCAACGATATTGGC .... 840

V. litorea cDNA ATCCAGGGTAACCTTGATCCAGCCGCCTTGTTTGCACCAAATGAAGTTATCAAGGAAAGG E. chlorotica cDNA ATCCAGGGTAACCTTGATCCAGCTGCCTTGTTTGCACCAAATGAAGTTATCAAGGAAAGG .... 900

V. litorea cDNA ACTGAAGAAATTTTGAGGGCATGCGGAGGGAGAAACCATGTCATGAATTTGGGCCATGGA E. chlorotica cDNA ACTGAAGAAATTTTGAGGGCATGCGGAGGGAGAAACCATGTCATGAATTTGGGCCATGGA .... 960

V. litorea cDNA ATCGAAGCGACGACTTCAGAAGAAAAGGCTGAATTTTTCATCAATACCGTAAAAAACTTC E. chlorotica cDNA ATCGAAGCGACGACTTCAGAAGAAAAGGCTGAATTTTTCATCAATACCGTAAAAAACTTC .... 1020

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

from some sort of long-term storage of massive amounts of enzyme in the symbiotic chloroplast. However, the presence and expression of Chl-D and –H in the slug, genes for the subunits of the initial enzyme in the Chla specific

pathway, as well as the terminal enzyme, Chl-G, indicate a good possibility that the genes for the intermediate steps have been transferred as well.

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. litorea transcriptome data. These gene fragments match exactly over the 975 bases. 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 AAAGAAATGAGCCTGAGCCGGAAAATCAGCCCGAAGATGACGAAGCCCCATCTGTACCCC E. chlorotica cDNA AAAGAAATGAGCCTGAGCCGGAAAATCAGCCCGAAGATGACGAAGCCCCATCTGTACCCC .... 60

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

V. litorea cDNA GGAAGAACAATGCCGGCAGGTCTGGGAAGAGGGGAATGATCTTTAACATGGAAAGAGGGC E. chlorotica cDNA GGAAGAACAATGCCGGCAGGTCTGGGAAGAGGGGAATGATCTTTAACATGGAAAGAGGGC .... 180

V. litorea cDNA GGAAGAACAATGCCGGCAGGTCTGGGAAGAGGGGAATGATCTTTAACATGGAAAGAGGGC E. chlorotica cDNA GATATATCAAGCCGATGCTTCCGAAAGGAAAAAAAGGGAAATTGGCGTTGGATGCGACGC .... 240

V. litorea cDNA TGAGATCAGCGGCGCCGTATCAATTGTCGAGAAGATCGCGCGCTCTCTCAAAGAACGACG E. chlorotica cDNA TGAGATCAGCGGCGCCGTATCAATTGTCGAGAAGATCGCGCGCTCTCTCAAAGAACGACG .... 300

V. litorea cDNA GGAATCCGACCAAAAGAACAGTCTTTGTCGAAAAGTCTGATCTGAGGGTCAAAAAGCTCG E. chlorotica cDNA GGAATCCGACCAAAAGAACAGTCTTTGTCGAAAAGTCTGATCTGAGGGTCAAAAAGCTCG .... 360

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

V. litorea cDNA ACCGAATGAACGCCGCGAAAGGCGCAGCAATGTCATTGCTGGGCGAAGCCTACAAAAGCA E. chlorotica cDNA ACCGAATGAACGCCGCGAAAGGCGCAGCAATGTCATTGCTGGGCGAAGCCTACAAAAGCA .... 480

V. litorea cDNA GGGACAAAGTGTGCCTCATACCTTTCCAGGGGGAAAGGGCTGAAGTCCTCCTCCCACCTT E. chlorotica cDNA GGGACAAAGTGTGCCTCATACCTTTCCAGGGGGAAAGGGCTGAAGTCCTCCTCCCACCTT .... 540

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

V. litorea cDNA CGCTCGCTCATGCAATCAACGTCGCTGTACGGACAGGGATTAACGCCATCAAATCACAGG E. chlorotica cDNA CGCTCGCTCATGCAATCAACGTCGCTGTACGGACAGGGATTAACGCCATCAAATCACAGG .... 660

