Ubiquity and quantitative significance ofdetoxification catabolism of chlorophyllassociated with protistan herbivoryYuichiro Kashiyamaa,1,2, Akiko Yokoyamab,1, Yusuke Kinoshitaa, Sunao Shojia, Hideaki Miyashiyac, Takashi Shiratorid,Hisami Sugae, Kanako Ishikawaf, Akira Ishikawag, Isao Inouyeb, Ken-ichiro Ishidab, Daiki Fujinumah, Keisuke Aokih,Masami Kobayashih, Shinya Nomotoi, Tadashi Mizoguchia, and Hitoshi Tamiakia,2

aGraduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; bFaculty of Life and Environmental Sciences and dGraduate Schoolof Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; cGraduate School of Human and Environmental Studies, KyotoUniversity, Kyoto 606-8501, Japan; eInstitute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa 237-0061,Japan; fSystem Analysis Division, Lake Biwa Environmental Research Institute, Otsu, Shiga 520-0022, Japan; gGraduate School of Bioresources, Mie University,Tsu, Mie 514-8507, Japan; hInstitute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan; and iDepartment of Chemistry, Universityof Tsukuba, Tsukuba, Ibaraki 305-8571, Japan

This Feature Article is part of a series identified by the Editorial Board as reporting findings of exceptional significance.

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved August 2, 2012 (received for review May 2, 2012)

Chlorophylls are essential components of the photosynthetic appa-rati that sustain all of the life forms that ultimately depend on solarenergy. However, a drawback of the extraordinary photosensitizingefficiency of certain chlorophyll species is their ability to generateharmful singlet oxygen. Recent studies have clarified the catabolicprocesses involved in the detoxification of chlorophylls in landplants, but little is understood about these strategies in aquaticecosystem. Here, we report that a variety of heterotrophic protistsaccumulate the chlorophyll a catabolite 132,173-cyclopheophorbidea enol (cPPB-aE) after their ingestion of algae. This chlorophyll de-rivative is nonfluorescent in solution, and its inability to generatesinglet oxygen in vitro qualifies it as a detoxified catabolite of chlo-rophyll a. Using a modified analytical method, we show that cPPB-aE is ubiquitous in aquatic environments, and it is often the majorchlorophyll a derivative. Our findings suggest that cPPB-aE metab-olism is one of the most important, widely distributed processes inaquatic ecosystems. Therefore, the herbivorous protists that con-vert chlorophyll a to cPPB-aE are suggested to play more significantroles in the modern oceanic carbon flux than was previously recog-nized, critically linking microscopic primary producers to the mac-roscopic food web and carbon sequestration in the ocean.

phototoxicity of chlorophylls | microbial herbivory | phagocytosis |biodiversity of eukaryotes | microbial loop

Chlorophylls are crucial to sustaining most life forms on Earth,the majority of which ultimately depend on solar energy.Photoexcitation of chlorophylls initiates various photosyntheticreactions that convert the energy of photons into chemical poten-tials, which in turn, drive the full range of metabolic reactionsthroughout the global ecosystem. Chlorophylls play a central rolein the photosynthetic apparatus by absorbing light and trans-ferring the excitation energy to the reaction centers of photo-systems before photosynthetic electron transport. However,without measures to contain the excited energy, chlorophylls canharm organisms because of their high photosensitizing potential.Photoexcited chlorophylls generate singlet oxygen, a highly re-active oxygen species that can cause severe cellular damage (1).Therefore, the phototoxicity of chlorophylls has been a continu-ous concern for the Earth’s ecosystem since the global rise inatmospheric oxygen about 2.3 billion y ago (2).Given its potential risks, chlorophyll metabolism is thought to

be carefully controlled in the cells of phototrophic organisms.Recent works have revealed that the biodegradation of chloro-phyll a (Chl-a) (Fig. 1) in land plants (embryophytes) is regulatedas carefully as its biosynthesis (3). The chlorin moiety found inChl-a is a highly π-conjugated tetrapyrrolic macrocycle that acts

