Does Formation of Multicellular Colonies by Choanoflagellates

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE Does Formation of Multicellular Colonies by Choanoﬂagellates Affect Their Susceptibility to Capture by Passive Protozoan Predators?

William E. Kumlera,b , Justin Jorgea,c , Paul M. Kima , Noama Iftekhara & M. A. R. Koehla a Department of Integrative Biology, University of California, Berkeley, California, 94720-3140 b School of Oceanography, University of Washington, Seattle, Washington, 98105 c Department of Biology, Duke University, Durham, North Carolina, 27708-0338

Keywords ABSTRACT Choanoflagellate; heliozoa; multicellularity; predation; videomicrography. Microbial eukaryotes, critical links in aquatic food webs, are unicellular, but some, such as choanoflagellates, form multicellular colonies. Are there con- Correspondence sequences to predator avoidance of being unicellular vs. forming larger colo- W.E. Kumler, School of Oceanography, nies? Choanoflagellates share a common ancestor with animals and are used University of Washington, 1501 NE Boat St, as model organisms to study the evolution of multicellularity. Escape in size Seattle, WA 98195, USA from protozoan predators is suggested as a selective factor favoring evolution Telephone number: +1-909-907-2385; of multicellularity. Heterotrophic protozoans are categorized as suspension e-mail: [email protected] feeders, motile raptors, or passive predators that eat swimming prey which bump into them. We focused on passive predation and measured the mecha- Received: 31 July 2019; revised 17 April nisms responsible for the susceptibility of unicellular vs. multicellular 2020; accepted May 14, 2020. choanoflagellates, Salpingoeca helianthica, to capture by passive heliozoan predators, Actinosphaerium nucleofilum, which trap prey on axopodia radiat- ing from the cell body. Microvideography showed that unicellular and colonial doi:10.1111/jeu.12808 choanoflagellates entered the predator’s capture zone at similar frequencies, but a greater proportion of colonies contacted axopodia. However, more colonies than single cells were lost during transport by axopodia to the cell body. Thus, feeding efficiency (proportion of prey entering the capture zone that were engulfed in phagosomes) was the same for unicellular and multicellular prey, suggesting that colony formation is not an effective defense against such passive predators.

MICROBIAL eukaryotes that eat bacteria are critical links unknown sources of variability involved in comparing uni- in aquatic food webs (Azam et al. 1983; Montagnes et al. cellular species with different colonial species can be 2008; Ohtsuka et al. 2015; Weisse et al. 2016). Hetero- avoided. trophic microbial eukaryotes are unicellular, but some can Studying the mechanisms that determine the vulnerabil- form multicellular colonies. Many of the organisms that ity to predation of unicellular vs. multicellular choanoﬂagel- eat microbial eukaryotes are size-selective predators (Mon- lates can not only help us understand how forming tagnes et al. 2008; Strom and Loukos 1998; Verity 1991; colonies affects trophic interactions of aquatic protozoans, Weisse et al. 2016), but the consequences to predator but may also shed light on the evolutionary origins of ani- avoidance of being unicellular vs. forming larger multicellu- mals. Multicellular animals evolved from a unicellular pro- lar colonies are not yet well understood. Many species of tozoan ancestor more than 600 million years ago choanoﬂagellates can be unicellular and can also form mul- (Armstrong and Brasier 2005; Knoll and Lipps 1993; ticellular colonies by cell division (King 2004; Leadbeater Schopf and Klein 1992), a profound step in the evolution 2015) (Fig. 1A); thus, they provide research systems that of life on Earth. The development of multicellularity was can be used to study the effects of multicellularity on the followed by the Cambrian explosion, when a diversity of susceptibility to predation within a single species so that complex animals representing most major phyla appeared