V. litorea cDNA ACGTGGGAAAAGTGGTGATTGTGATGGTAAGCGATGGTCGAGCGAATGTGCCCCTCGCGG E. chlorotica cDNA ACGTGGGAAAAGTGGTGATTGTGATGGTAAGCGATGGTCGAGCGAATGTGCCCCTCGCGG .... 720

V. litorea cDNA TCAGTAACGGCACGCAGCTCCCTGAAGATGAGAAGATGTCCAGGGAGGAGTTGAAGGAGG E. chlorotica cDNA TCAGTAACGGCACGCAGCTCCCTGAAGATGAGAAGATGTCCAGGGAGGAGTTGAAGGAGG .... 780

V. litorea cDNA AGGTGTTGAACACTGCGAAGGCGCTGAGGGAGTTGCCGGCCTTTAGCTTGGTGGTGTTGG E. chlorotica cDNA AGGTGTTGAACACTGCGAAGGCGCTGAGGGAGTTGCCGGCCTTTAGCTTGGTGGTGTTGG .... 840

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

V. litorea cDNA GGAGATATCATTATATTCCAAAAGCAACGGATCAAGCGATGGCGAAGGTGGCCAGCGAAG E. chlorotica cDNA GGAGATATCATTATATTCCAAAAGCAACGGATCAAGCGATGGCGAAGGTGGCCAGCGAAG .... 960

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

Each generation of E. chlorotica must take up

chloroplasts from the algal food. However, once ensconced into the digestive cells, the symbiotic plastids encounter an array of algal genes transmitted from the parent slugs, which are sufficient to keep photosynthesis operating. During the life cycle, the slugs will feed and take up chloro-

plasts as long as V. litorea is available. However, under field or lab starvation conditions, photosynthesis continues for months at a level sufficient to sustain slug reproduction without the input of fresh plastids. The remarkable longevity of the chloroplast symbiosis in E. chlorotica 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 magnesium chelatase) in cDNA (GU068610) and genomic DNA (GU068611) from V. litorea and also cDNA from E. chlorotica adults (GU068612) and genomic DNA from pre-hatched E. chlorotica veliger larvae (GU068613). The sequences differ in 1 nucleotide between algae 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 AGGCTTTGTATGCCAGAACCAAACTCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA

V. litorea genomic DNA AGGCTTTGTATGCCAGAACCAAACTCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA

E. chlorotica cDNA AGGCTTTGTATGCCAGAACCAAACTCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA

E. chlorotica larval genomic DNA AGGCTTTGTATGCCAGAACCAAACTCTTGAACCCGAAGTTCTACGAGGGGATGTTGAACA .... 60

V. litorea cDNA GTGGGTACGAGGGCACAAGGGAGATCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG

V. litorea genomic DNA GTGGGTACGAGGGCACAAGGGAGATCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG

E. chlorotica cDNA GTGGGTACGAGGGCACAAGGGAGATCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG

E. chlorotica larval genomic DNA GTGGGTACGAGGGCACAAGGGAGATCACCAAAAGGCTGAGAAATACCATGGGATGGTCTG .... 120

V. litorea cDNA CCACTGCAGGGGAGGTGGACAACTTTATCTACGAAGATGCGAACGATGTTTTCATCAAAG

V. litorea genomic DNA CCACTGCAGGGGAGGTGGACAACTTTATCTACGAAGATGCGAACGATGTTTTCATCAAAG

E. chlorotica cDNA CCACTGCAGGGGAGGTGGACAACTTTATCTACGAAGATGCGAACGATGTGTTCATCAAAG

E. chlorotica larval genomic DNA CCACTGCAGGGGAGGTGGACAACTTTATCTACGAAGATGCGAACGATGTGTTCATCAAAG .... 180

V. litorea cDNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA

V. litorea genomic DNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA

E. chlorotica cDNA ATGAAGCCATGAGGGAGAGACTGCTCAATACCAATCCGAACGCCTTCCGCGACATGATCA

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

V. litorea cDNA CCACTTTTCTGGAGGCCAATGGAAGGGGCTACTGGGACACCTCGGATGATAATATAGAAC

V. litorea genomic DNA CCACTTTTCTGGAGGCCAATGGAAGGGGCTACTGGGACACCTCGGATGATAATATAGAAC

E. chlorotica cDNA CCACTTTTCTGGAGGCCAATGGAAGGGGCTACTGGGACACCTCGGATGATAATATAGAAC

E. chlorotica larval genomic DNA CCACTTTTCTGGAGGCCAATGGAAGGGGCTACTGGGACACCTCGGATGATAATATAGAAC .... 300

V. litorea cDNA TGTTGCAGGATCTGTACCAAGAGGTGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT

V. litorea genomic DNA TGTTGCAGGATCTGTACCAAGAGGTGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT

E. chlorotica cDNA TGTTGCAGGATCTGTACCAAGAGGTGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT

E. chlorotica larval genomic DNA TGTTGCAGGATCTGTACCAAGAGGTGGAAGATAAAATCGAGGGAGTTTGAGGAAAAT ...... 357

than we have uncovered so far-perhaps even pieces of, or even entire, algal chromosomes are involved. Nevertheless, these results clearly show that the successful transfer of functional nuclear genes between multicellular species not only can occur, but also can involve many genes which are expressed producing alien proteins that reach cellular targets and are capable of function.

These results seem important at several levels. First, the phenomenon of chloroplast symbiosis has been of interest for almost half a century. Although many species of elysiid sea slugs are capable of intracellular sequestration of chloroplasts, some protists do it as well (Gast et al., 2007; Johnson et al., 2007). The division of the symbiotic chloroplasts has not been seen so far in any kleptoplastic species, including E. chlorotica. Also, while E. chlorotica may hold the longevity record for plastid maintenance, the symbiotic organelles in other species persist from but a few days [for example, Elysiella pusilla (Evertson et al., 2007)] to several months [E. clarki (Pierce et al., 2006)] and involve a variety of algal taxa, often, unlike E. chlorotica, with more than one species of alga per species of sea slug (Curtis et al., 2006). While the mechanism of sequestration

is likely similar amongst the slug species (but see below), the differences in chloroplast source and longevity suggest that the transferred gene array is different between slug species. This specificity of transferred gene arrays within sea slug species suggests, in turn, that gene movements have occurred many times across species and in different amounts. Second, on a broader scale, our results here and previously (Pierce et al, 2007) clearly show that completely unrelated organisms can transfer genes between them, integrate the transfers into the host genome and, not only express the genes, but also successfully use the gene products. Thus, at least some multicellular species do not have to wait for a mutation to occur for an evolutionary change to take place. Much as in prokaryotes and protists, mechanism(s) for a successful swap of DNA between even distantly related species is both present and active in metazoans.

Clearly, genome sequencing is necessary to determine the entire scope of algal genes in the genome of E. chlorotica. In addition to the pathway we have found, to work efficiently, the chlorophyll biosynthesis requires retrograde signaling by the chloroplasts to help regulate

128 S.K. PIERCE ET AL.

Table 4. Comparison of consensus sequences of the chlorophyll synthase gene (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 sequences 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 TCACACCTGGAATCCATTCGCAGGGCCAGATGCAGTTGATTTACAAGATGCTGGGATTGA E. chlorotica cDNA TCACACCTGGAATCCATTCGCAGGGCCAGATGCAGTTGATTTACAAGATGCTGGGATTGA .... 60

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

V. litorea cDNA AACCATCAACGATTGGTATGACCGAGATATTGATGCGATCAATGAGCCATATCGACCCAT E. chlorotica cDNA AACCATCAACGATTGGTATGACCGAGATATTGATGCGATCAATGAGCCATATCGACCCAT .... 180

V. litorea cDNA TCCTTCTGGAGCTATTTCTGAGGGTCAAGTGAAAGCGCAAATTGCCTTTCTTCTAGTTGG E. chlorotica cDNA TCCTTCTGGAGCTATTTCTGAGGGTCAAGTGAAAGCGCAAATTGCCTTTCTTCTAGTTGG .... 240

V. litorea cDNA TGGATTGGCTTTGTCGTATGGTTTGGATCTATGGGCAGGGCACCAAATGCCCACTGTTTT E. chlorotica cDNA TGGATTGGCTTTGTCGTATGGTTTGGATCTATGGGCAGGGCACCAAATGCCCACTGTTTT .... 300