as a potentially phototoxic chromophore or fluorophore. It is con-verted stepwise into an unconjugated and hence, colorless andnonfluorescent linear tetrapyrrole (SI Text, section 1.1). However,many algae and cyanobacteria apparently lack the programmeddetoxifying catabolism of chlorophyll observed in embryophytes (SIText, section 1.2). The inability of unicellular phototrophs to detoxifychlorophyll can be rationalized by their lack of the need to remo-bilize nutrients before death, unlike multicellular land plants. Re-garding heterotrophs, the digestive systems of most terrestrialherbivores are dark, whereas the digestive systems of most aquaticherbivores, such as multicellular and unicellular zooplankton, aresmall and translucent. This finding renders the microscopic aquaticherbivores susceptible to damage by the singlet oxygen generatedwhen ingested chlorophylls are exposed to light (SI Text, section 1.3).Thus, strategies to protect against the accumulation of phototoxicchlorophyll derivatives should be critical for the survival of the mi-croscopic aquatic herbivores feeding under illumination.We conducted feeding experiments on several microorganisms

to screen for potentially detoxified chlorophyll catabolites. Here,we report that 132,173-cyclopheophorbide a enol (cPPB-aE) (Fig.1) is the dominant product derived from Chl-a that accumulatesin cells of various aquatic heterotrophic protists (i.e., unicellulareukaryotes) that feed on algae and that cPPB-aE is a virtuallynonfluorescent and nonphotosensitizing chlorophyll derivativeincapable of generating singlet oxygen. These results stronglysuggest that herbivorous protists generate cPPB-aE as a detoxi-fied catabolite of Chl-a. We also show that cPPB-aE is ubiqui-tous in all of the aquatic environments that we tested, frequentlyas the most abundant Chl-a derivative in the surface sediments,and that it is actively generated in the water near illuminatedsurfaces.

Author contributions: Y. Kashiyama, A.Y., H.M., H.S., K.I., A.I., I.I., K.-i.I., M.K., S.N., T.M., andH.T. designed research; Y. Kashiyama, A.Y., Y. Kinoshita, S.S., H.M., T.S., D.F., K.A., and S.N.performed research;Y. KashiyamaandA.Y. analyzeddata; andY. Kashiyamawrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

See Commentary on page 17311.1Y. Kashiyama and A.Y. contributed equally to this work.2To whom correspondence may be addressed. E-mail: chiro@fc.ritsumei.ac.jp or tamiaki@fc.ritsumei.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1207347109/-/DCSupplemental.

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Results and DiscussionFeeding Experiments. We showed that three genetically distantheterotrophic protists—a stramenopile, a cercozoan (Filosa),and a heliozoan (Centrohelida)—accumulated cPPB-aE as theonly major chlorophyll derivative associated with herbivory. Weestimated that levels of cPPB-aE in extracts from these protistsafter they were fed on fresh unialgal cells are as high as 80 mol%of total amounts of chlorins derived from Chl-a (Fig. 2). Howe-ver, a heterotrophic discicristoidean did not accumulate any cPPB-aE. No trace of cPPB-aE was detected in control experiments,where the unialgal cultures fed to the protists were incubatedunder conditions identical to those conditions used for thefeeding experiments (Table S1). Organic synthesis of cPPB-aErequires a strong base to form the C–C bond between 132 and173 carbons (4, 5), suggesting that it is difficult to generate cPPB-aE in vitro under normal conditions. In fact, the generation ofcPPB-aE has not been reported in so-called dark incubationexperiments involving pure unialgal cultures sealed and pre-served in darkness for months to years (6–8). Therefore, cPPB-aE detected in the present experiments must be conclusivelyattributed to metabolic processes in the heterotrophic protists.Minor products of Chl-a degradation are pyropheophytin a

(pPhe-a) (Fig. 1) as well as various polar chlorins, including (132R)-and (132S)-hydroxychlorophyllones a [(R/S)-hCPLs-a] (Fig. 1). Darkincubation experiments of unialgal cultures revealed substantialaccumulation of pPhe-a only after the incubations had proceededfor several months (6–8). Given that pPhe-a is absent from ourcontrol experiments involving unialgal cultures, its accumulation canalso be attributed to the heterotrophic process. However, (R/S)-

hCPLs-amay be a product of biotic processing (9, 10) and/or abioticoxidation of cPPB-aE (11). All samples of heterotrophic protistsalso contain variable amounts of intact Chl-a, which may have beenderived from undigested algal materials in phagosomes.Our experiments, thus, suggest qualitatively that the herbivo-

rous protists studied actively modify Chl-a into cPPB-aE duringtheir course of phagotrophic digestion (Fig. 3). Given that het-erotrophic and phototrophic cells grew together in the culturesused in our experiments, quantitative estimations of the rate ofcPPB-aE production from Chl-a as well as the stability of cPPB-aE in vivo were beyond the scope of the present work. None-theless, it seems reasonable to conclude that cPPB-aE is a majorChl-a catabolite produced by the herbivorous protists that westudied. Therefore, cPPB-aE could potentially emerge as a bio-chemical marker of protistan herbivory in aquatic environments.