Figure 1 Study organisms. (A) Diagram of a unicellular choanoflagellate, Salpingoeca helianthica, and a multicellular colony that forms when daughter cells remain attached to each other after mitosis. (B) Plot of the maximum length of S. helianthica plotted as a function of the number of cells in the choanoflagellate. (C) Actinosphaerium nucleofilum capturing multicellular colonies of S. helianthica.(D) A. nucleofilum capturing unicellular S. helianthica. Prey are captured on the long, slender axopodia, transported to the cell body along an axopod, and engulfed into a phagosome at the cell surface. in the fossil record (Butterfield 1997; Stanley 1973). Geno- also been argued that an important selective factor leading mic and molecular phylogenetic analyses indicate that ani- to the evolution of multicellularity in the protozoan ancestors mals and choanoflagellate protozoans shared a common of animals was the relative difficulty that protozoan preda- ancestor (King et al. 2008). Because colony formation is tors may have had in capturing larger multicellular colonies found in diverse choanoflagellate lineages, it has been than smaller unicellular prey (Boraas et al. 1998; Richter and suggested that colony formation was present in the last King 2013; Stanley 1973). Studies of living heterotrophic common ancestor of animals and choanoflagellates (Carr protozoans have shown that some have difficulty consum- et al. 2017). Therefore, studying living choanoflagellates to ing large prey (Fenchel 1986; Jonsson 1986; Verity 1991). help us understand how the extinct ancestors of Fossils, chemical biomarkers, and molecular analyses choanoflagellates and animals might have worked enables indicate that diverse heterotrophic protozoans (including us to make informed inferences about possible selective ciliates, flagellates, and various amoeboid forms and Rhi- pressures at the time of animal origins. Choanoflagellates zaria) evolved before multicellular animals (Armstrong and in the genus Salpingoeca, which have both unicellular and Brasier 2005; Parfery et al. 2011; Schopf and Klein 1992), multicellular life stages, are used as model organisms to so the predators on the ancestors of animals and study the evolution of multicellularity in the ancestors of choanoflagellates were most likely protozoans from these animals (Brunet and King 2017; King 2004; King et al. groups. The diverse mechanisms by which swimming, 2008; Richter and King 2013). crawling, and sessile protozoan predators of different mor- Several selective factors have been proposed that might phologies capture prey (Arndt et al. 2000; Fenchel 1986; have favored the evolution of multicellularity in the ances- Sleigh 1991) have been categorized by Sleigh (2000) into tors of choanoflagellates and animals (reviewed by Koehl functional types. Suspension or filter feeders create a 2020). One suggestion is that multicellularity may have feeding current and intercept prey on capture surfaces, enhanced the rates at which cells in colonies captured bac- motile raptors actively catch prey with pseudopodia or ten- terial prey (Kirkegaard and Goldstein 2016; L’Etoile and tacles, and passive predators feed by intercepting prey King-Smith 2020; Roper et al. 2013; Stanley 1973). It has that swim or drift nearby.