V. litorea cDNA TTTGTTGTCATTGTTTGGGACTTTCATTTCATACATATACTCAGCCCCGCCACTGAAATT E. chlorotica cDNA TTTGTTGTCATTGTTTGGGACTTTCATTTCATACATATACTCAGCCCCGCCACTGAAATT .... 360

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

V. litorea cDNA GTGGTGTGGTCAGGCTATGTTTGGTGAGCTCAACTTGCAAGTTGTGGTCCTAACTTTGCT E. chlorotica cDNA GTGGTGTGGTCAGGCTATGTTTGGTGAGCTCAACTTGCAAGTTGTGGTCCTAACTTTGCT .... 480

V. litorea cDNA GTATTCTTGGGCAGGCCTTGGAATTGCAATAGTAAATGACTTCAAATCAGTTGAGGGGGA E. chlorotica cDNA GTATTCTTGGGCAGGCCTTGGAATTGCAATAGTAAATGACTTCAAATCAGTTGAGGGGGA .... 540

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

V. litorea cDNA ATGTGTGAGTAGCATTGACATTACTCAATTGGGCATAGCCGCATGGCTATATTATATTGG E. chlorotica cDNA ATGTGTGAGTAGCATTGACATTACTCAATTGGGCATAGCCGCATGGCTATATTATATTGG .... 660

V. litorea cDNA AGAGCCTACCTATGCATTCGTTTTATTGGGCCTCATTCTTCCTCAGATATATGCACAATT E. chlorotica cDNA AGAGCCTACCTATGCATTCGTTTTATTGGGCCTCATTCTTCCTCAGATATATGCACAATT .... 720

V. litorea cDNA TAAGTATTTTTTGCCGGATCCAGTTGAGAATGATGTCAAATACCAAGGATTTGCTCAGCC E. chlorotica cDNA TAAGTATTTTTTGCCGGATCCAGTTGAGAATGATGTCAAATACCAAGGATTTGCTCAGCC .... 780

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

nuclear gene expression (Green et al., 2000). It is not yet clear how refined this control system is in the symbiotic plastids in the E. chlorotica digestive cells, but algal nuclear genes that code for the proteins that signal between the chloroplast and nucleus as well as proteins involved in targeting and trafficking are obvious candidates. This especially long-lived chloroplast symbiosis in E. chlorotica presents a unique opportunity to study the evolution of intracellular organelles as it is occurring.

Finally, from both theoretical and applied perspectives understanding the gene transfer mechanism may be of considerable significance. The uptake of the chloroplasts has only been examined in a few instances. However, the ancient literature makes clear that digestion in herbivorous

gastropods is largely accomplished using intracellular vacuoles (Owen, 1966). During ingestion, food material is mechanically broken down into small pieces or sucked up, in the case of the sacoglossan. It then passes into the digestive tubules where the epithelial cells phagocytize the pieces into lysosomal vacuoles where digestion proceeds. The chloroplasts are engulfed from the lumen of the tubules by either the same phagocytic mechanism, or an analogous process, according to the few studies that have examined it (McLean, 1976; Mondy and Pierce, 2003). Although some other reports have incorrectly stated that the symbiotic chloroplasts are naked in the cytoplasm of the digestive tubule cell (Graves et al., 1979; Rumpho et al., 2001) 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 radioactivity (14C) in fractions collected from the HPLC column eluant also as described in the methods. The large peak in radioactivity at approximately 43.5 min coelutes exactly with the Chla peak in the upper chart. Although we did not identify them, the smaller radioactive peaks just preceding the Chla location are most likely intermediates in the Chla synthesis pathway (see Pinckney et al., 1996). The large peak of radioactivity starting at about 4 min, which was not detected on the HPLC chromatogram, is right at the column void volume. membrane (Mondy and Pierce, 2003; Curtis et al., 2006). 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 endogenous retroviruses could be the transfer agent, at least in E. chlorotica (Pierce et al., 1999; Mondy and Pierce, 2003) although the increasing number of transferred genes being found in this species may make viral transfer a less attractive hypothesis.