Photochemical Significance. We determined various properties ofcPPB-aE by analyzing an authentic sample that was semisynthesizedfrom Chl-a (Materials and Methods). The most striking propertyof cPPB-aE is that it is essentially nonfluorescent (4), despite itshighly π-conjugated structure (Fig. 4 A and B, SI Text, section 1.5,Fig. S1, and Table S2). This finding is consistent with our

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Fig. 1. Chemical structures of Chl-a and its derivatives discussed in thepresent work.

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Fig. 2. Protistan herbivory and associated chlorophyll catabolism/degrada-tion. Relative abundances of chlorophyll derivatives in extracts of culturedheterotrophic protists. Four experiments involved feeding a stramenopile,a cercozoan, a heliozoan, and a discicristoidean with the diatoms Nitzschia sp.and Skeletonema sp., a prasinophyte Pyramimonas sp., and a pannate diatom,respectively.We observed that, whereas cPPB-aEwas the dominant componentin the cultures of the stramenopile, the cercozoan, and the heliozoan, it wasabsent from the culture of the discicristoidean. Crustacean zooplankton(Daphnia pulex) was fed a cryptophyte, Cryptomonas tetrapyrenoidosa, fromwhich we did not identify any cPPB-aE production. When the γ-proteobacte-rium Entrobacter aerogenes was grown on a media containing green juicepowder made from young barley leaves, we did not identify any cPPB-aE pro-duction. None of the unialgal control cultures contained any cPPB-aE either,suggesting that the Chl-a metabolite was only produced after phagotrophicfeeding by the three heterotrophic protists. Additional details are provided inTable S1, and additional discussions are in SI Text, section 1.4.

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microscopic observation that the autofluorescence of chlor-ophylls in the plastids of ingested algae rapidly disappears duringtheir phagocytosis by herbivorous protists (Fig. 3 and Fig. S2).This observation suggests very rapid and nonradiative quenchingof the photoexcited singlet state of cPPB-aE, which is ascribed tointernal conversion(s) to the ground singlet state (12) and/or in-tersystem crossing to the excited triplet state. Considering thebiochemical significance of producing cPPB-aE by some protists,the latter possibility seems unreasonable owing to the highly toxicsinglet oxygen that is readily produced from the triplet state(Fig. S3).Fig. 4C shows that production of singlet oxygen by photoex-

cited cPPB-aE is negligible relative to the levels generated fromChl-a and Phe-a. Here, the generation of singlet oxygen was de-tected using Singlet Oxygen Sensor Green (SOSG), a commerciallyavailable fluorescent probe. No change in SOSG fluorescencewas observed over the course of the 180-min experiment withcPPB-aE, which recapitulated the result of the control experi-ment. This finding contrasts strikingly with the results for Chl-a and Phe-a. The increase in the SOSG fluorescence caused by

rapid generation of singlet oxygen was approximately three timesmore rapid for Chl-a than Phe-a. The observed difference be-tween Chl-a and Phe-a should partially reflect differences in theabsorption intensities of Qy bands (Table S3). In conclusion,cPPB-aE is likely to be a colored but nonphotosensitizing Chl-acatabolite, which is comparable with the colorless nonfluores-cent chlorophyll catabolites (NCCs) of higher plants (SI Text,section 1.1) insofar as its generation during the detoxification ofchlorophylls protects against the accumulation of singlet oxygenin vivo.Previous studies have frequently regarded cPPB-aE as an an-

tioxidant that accumulates in marine macrofauna (11, 13–16).However, other workers have failed to highlight the possibilitythat the primary evolutionary advantage of cPPB-aE productionby single-celled organisms is the detoxification of the phototoxicchlorophyll derivatives that are inevitably ingested on feedingchlorophyll-containing diets to protists in an illuminated andaerated environment. The reported antioxidant function ofcPPB-aE accumulated in the intestines of metazoans would, thus,be a secondary adaptation if the derivative, indeed, functions asan antioxidant. We note that its antioxidant activity, which shouldprotect against the oxidative damage exerted by reactive oxygenspecies, has not been shown experimentally. Furthermore, nodirect evidence has yet been presented that metazoans producecPPB-aE. Geochemical evidence that is consistent with the abilityof microscopic plankton to produce cPPB-aE is discussed below.

