LETTER LETTER rubrum:Thesymbiosisthatwasn’t

Matthew D. Johnsona,1, Erica Lasek-Nesselquistb, Holly V. Moellerc, Andreas Altenburgerd, Nina Lundholmd, Miran Kime, Kirstine Drumme, Øjvind Moestrupe, and Per Juel Hansene

Qiu et al. (1) report that a red tide of the photosynthetic genes in an Antarctic M. rubrum culture (clade A) (8). In Mesodinium rubrum in Long Island Sound “farms” addition, because genomes or transcriptomes of the symbiotic Teleaulax amphioxeia cells within its cyto- target organisms were not used for annotation (1) there plasm. M. rubrum has long been studied for causing is a high degree of uncertainty in assigning transcript red tides (2–5), and laboratory culture work on multiple identity. strains from around the world has shown that M. rubrum Second, Qiu et al. (1) report intact cryptophyte cells extracts from ingested cryptophyte algae, in- inside M. rubrum. However, their TEM images are incon- cluding chloroplasts, mitochondria, cytoplasm, and a clusive due to (i) low resolution (ii), extraordinarily poor transcriptionally active nucleus, or kleptokaryon (6, 7). fixation quality, and (iii) unusually small cryptophyte or- M. rubrum functions like a true phototroph, with the ganelles, compounding the interpretation of the low-res- ability to regulate and divide chloroplasts (7). olution images. No clear cell membrane, which would The conclusions of Qiu et al. (1), based on a single include cytoplasm completely surrounding the chloro- field sample, contrast sharply with these previously plast, is visible around the cryptophyte organelles in their published studies of M. rubrum. Their conclusions images. Rather, they seem to be complexes are based on (i) their inference that “complete” prey that are packed into a membrane, consistent with pre- metatranscriptomes indicate metabolically intact prey vious observations (9). Furthermore, in other M. rubrum cells and (ii) their visual observation, using transmis- recently ingested intact cryptophytes seem to be in a sion electron microscopy (TEM), of intact prey cells. vacuole before organelle extraction (Fig. 1) (10). However, we believe that these findings do not pro- M. rubrum-like are complex organisms that vide sufficient evidence to support the extraordinary do not fit into established “boxes” for trophic modes claim by Qiu et al. (1) that M. rubrum farms prey cells. or cellular organization. However, we have previously First, the authors argue that the expression of shown that their unique mode of acquired phototro- genes involved in membrane transporters, nucleus- phy is capable of “farming” cryptophyte organelles to-cytoplasm RNA transporters, and all major meta- when the kleptokaryon is present (7), and there is no bolic pathways is evidence of intact cryptophyte evidence for maintenance of intact symbionts within symbionts. Here we show data from a temperate strain any cultures of the ciliate. We firmly believe that the of M. rubrum (clade G) that indicate that many crypto- conclusions of Qiu et al. (1) do not represent a new phyte gene pathways are expressed at levels equal to association in M. rubrum but rather illustrate the actual or greater than in T. amphioxeia even when only prey difficulties of accurately interpreting “snapshots” of organelles remain (Table 1 and Fig. 1). One exception natural populations. is expression levels of ABC-like transporters, which were observed to be at even lower numbers in Qui Acknowledgments et al. (1). Furthermore, we have previously shown similar M.D.J. and E.L.-N. were supported by National Science Founda- transcriptional patterns of highly expressed cryptophyte tion Integrative and Organismal Systems Award 1354773.

aBiology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543; bWadsworth Center, New York State Department of Health, Albany, NY 12208; cDepartment of Ecology, Evolution & Marine Biology, University of California, Santa Barbara, CA 93106; dNatural History Museum of Denmark, University of Copenhagen, 1350 Copenhagen, Denmark; and eMarine Biological Section, Department of Biology, University of Copenhagen, 2200 Copenhagen, Denmark Author contributions: M.D.J. designed research; M.D.J. and E.L.-N. performed research; M.D.J. and E.L.-N. analyzed data; and M.D.J., E.L.-N., H.V.M., A.A., N.L., M.K., K.D., Ø.M., and P.J.H. wrote the paper. The authors declare no conflict of interest. 1To whom correspondence should be addressed. Email: [email protected].

E1040–E1042 | PNAS | February 14, 2017 | vol. 114 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1619247114 Downloaded by guest on September 24, 2021 Fig. 1. Transmission electron micrograph of Mesodinium rubrum (CBJR05; clade G) fed T. amphioxeia (GCEP01). The image shows a lateral cross- section of an M. rubrum cell revealing at least nine complexes and a recently ingested T. amphioxeia cell (also a cross-section) within a vacuole (white arrow) in the center. Note the ingested cell’s periplast membrane and cytoplasm surrounding the chloroplast, and the vacuolar space surrounding it. In this image the cytoplasm of the cryptophyte organelle complexes is lighter than that of the ciliate, revealing that large portions of M. rubrum cells are devoted to hosting stolen organelles. Ciliate cytoplasm and many of the organelles in the image of Qiu et al. (1) are missing or unrecognizable, respectively.

Johnson et al. PNAS | February 14, 2017 | vol. 114 | no. 7 | E1041 Downloaded by guest on September 24, 2021 Table 1. Comparison of key metabolic pathways (reads per kilobase of transcript per million mapped reads) in the T. amphioxeia-derived kleptokaryon of M. rubrum (KN) and free-living T. amphioxeia (TA) KEGG gene orthology groups* Metabolic pathway KN TA KN/TA

Metabolism ko01100 Metabolic pathways 223,716 169,969 1.32 ko00195 64,880 53,849 1.20 ko00196 Photosynthesis-antenna proteins 34,946 4,731 7.39 ko00860 Porphyrin and chlorophyll metabolism 10,007 13,685 0.73 Transport and protein processing ko04141 Protein processing in 59,957 62,823 0.95 ko04130 SNARE interactions in vesicular transport 1,670 1,451 1.15 ko02010 ABC transporters 699 3,686 0.19 DNA and RNA pathways ko03010 Ribosome 288,272 714,136 0.40 ko03040 Spliceosome 29,099 27,447 1.06 ko03013 RNA transport 29,037 18,314 1.59 ko03030 DNA replication 10,621 1,583 6.71 ko03015 mRNA surveillance pathway 10,253 6,909 1.48 ko03020 RNA polymerase 10,124 7,932 1.28 Cell cycle and cytoskeleton ko04110 Cell cycle 17,241 5,403 3.19 ko04810 Regulation of actin cytoskeleton 5,460 4,578 1.19 Signaling ko04010 MAPK signaling pathway 19,891 18,091 1.10 ko04020 Calcium signaling pathway 17,312 8,949 1.93

*Kyoto Encyclopedia of Genes and Genomes.

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E1042 | www.pnas.org/cgi/doi/10.1073/pnas.1619247114 Johnson et al. Downloaded by guest on September 24, 2021