Choanoflagellates and their protozoan predators are so 2016; Richter and King 2013; Roper et al. 2013). We used small that inertial forces can be ignored and the viscous S. helianthica as the prey in our study because it is a resistance of water to being sheared determines the flow freshwater choanoflagellate with both unicellular and colo- around them and hydrodynamic forces on them (reviewed nial forms (Fig. 1A, B) that is readily eaten by A. nucle- by Koehl 2020). In such viscous flow regimes, it can be ofilum in the laboratory. A choanoflagellate cell (Fig. 1A) difficult for microscopic bodies to approach each other. A bears one flagellum which propels the cell through the mathematical model of a motile sphere approaching a sta- water and creates a feeding current past a collar of micro- tic sphere operating in this viscous regime showed that a villi, where bacterial prey are caught (Dayel and King 2014; sphere pushed by a flagellum behind it (as is the case for Leadbeater 2015). Rosette colonies are formed when choanoflagellates) can approach a nonmotile target (e.g. a dividing cells of S. helianthica remain attached to each passive protozoan predator) that is the same size or bigger other (Fig. 1A). Choanoflagellates such as S. helianthica than the motile sphere (Jabbarzadeh and Fu 2018). There- are commonly found in freshwater habitats, where they fore, basic models of encounter rates between passive can be an integral part of aquatic food webs (Leadbeater predators and motile prey that do not include hydrodynam- 2015). ics are applicable to choanoflagellates and passive protozoan predators. These models predict that the encounter Objectives of this study rate of passive protozoan predators with motile prey depends on the swimming speeds and paths of the prey The goal of this study was to determine the organismal- and that large motile prey are more likely than small ones level mechanisms responsible for the susceptibility of uni- to contact a predator (Fenchel 1982, 1984; Rubenstein cellular vs. multicellular choanoflagellates, S. helianthica, and Koehl 1977; Shimeta and Jumars 1991). This suggests to capture by the passive heliozoan predator A. nucle- that multicellularity might increase rather than reduce the ofilum. The specific questions addressed were as follows: susceptibility to predation by passive protozoan predators. 1 Is the swimming behavior of unicellular S. helianthica To address these conflicting suggestions, the focus of this different from that of multicellular colonies? Does the study is on the susceptibility of multicellular vs. unicellular behavior of unicellular or multicellular choanoflagellates choanoflagellates to predation by a passive protozoan. change when near an A. nucleofilum? 2 What are the mechanisms of capture of S. helianthica by A. nucleofilum, and do they differ between prey of dif- Research system ferent size (i.e. unicellular choanoflagellates and colonies The passive protozoan predator Actinosphaerium nucle- composed of different numbers of cells)? ofilum (Fig. 1C) was chosen for this study because it is 3 Do the frequencies of encounters with the capture zone ecologically important, is easy to maintain in culture, and (Fig. 2A) or contacts with the axopodia differ between uni- readily eats choanoflagellates in the laboratory. This helio- cellular and colonial choanoflagellates? zoan (“sun animalcule”) is found in freshwater habitats 4 Does the size of an A. nucleofilum affect its feeding worldwide where it consumes a wide variety of other pro- efficiency (number of prey captured per number encoun- tozoans (Barrett 1958; Leidy 1879). Heliozoans can be tered) on S. helianthica? found in concentrations of more than 5,000 cells/liter and 5 Does the feeding efficiency of A. nucleofilum on S. he- likely exert a large grazing pressure on protozoans in lakes lianthica depend on prey size or differ between unicellular and ponds (Pierce and Coats 1999). Prey are trapped and multicellular prey? when they contact the long axopodia of a heliozoan and then are transported along the axopodia to the predator’s MATERIALS AND METHODS cell body, where prey items are phagocytosed (Bovee and Cordell 1971; Nikolaev et al. 2004; Suzaki et al. 1980). The Culture of protozoans Rhizaria (a group of eukaryotes that includes radiolarians, foraminiferans, and heliozoans) are estimated, based on Salpingoeca helianthica were purchased from the Ameri- molecular data, to have originated ~620 mya (Cavalier- can Type Culture Collection (Manassas, Virginia). The origi- Smith et al. 2018), and foraminifera 650–920 mya (Cava- nal culture was frozen at 70 °C for one week and kept lier-Smith et al. 2018; Pawlowski et al. 2003), although the on liquid nitrogen until needed. Three different cultures of oldest fossils of cells are from 545 mya (Groussin et al. S. helianthica were revived and cultured at 22 °C using 2011). Radiolarians and foraminiferans use the same prey- the protocols described in detail by King et al. (2009) and capturing mechanism as heliozoans, so A. nucleofilum is a available at http://live-king-lab.pantheon.berkeley.edu/wp- good model organism for study of a passive amoeboid content/uploads/2018/08/King-Lab-Choanoflagellate-Protoc type of predator that may have been present in premeta- ol-Handbook-April-2015.pdf. After a culture was revived, it zoan oceans. was passaged every 3–4 d by pipetting 1 ml of culture Choanoflagellates in the genus Salpingoeca that have into 9 ml of new media (25% cereal germ; King et al. both unicellular and colonial life stages are used as model 2009). The proportion of colonies in a culture decreased systems to study the functional consequences of and evo- as the number of passages increased. Therefore, one cul- lution of becoming multicellular (Brunet and King 2017; ture was passaged 180 times before the culture was used King 2004; King et al. 2008; Kirkegaard and Goldstein in the experiment to encourage the formation of single