Ubiquity and Geochemical Significance of cPPB-aE in Aquatic Environ-ments. To determine the distribution of cPPB-aE metabolismamong various aquatic settings, we used a geochemical approachthat involved detecting cPPB-aE directly from particulate organicmatter (POM) in water columns as well as surface sedimentarysamples just beneath the columns. We analyzed extracts of glassmicrofiber filters (GF/F grade;Whatman) that presumably collectedany particulate matters larger than 0.7 μm in diameter from thewater samples examined, and therefore, they should have includedany eukaryotic cells and most prokaryotic cells present as well asfecal materials and any organic aggregates.The results showed that cPPB-aE is ubiquitous and abundant in

all of the aquatic environments tested, confirming the ecologicaland biogeochemical significance of cPPB-aE metabolism (Fig. 5).In summary, we detected cPPB-aE in all POMsamples fromdiverseenvironments, although its abundance relative to the other Chl-aderivatives ranged from5mol% (samples collected from Ise Bay) to42 mol% (samples collected from a dammed creek). These valuescorrespond to ranges in the cPPB-aE/Chl-a ratios from 0.07 to 1.53.However, the relative concentrations of cPPB-aE in the surfacesediments were generally very high (51–81%), representing vir-tually the most abundant Chl-a derivatives. In fact, the presenceof cPPB-aE in sediments has been occasionally reported in pre-vious works (11, 17–19), although it has been probably completelymissed in other reports because of analytical artifacts (20).The abundance of cPPB-aE relative to Chl-a in water columns

implies that a considerable proportion of photosynthetic primaryproduction should initially be processed by herbivorous protiststhat produce cPPB-aE. Given that Chl-a is generally regarded asstanding biomass of phototrophic plankton, we similarly expectthat the cPPB-aE content in POM should represent the mass ofherbivorous protists as well as their excreta in water. Depthprofile patterns of cPPB-aE and the other chlorophyll derivatives(Fig. 6) are perhaps best explained by (i) in situ feeding activitiesof herbivorous protists in the upper parts of water columns and/or (ii) selective preservation into deeper water and sediments,which would explain the elevated relative concentration of cPPB-aE in the surface sediments.Evaluations of those data in terms of either the rate of protistan

herbivory or the rate of cPPB-aE production require additionalinvestigation, including investigation of the rate of cPPB-aE pro-

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Fig. 3. A typical differential interference image (A) and a correspondingfluorescent image (B; excitation light = 400–440 nm) of cercozoans in dif-ferent stages of phagocytosis. The cercozoan cells are indicated by the dashedlines on the fluorescent image. EP, a cell in an earlier stage of phagocytosiscontaining two chains of Skeletonema sp. in different stages of digestion;LP, a cell in a later stage of phagocytosis containing a chain of Sketetonemasp. in a later stage of digestion displaying no autofluorescence; NP, a cellexhibiting no phagosome formation. (Scale bars: 50 μm.) In EP cell, the longerchain of diatoms on the left is partially digested, and therefore, the plastids ofseveral diatom cells still display the autofluorescence of chlorophyll (arrows),whereas the shorter chain on the right is in a later stage of digestion andcontains remnant plastids (dark brown grains) displaying no autofluores-cence. Additional information is given in Fig. S2.

17330 | www.pnas.org/cgi/doi/10.1073/pnas.1207347109 Kashiyama et al.

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duction by various herbivorous protists, its lifetime in their cells andthe environment (e.g., in the excreta), and the sinking rate of POMin each environment. In particular, a better understanding of thechemical properties of cPPB-aE in vivo is required to explain itsapparently high preservation potential in environmental samples,despite its instability in organic solutions. In fact, we do not exclude

the possibility that protistan catabolism responsible for producingcPPB-aE would occur even in darkness (e.g., in deep water columnsand sediments), despite our hypothesis that the process evolvedprimarily to avoid the phototoxicity of chlorophyll derivatives.