Figure. 2. Typical HPLC chromatogram (upper chart) of Chla extracted from E. chlorotica and separated by the same protocol that produced the V. litorea results in Fig. 1. The peak at approximately 43.5 min corresponds to the elution time of both standard Chla (Sigma Chemicals) as well as the Chla peak in the V. litorea chromatogram. The lower chart represents the (14C) radioactivity profile in fractions of eluant collected during the HPLC run. The large peak of radioactivity at approximately 43.5 min corresponds exactly with the elution of the Chla peak. The 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 13–15 min is in the region where chlorophyll c and fucoxanthin elute in this HPLC protocol (Llewellyn et al., 1990).

Acknowledgements

We gratefully acknowledge the generous financial

support of a private donor, who wishes to remain anonymous. The work reported here could not have been done without that donor’s help.

130 S.K. PIERCE ET AL.

Figure 3. Typical HPLC chromatogram of Chla and associated radioactivity following incubation of slugs with 14C ALA in the dark and extraction as described in the methods. As in the other figures, Chla is the peak at 45 min labeled “chlorophyll a”. The histogram inset displays the small amount of radioactivity that was incorporated in Chla by slugs and algal filaments during an 18 hr incubation in the dark, indicating that almost no Chla synthesis occurs in either the algal filaments or the symbiotic chloroplasts without the presence of light (compare to Figs. 1 and 2).

Figure 4. Typical chromatogram showing the results of conversion of radioactive chlorophyll purified from E. chlorotica to phaeo-phytin by acid treatment as described in the methods. Initially, radioactive Chla was collected as usual from the HPLC. Neither peak nor radioactivity was recovered in the region where phaeophytin elutes (55.8 min). The collected Chla was acid treated as described, the extract was spiked with non-radioactive Chla to mark its elution point (arrow) and rechromatographed. As shown here, a new radioactive peak (inset) has appeared which co-elutes with phaeophytin (Llewellyn et al., 1990) (arrow) and is well separated from the Chla spike.

REFERENCES Curtis, N.E., Massey, S.E., and Pierce, S.K. 2006. The symbiotic

chloroplasts in the sacoglossan Elysia clarki are from several algal species. Journal of Invertebrate Biology 125: 336–345.

Eberhard, S., Finazzi, G., and Wollman, F.-A. 2008. The dynamics of photosynthesis. Annual Review of Genetics 42: 463–515.

Evertsen, J., Burghardt, I., Johnsen, G., and Wagele, H. 2007. Retention of functional chloroplasts in some sacoglossan from the Indo-Pacific and Mediterranean. Marine Biology 151: 2159–2166.

Gast, R.J., Moran, D.M., Dennett, M.R., and Caron, D.A. 2007. Kleptoplasty in an Antarctic dinoflagellate: caught in evolutionary transition? Environmental Microbiology 9: 39–45.

Graves, D.A., Gibson, M.A., and Bleakney, J.S. 1979. The digestive diverticula of Alderia modesta and Elysia chlorotica (Opisthobranchia : Sacoglossa). Veliger 21: 415–422.

Green, B.J., Li, W.-Y., Manhart, J.R., Fox, T.C., Summer, E.J., Kennedy, R.A., Pierce, S.K., and Rumpho, M.E. 2000. Mollusc-algal chloroplast endosymbiosis, photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus. Plant Physiology 124: 331–342.

Hanten, J.J. and Pierce, S.K. 2001. Synthesis of several light-harvesting complex I polypeptides is blocked by cycloheximide in symbiotic chloroplasts in the sea slug, Elysia chlorotica (Gould): A case for horizontal gene transfer between alga and animal? Biological Bulletin 201: 33–44.

Johnson, M.D., Oldach, D., Delwiche, C.F., and Stoecker, D.K. 2007. Retention of transcriptionally active cryptophyte nuclei by the ciliate Myrionecta rubra. Nature 445: 426–428.

Llewellyn, C.A, Mantoura, R.F.C., and Brereton, R.G. 1990. Products of chlorophyll photodegradation -1 Detection and separation. Photochemistry and Photobiology 52: 1037–1041.

McLean, N. 1976. Phagocytosis of chloroplasts in Placida dendritica (Gastropoda: Sacoglossa). Journal of Experimental Zoology 197: 321–330.