© 2020 International Society of Protistologists Journal of Eukaryotic Microbiology 2020, 0, 1–11 3 Predation on Uni- vs. Multicellular Choanoflagellates Kumler et al. cells. The second and third cultures were revived later, the cultures in an opaque box when not actively being were passaged every 3–4 d, and were used after 75 pas- used. sages when the cultures were still rich in colonies. Ali- quots of each of the three cultures were used during the Videomicrography first two weeks following passaging. Cultures of Actinosphaerium nucleofilum were pur- Predator–prey interactions were observed at room temper- chased from Carolina Biological Supply Company (Burling- ature (20 °C) in the flat-bottomed well (0.7 mm depth; ton, NC) and were kept in their original culture jars at 15 mm diameter) of a depression slide. For each experi- room temperature (20 °C). No passaging or sub-culturing ment, one individual A. nucleofilum was pipetted directly of these organisms was necessary, as all were used from the culture into the well, followed by the addition of within four weeks of delivery and the original containers enough choanoflagellate culture to fill the well, which was included wheat media that provided sustenance to the then covered by a coverslip (total volume in predators. Exposure to light was minimized by keeping well = 0.124 ml). After 30 min, the protozoans were observed using a Leica DMLS microscope with fiber-optic lighting so that illumination did not affect stage temperature. Videos were taken at a magnification of 10X to mea- A 1400 sure the swimming speeds of the choanoflagellates, and 30 at 40X to record the process of prey capture and inges- 1200 tion. To minimize wall effects on the motions of the proto- 24 1000 zoans, the microscope objective lenses had long working distances so that the plane of a video was at least mid- 800 18 depth (150 lm below the cover slip) in the well for the

µm l µm/s 10X videos, and at least 120 m below the coverslip for 600 12 the 40X videos. Videos were made using a Fastec HiSpec – 400 1 camera. The videos at 40X of predator prey interactions 6 were taken at 5 frames/s for a duration of 120–300 s and 200 the videos of choanoﬂagellate swimming at 10X were 0 taken at 5 frames/s for a duration of 327 s. 0 0 200 400 600 800 1000 1200 1400 1600 The concentrations of choanoﬂagellates in the well was µm determined by counting the number of colonies and of single cells in sharp focus in the region of water outside the B 12 outside capture zone inside capture zone

10 Figure 2 Salpingoeca helianthica movement patterns near an Acti- nosphaerium nucleofilum. (A) Swimming trajectories of S. helianthica, 8 with higher speeds denoted by warmer colors. The capture zone (CZ) is indicated by the bright red circle. (B) Instantaneous speed of unicel- 6 lular (black circles) and multicellular (white circles) S. helianthica outside and inside the CZ. For each replicate predator individual, we Speed (µm/s) 4 calculated the mean velocity of each choanoflagellate and then calculated the mean velocity outside and mean velocity within the capture 2 zone of all unicellular choanoflagellates and of all colonial choanoflagellates. The mean of those means for all replicate predators is plotted 0 here (n = 6 predators). Error bars indicate one standard deviation. unicellular multicellular unicellular multicellular (C) Straightness index (ratio of the distance between the position of the choanoflagellate at the start and the end of the trajectory, to the C outside capture zone inside capture zone length of the path that the choanoflagellate followed along its trajectory) of unicellular and multicellular S. helianthica outside and inside the capture zone. The median of the straightness indices outside and the median within the capture zone were calculated for all the unicellular choanoflagellates or for all the colonies for an individual predator, and then, the median of those median straightness indices was calculated for each case for all the predators (n = 6 predators for all cases except unicellular choanoflagellates outside the CZ, for which n = 5 predators). Black bars indicate medians, boxes the first quartiles about the median, and error bars the range. For B and C, 12 videos of unicellular S. helianthica and 11 videos of colonies were used for this analysis, with 1–266 choanoflagellates per video; 6 predators fed on unicellular prey and 6 fed on colonies, and data from multiple videos unicellular multicellular unicellular multicellular of a single predator were pooled for that predator.