Evolutionary Significance of Protistan Herbivory. We postulate thatthe ingestion of Chl-a by small, transparent, or translucent microbesin illuminated and oxygenated environments places an evolutionaryconstraint on microbial herbivory by necessitating appropriatebiochemical strategies to contain levels of singlet oxygen-gener-ating molecules after feeding on microalgae, cyanobacteria, orphototrophic bacteria. The evolution of aquatic microbial her-bivory should have been accompanied by the establishment ofsome enzymatic pathways that catabolize chlorophylls into non-fluorescent substances. Unlike the NCCs of higher plants, cPPB-aE is a colored catabolite, despite its nonphotosensititizing prop-erties. Given that conversion of Chl-a to cPPB-aE apparentlyrequires fewer enzymatic steps than the generation of NCCs, ithas emerged as a simple and efficient strategy for rapid detoxifi-cation of chlorophyll among aquatic protists.Our evidences regarding the synthesis of cPPB-aE by protistan

herbivores are comparable with the results of similar feeding ex-periments in the work by Goericke et al. (17) using heterotrophicprotists. The work by Goericke et al. (17) also identified cPPB-aEas a major Chl-a derivative in the fecal material of three protists,the ciliate Strombidinopsis acuminatum and the heterotrophicdinoflagellates Amphidinium sp. and Noctiluca scintillans. Fig. 7illustrates that cPPB-aE producers, thus, distribute widely amongthe protistan lineage, at least in two supergroups. These are theStramenopile-Alveolate-Rhizaria (SAR) clade and the Cryptophyte-Centrohelid-Telonemid-Haptophyte (CCTH) clade (21, 22). Incontrast, one species belonging to the opistkonta clade lacks theability to synthesize cPPB-aE. Significantly, over 70% of hetero-trophic protists in the marine environment are known to belong

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Fig. 5. Relative abundances of chlorophyll derivatives in extracts of POMfrom various aquatic samples and surface sediments. The samples analyzedare summarized in Table S4.

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to either the SAR or CCTH clade (23), which is consistent withour geochemical data.The SAR and CCTH clades include various phototrophic and

mixotrotrophic protists other than the obligate heterotrophsanalyzed here. Many of these phototrophic and mixotrophicprotists possess plastids that had been derived from ancestral redalgae through secondary symbiosis (Fig. 7). They include majormarine algae, such as diatoms and dinoflagellates, as well as di-verse picophytoplankton species (24). The rest of the SAR andCCTH protists are colorless and lack plastids, and thus, they are

presumably heterotrophic. Nonetheless, it is still not certainwhether these protists lost plastids during the course of evolution(i.e., secondary evolution of heterotrophy) or had never possessedplastids (i.e., multiple origins of secondary phototrophy). Our evi-dence implies that the generation of cPPB-aE, a key metabolicstep in the heterotrophic lifestyle, is likely to have evolved atthe root of the two clades, if not at an earlier evolutionarystage. The ability to synthesize cPPB-aE must, thus, be retainedin heterotrophic protists of distant lineages, regardless of thereason for the absence of plastids.

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Fig. 7. Unrooted tree of eukaryotes comprising six distinctive supergroups. Yellow stars beside taxonomic groups denote that they contain protists thatproduce cPPB-aE, which was reported in this study or a previous report (17). Examined protists belonging to discicristoidea did not produce cPPB-aE (red x).The taxonomic groups with green circles contain phototrophs with true chloroplasts. Note that the stramenopiles include not only the cPPB-aE–producingheterotrophs but also phototrophic diatoms used as diets that did not produce any trace of cPPB-aE (Fig. 2). This finding suggests that cPPB-aE metabolismevolved primarily for heterotrophy. Modified from Walker et al. (22).