Mondy, W.L. and Pierce, S.K. 2003. Apoptotic-like morphology is associated with the annual synchronized death of a population of kleptoplastic sea slugs (Elysia chlorotica). Journal of Invertebrate Biology 122: 126–137.

Nayar, S., Goh, B.P.L., and Chou, L.M. 2003. Interference of chlorophyll a in liquid scintillation counting of phytoplankton productivity samples by the 14C technique. Estuarine and Coastal Shelf Science 56: 957–960.

Obornik, M. and Green, B.R. 2005. Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes. Molecular Biology and Evolution 22: 2343–2353.

Owen, G. 1966. Digestion. In: Physiology of Mollusca II. Wilbur, K.M. and Yonge, C.M., eds. Academic Press, NY, pp. 53–96.

Pierce, S.K., Biron, R.W., and Rumpho, M.E. 1996. Endosymbiotic chloroplasts in molluscan cells contain proteins synthesized after plastid capture. Journal of Experimental Biology 199: 2323–2330.

Pierce, S.K., Curtis, N.E., Hanten, J.J., Boerner, S.L., and Schwartz, J.A. 2007. Transfer, integration and expression of functional nuclear genes between multicellular species. Symbiosis 43: 57–64.

Pierce, S.K., Curtis, N.E., Massey, S.E., Bass, A.L., Karl, S.A., and Finney, C. 2006. A morphological and molecular comparison between Elysia crispata and a new species of kleptoplastic sacoglossan sea slug (Gastropoda: Opisthobranchia) from the Florida Keys USA. Molluscan Research 26: 23–38.

Pierce, S.K., Maugel, T.K., Rumpho, M.E., Hanten, J.J., and Mondy, W.L. 1999. Annual viral expression in a sea slug population: Life cycle control and symbiotic chloroplast maintenance. Biological Bulletin 197: 1–6.

CHLOROPHYLL SYNTHESIS BY AN ANIMAL 131

Pinckney, J.L., Millie, D.F., Howe, K.E., Paerl, H.W., and Hurley, J.P. 1996. Flow scintillation counting of 14C-labeled microalgal photosynthetic pigments. Journal of Plankton Research 18: 1867–1880.

Pinckney, J.L., Paerl, H.W., Harrington, M.B., and Howe, K.E. 1998. Annual cycles of phytoplankton community-structure and bloom dynamics in the Neuse River Estuary, North Carolina. Marine Biology 131: 371–381.

Reith, M. 1995. Molecular biology of rhodophyte and chromophyte plastids. Annual Review of Plant Physiology and Molecular Biology 46: 549–575.

Riper, D.M., Owens, T.G., and Falkowski, P.G. 1979. Chlorophyll turnover in Skeletonema costatum, a marine plankton diatom. Plant Physiology 64: 49–54.

Rumpho, M.E., Summer, E.J., Green, B.J., Fox, T.C., and Manhart, J.R. 2001. Mollusc/algal chloroplast symbiosis: How can isolated chloroplasts continue to function for months in the cytosol of a sea slug in the absence of an algal nucleus? Zoology 104: 303–312.

Rumpho, M.E., Worful, J.M., Lee, J., Kannan, K., Tyler, M.S., Bhattacharya, D., Moustafa, A., and Manhart, J.R. 2008. Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proceedings of the National Academy of Science, USA 105: 17867–17871.

Schwartz, J.A., Curtis, N.E., and Pierce, S.K. Transcriptome analysis reveals several horizontally transferred algal nuclear genes in the genome of the sea slug Elysia chlorotica. Journal of Molecular Evolution (submitted).

Tanaka, R. and Tanaka, A. 2007. Tetrapyrrole biosynthesis in higher plants. Annual Review of Plant Biology 58: 321–346.

Wamsley, J. and Adamson, H. 1994. Chlorophyll turnover in etiolated greening barley transferred to darkness: Isotopic (1-14C glutamic acid) evidence of dark chlorophyll synthesis in the absence of chlorophyll accumulation. Physiologia Plantarum 93: 435–444.

West, H.H., Harrigan, J., and Pierce, S.K. 1984. Hybridization of two populations of a marine opisthobranch with different developmental patterns. Veliger 26: 199–206.