© 2020 International Society of Protistologists 4 Journal of Eukaryotic Microbiology 2020, 0, 1–11 Kumler et al. Predation on Uni- vs. Multicellular Choanoflagellates perimeter of the A. nucleofilum in the first frame of videos prey “outside” vs. “within” the CZ, where straightness taken at 10X. The area of water in the frame in which the index (also called “net-to-gross-displacement ratio”) is the choanoflagellates were counted was measured using Ima- ratio of the distance between the position of the choanoflag- geJ (Rasband 2016) and was multiplied by the depth of ellate at the start of the trajectory and the end of the trajec- the focal plane (11.4 lm) to determine volume. The mean tory, to the length of the path that the choanoflagellate concentration of single cells was 3.3 9 107 cells/ml followed during its trajectory (Hadfield and Koehl 2004). (SD = 4.3 9 107, n = 5) and of colonies was 2.9 9 107 Because the straightness index can differ between short tra- colonies/ml (SD = 3.9 9 107, n = 5). Although concentra- jectories and longer ones, we only determined straightness tions of protozoans in natural waters vary greatly (Fenchel index for trajectories lasting ≥ 50 s. Straightness indices 1987), we chose to use high concentrations of choanoflag- close to one denote nearly linear swim paths, while lower ellate prey in our brief experiments to assure that encoun- indices indicate paths characterized by turns or circling. For ters would occur between the predator and prey in our each individual predator, we calculated the mean of veloci- small field of view (e.g. Fig. 1C, D). ties outside and the mean of velocities within the capture zone of all unicellular choanoflagellates and of all colonial choanoflagellates, and then, the mean of those mean veloci- Video analysis of choanoflagellate swimming ties was calculated for all predators. Similarly, for each indi- Choanoflagellate motions near A. nucleofilum were vidual predator, the median of the straightness indices recorded in videos made at a magnification of 10X and outside and the median within the capture zone was calcu- were analyzed using in-house software written to use lated for all the unicellular choanoflagellates and for all the Python (version 2.7) bindings to the OpenCV (version 2.4) colonies, and then, the median of those median straightness Computer Vision Library (https://opencv.org/) (Bradski and indices was calculated for all the predators. Kaehler 2008). The capture zone (CZ, a circle with a radius that is the distance between the cell center and the tip of Video analysis of prey contact, transport, and capture the longest axopod of the heliozoan; Fig. 2A) was determined for each A. nucleofilum. The positions of an A. nu- Each video made at a magnification of 40X was saved into cleofilum in each video were tracked using a combination digital.avi format and imported into ImageJ software of pyramid smoothing and blob detection filters that were (Rasband 2016) for analysis. For each video, the diameter thresholded by pixel brightness (Bradski and Kaehler 2008). of the cell body of each A. nucleofilum was measured on Thus, the CZ of each predator was defined relative to the a single frame of a video to the nearest 10 lm and the CZ moving predator. The smaller choanoflagellates were identi- was determined. fied using the cvGoodFeaturesToTrack function of the All unicellular and colonial choanoflagellates that were in OpenCV library (Shi and Tomasi 1994), and their positions sharp focus and that entered the CZ were tracked by were followed through time using the Lucas–Kanade opti- hand. Colonies and single cells were easily distinguished cal flow methods of the cvCalcOpticalFlowPyrLK function from each other while they were swimming freely (Bradski and Kaehler 2008; Lucas and Kanade 1981). Track- (Fig. 1C, D), and the number of cells in each S. helianthica ing of an individual was terminated when the feature being colony was counted while it was swimming freely. The tracked was no longer discernable by the algorithm, either longest dimension of freely swimming single-celled due to swimming out of field or out of the focal plane. choanoflagellates and of freely swimming colonies were After tracking, only those tracked features that were clearly measured to the nearest 1 lm on single frames of videos. choanoflagellates (not suspended detritus) were manually The edges of cell bodies were clearly visible, while collars chosen and used for further analysis. Each choanoflagellate and flagella were not always in sharp focus, so only cell was also identified as either unicellular or colonial at this bodies were used to measure choanoflagellate lengths. stage in the analysis. Contacts with the axopodia of a predator by choanoflag- These choanoflagellate trajectories were used to deter- ellates that were in the CZ were determined. Because the mine the encounters of prey with the CZ of the predators. depth of field was thicker than an axopod, a contact was The proportion of unicellular and of colonial choanoflagel- only counted if the choanoflagellate near an axopod lates that were swimming outside the CZ near an A. nu- stopped when it overlapped with the axopod and then cleofilum (within a radius of 400 lm from the center of was transported along the axopod. The proportions of uni- the predator) that subsequently entered the CZ was deter- cellular and colonial S. helianthica in the CZ that contacted mined for each A. nucleofilum. an axopod were determined for individual A. nucleofilum. The trajectories of the choanoflagellates were split into The instantaneous speed at which each prey on an axo- two categories for analysis of swimming behavior: “outside” pod was transported toward the cell body of the predator the CZ of the predator or “within” the CZ. Central differ- was measured to the nearest 0.1 lm/s, and the mean ences were used to calculate instantaneous swimming speed for each prey item was calculated. speeds from the positions of a choanoflagellate in succes- The feeding efficiency (number of prey caught per num- sive frames of the video. Then, for each choanoflagellate, ber encountered) of each predator at capturing choanoflag- the mean of its instantaneous velocities when “outside” and ellates was determined. Encountered prey were all those the mean when “within” the CZ were calculated. We also that entered the CZ of the predator, and captured prey determined a straightness index for the entire trajectory for were only those that were eventually engulfed into a