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Ecological Significance of Protistan Herbivory. We postulate thatherbivory, particularly of the picophytoplankton, by cPPB-aE–producing protists is one of the most important processes in themodern aquatic food web, but it has been poorly understooduntil now (Fig. 8). Importance of protistan herbivory as a part ofthe microbial loop in aquatic environments has been recognizedfor nearly three decades (25). During this time, the quantitativesignificance of picophytoplankton (ϕ ≤ 3 μm) has also beenrevealed (23, 24). Picophytoplankton includes coccoidal cyano-bacteria and eukaryotic picophytoplankton. The former belongto the two major genera Prochlorococcus and Synechococcus,which account for up to 50% of pelagic marine oxygenic pho-tosynthesis (26–28). Eukaryotic picophytoplankton are believedto account for 20–50% of aquatic photosynthesis (29–32). Theuse of the PCR to analyze environmental samples has alsorevealed the amazingly wide diversity and activity of eukaryoticpicophytoplankton species (33, 34).However, we are still largely ignorant of abundance as well as

ecological and biogeochemical roles of colorless protists in theoceans, in large part because the majority of these protists has

never been studied because of an inability to maintain them inculture. Indeed, only recent advances showed the tremendousdiversity of protists in aquatic environments (32, 35, 36), thankslargely to molecular biological approaches, such as PCR surveysof the diversity of 18S rRNA sequences in environmental samples.Therefore, the ecological functions of aquatic colorless protistsremain largely unexplored (37, 38).Our evidence suggests that picophytoplankton-based protistan

herbivory is a quantitatively important process in aquatic envi-ronments. In analyses of POM samples from the water column ofLake Biwa, cPPB-aE was substantially enriched in the fine POMfractions, which contained POM with diameters of 5–0.7 μm(Fig. S4), which indicates that cPPB-aE was either accumulatedin picozooplanktonic protists (ϕ ≤ 3 μm) and/or concentrated invery fine particulate excreta most likely derived from variouslysized zooplanktonic protists. Therefore, picophytoplankton areprobably consumed by these protistan herbivores (Fig. 8). In fact,the small size of picophytoplankton precludes their consumptionby metazoan zooplankton such as copepods, suggesting that theyare instead likely to be preyed on by protists (23, 39).

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ConclusionsThe singlet oxygen generated when oxygen is photosensitized bychlorophylls is probably a major concern among the organismsliving on the oxygenated and illuminated surface of the Earth.However, all organisms have evolved to somehow protect them-selves against the potential phototoxicity of the chlorophylls intheir respective environments. However, the significance of thesedetoxification processes is only readily apparent in such strikingmanifestations as autumnal tints. In the present work, we haveshown a cryptic process of chlorophyll detoxification that is widelydistributed among the SAR and CCTH protists, where colored butnonfluorescent cPPB-aE is the catabolite of Chl-a. The ubiquitousoccurrence of cPPB-aE in all aquatic environments indicates thatcPPB-aE catabolism is one of the major chlorophyll detoxificationmechanisms in the modern global ecosystem.If we consider cPPB-aE a biomarker of protistan activity, our

results suggest the quantitative importance of herbivory by theSAR and CCTH protists in aquatic environments. Picophyto-planktons are particularly likely to be an important prey. Althoughthe microbial loop tends to be pictured as a material flow startingfrom bacteria and archaea that are fed on POM or dissolved or-ganic matter (i.e., protistan bacterivory), nano- and microplanktonicprotists should also play primary roles in the picophytoplankton-based microbial loop (37, 40, 41) that, hence, makes substantialcontributions to the flow of carbon and energy in aquatic envi-ronments (42). Additional studies of cPPB-aE, including its suit-ability as a proxy for protistan herbivory, may provide approachesfor quantifying the contribution of picophytoplankton to flux inthe aquatic geochemical cycle.A better understanding of cPPB-aE metabolism within and

outside the SAR and CCTH clades of protists may provide valu-able insights into the evolutionary origin of the modern aquaticecosystem, founded largely on protistan herbivory. Feeding onphotosynthetic organisms in situ in the presence of molecularoxygen and light, would have enabled much more efficient bio-geochemical recycling and bioenergetic redistribution within envi-ronments near the water surface. Interestingly, chemical structuresof fossil biomarkers that could be derived from cPPB-aE have beenreported from various aquatic sedimentary rocks as old as the EarlyJurrasic (ca. 183 Ma) (Fig. S5) (10, 43–50). Interestingly, fossil-based evidence suggests that this timing correlates roughly withthe emergence of secondary algae containing red algae-derivedplastids. These plastids include phototrophic protists within theSAR and CCTH clades, including dinoflagellates, coccolitho-phores, and diatoms (51). An understanding of aquatic microbialherbivory gleaned from combining biochemical, molecularbiological, and geochemical evidences would provide criticalinsights into the evolutionary dynamics of the aquatic protistanlineages as well as the global geochemical cycles.

Materials and MethodsPreparation of Authentic Samples. Schemes for preparation of authenticsamples of Chl-a, Phe-a, pPhe-a, pPPB-a, cholesteryl pPPB-a, cPPB-aE, and (R/S)-hCPLs-a are summarized in Fig. S6. Additional details are described in SI Text,section 2.1.

Development of HPLC Methods. We developed improved analytical methodsthat enable quantitative identification of unstable cPPB-aE from microbio-logical and environmental samples with high sensitivity. Analytical difficul-ties arose as a consequence of the instability of cPPB-aE during handlingduring extraction and analysis involving HPLC (11, 13, 17). Given that cPPB-aE is rather unstable in most organic solvents in the presence of molecularoxygen, it was rapidly degraded, especially when present at low concen-trations. Our results, thus, depend largely on the development of improvedanalytical methods that required the availability of the semisynthesizedauthentic standard. In short, the analysis required (i) careful removal ofmolecular oxygen from the solvents for extraction and the mobile phases ofHPLC, (ii) use of an end-capped reverse-phase HPLC column as well as itsdeactivation by preconditioning using mobile phase with 0.5% (wt/vol)

trifluoroacetic acid, and (iii ) use of stabilizing agents. In particular, theaddition of imidazole in the mobile phase dramatically improved the quanti-tative analysis of cPPB-aE using HPLC. In addition, cPPB-aE was found to beparticularly stabilized in anisole solution. Therefore, the use of the standardsolution in anisole permitted accurate calibration on the HPLC analysis (SIText, section 2.2). Consequently, the current methods significantly improvedthe quantitative analysis of cPPB-aE (Fig. S7, Fig. S8, and SI Text, section 2.3).Detection limit of cPPB-aE was ∼30 fmol per injection on the currentHPLC method.

Analytical HPLC (Fig. S9) was performed using a Shimadzu Prominence liquidchromatograph system, which comprised a CBM-20A communications busmodule, a DGU-20A3 degasser, two LC-20AD pumps constituting a binarypumping system, an SIL-20AAC auto sampler, a CTO-20AC column oven, and anSPD-M20Avp diode array detector. The system was coupled to a personalcomputer configured to run Shimadzu LC Solution software. All solvents usedfor the analytical HPLC were of HPLC-grade quality, and they were purchasedfromNacalai Tesque. The standard solutions of various Chl-a derivatives used inthe following analytical HPLCswereprepared using anisole (SI Text, section 2.2).Analytical HPLC involved the use of a reverse-phase monomeric columnZORBAX Eclipse Plus C18 (4.6 × 30 mm, 1.8-μm silica particle size; Rapid Reso-lution HT). The solvent gradient program used is summarized in Table S5, in-cluding the conditions used to precondition the column. Because only a binaryautomated programming techniquewith two pumps is available in our system,solvents B and C are introduced to the same second pump using a switchingvalve in the line. Therefore, switching of the solvents and purging of the pumpsystem by the solvent were performed promptly within 1 min after pre-conditioning of the column. All three solvents were degassed in vacuo withultrasonication and sealed under argon. The solvent reservoir bottles werespecially customized for the convenience of degassing or sealing with argon aswell as preventing the solvent from contacting the air during analysis. The flowrate of the mobile phase was 1.00 mLmin−1. The column ovenwas set to 25 °C.The auto sampler tray was set to 15 °C. Given that standard solutions andsamples were prepared in anisole, which is not a component of the eluent, theinjection volume was generally 1.00 μL or smaller.

Feeding Experiments. Four combinations of heterotrophic protists with algae,a combination of a crustacean zooplanktonwith an alga, and a combination ofabacteriumwithplantmaterialswereexamined. Experiments, thus, comprise sixfeeding experiments as well as six control experiments without heterotrophs/crustacean zooplankton/bacteria. All protistan strains as well as the copepodDaphnia magna were maintained by feeding unialgal clones (Table S1). Aftercertain incubation periods, these strains were collected on sterile glass micro-fiber filters (GF/F grade; 47 mm ϕ; Whatman), which were then extracted andanalyzed according to the analytical procedures used for natural samples de-scribed below. Information including specific names of organisms examined,their phylogenetic position, strain identities, and experimental conditions aresummarized in Table S1.

Optical Spectrometry and Singlet Oxygen Detection Experiments. Electronicabsorption spectraweremeasured using aHitachi U-3500 spectrophotometer.Fluorescence spectraweremeasuredusing aHitachi F-4500 spectrophotometer,and fluorescence quantum yields were obtained using a photoluminescencemethod with an absolute photoluminescence quantum yield measurementsystem model C9920-02 comprising an excitation xenon light source, amonochromator, an integral sphere, and a multichannel CCD spectrometer(Hamamatsu Photonics). OD was about 1.0/10 mm at the Soret absorptionmaximum in both anisole and tetrahydrofuran (THF) for electronic absorptionmeasurements. THF used for spectroscopy was distilled from a regent pur-chased from Nacalai Tesque. Anisole (RegentPlus grade) and tert-butylmethyl ether (ACS regent grade) were purchased from Sigma-Aldrich. All ofthe other solvents used herein were spectrometry-grade regents purchasedfrom Nacalai Tesque, and they were used without additional purification. Inoptical spectroscopy, absorption and emission properties of chlorophyllderivatives in THF and anisole are listed in Table S2. In experiments involvingcPPB-aE dissolved in THF, we, therefore, prepared the solution under argonin freshly distilled THF and measured all optical properties immediately. Insinglet oxygen detection experiments, generation of singlet oxygen was de-tected using SOSG (Invitrogen), a commercially available fluorescent probe(52). A chlorophyll derivative (10 μM) and SOSG (1 μM) were dissolved inanisole and methanol (1:1, vol/vol), placed in a quartz cell, irradiated withred light that was provided by a 250 W metal halide lamp (LS-250–7500;Sumita Optical Glass), and passed through a colored glass filter that failed totransmit light with a wavelength shorter than ∼630 nm (AGC Techno Glass).Therefore, the irradiated light selectively excited chlorophyll derivatives butnot SOSG. The aerated solutions were continuously stirred during irradiation

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experiments using a magnetic stirrer. The increase in fluorescence intensity(i.e., initial fluorescence ascribable to the purchased SOSG was subtracted)was plotted against the irradiation time to show and compare the relativeabilities of singlet oxygen generation between different Chl-a derivatives aswell as a control SOSG solution without the addition of any photosensitizers(Fig. 4C).

Analytical Procedures Used for Natural Samples. Water samples were filteredusing a glass microfiber filter (GF/F grade; 47 mm ϕ; Whatman). The filterswere then stored below −20 °C before analysis. Sediment samples werestored at 4 °C before analysis. A wet filter sample (containing POM) or a wetsediment sample was extracted in acetone and ultrasonicated for 5 min at0 °C in the dark, with the extraction and sonication procedures repeateda total of three times. The combined extracts were then dried undera stream of argon in the dark. The residue was then dissolved in anisole. Allof the above operations were performed in an argon atmosphere using

a glove bag. Acetone and anisole were carefully degassed and purged withargon gas before use. The sample dissolved in anisole was then transferredto the vial for the autosampler of the HPLC system.

ACKNOWLEDGMENTS. We thank the crew and scientific party of theTraining/Research Vessel Seisui-maru (cruise number 1127) and ResearchVessel Hakken. We also thank Mr. Hokuto Tsutamoto and Dr. NobutakaImamura for kindly supplying the clone of D. pulex and Dr. Hiroshi Endohfor favorably providing the clone of E. aerogenes; Mr. Ryuzou Narita, Dr.Satoshi Ohkubo, Ms. Hiroko Usui, Mr. Koichi Fujita, and Dr. ShinnosukeMachida for technical assistance; and Dr. Jun Yokoyama and Dr. JonathanJ. Tyler for critical comments. This study was supported in part by Grants-in-Aid for Scientific Research 23870028 (to Y. Kashiyama), 20570081 (to A.Y.),24657060 (to A.Y.), 21247005 (to H.M.), and 21247010 (to I.I.), a ResearchFellowship for Young Scientists (to Y. Kashiyama), and a grant from theRitsumeikan Global Innovation Research Organization (to Y. Kashiyamaand H.T.).

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