© 2020 International Society of Protistologists Journal of Eukaryotic Microbiology 2020, 0, 1–11 5 Predation on Uni- vs. Multicellular Choanoflagellates Kumler et al. phagosome (Fig. 1C). Cells that were already contained different mechanisms used by A. nucleofilum to transport within a phagosome at the start of the video were not adhered prey to the cell body, named “axopodial flow” counted because single cells and colonies could not be and “rapid axopodial contraction” by Suzaki et al. (1980) distinguished when in phagosomes. These data were when reporting on heliozoans eating other types of prey. used to calculate the feeding efficiency of an A. nucle- When prey reached the cell body of the predator, they ofilum for each size category (number of cells) of were engulfed in phagosomes that formed outwards from choanoflagellate prey: the surface of the cell body (Fig. 1C). These phagosomes were occasionally observed to combine with other phago- n Feeding efficiency ¼ consumed ; somes to form a single, large phagosome. The phago- nencountered some and the prey item were then moved into the cell body of the predator. where n is the number of individual prey (single-celled Axopodial flow (indicated by red in Fig. 3A) is the slow choanoflagellates or multicellular colonies) in a size cate- movement (Fig. 3C) of the axopodial plasma membrane, gory. If several videos of the same individual A. nucle- which draws a prey item toward the cell body (Suzaki ofilum were made, the data from those videos were et al. 1980). We observed axopodial flow velocities rang- pooled to give a single feeding efficiency for each size of ing from 1.1 to 21.5 lm/s (median = 2.4 lm/s, n = 7 choanoflagellate prey for that individual predator. A. nucleofilum individuals). This speed was not significantly different between unicellular choanoflagellate prey Statistical analyses and colonies (Mann–Whitney U, P = 0.63, n = 7 preda- Swimming speeds of S. helianthica of different sizes were tors), and no trend was found between axopodial flow compared using ANOVA–Bonferroni tests performed using velocity and number of cells in a choanoflagellate colony Python (version 2.7) and packages from the SciPy soft- (Kendall’s tau-b, P = 0.53, n = 7 predators). ware stack (https://www.scipy.org/). Nonparametric tests Rapid axopodial contraction (indicated by red in Fig. 3B) performed using R software (R Core Team 2017) (Mann– occurs when the microtubule skeleton of the axopod Whitney U, Kruskal–Wallis, Kendall’s tau-b, and Kol- breaks down at high speed, triggered by the contact of a mogorov–Smirnov tests) were used to analyze data that prey item with the axopod (Suzaki et al. 1994). were ratios (encounter with CZ, contact with axopodia, When A. nucleofilum transported S. helianthica prey by feeding efficiency, straightness index) or percentages, and rapid axopodial contraction, the entire motion occurred data for which the sample sizes of some cases were between two successive frames of the video (i.e. the small (e.g. speeds of axopodial flow). motion was completed in a period of ≤ 0.2 s), so speeds were measured in excess of 1,500 lm/s (Fig. 3D). The rapid axopodial contraction occurred more fre- RESULTS quently than did axopodial flow (median percent of axopodial transports done by rapid contraction = 75%, Choanoflagellate behavior near Actinosphaerium n = 8 predators). The distribution of the percent of nucleofilum transports by rapid contraction was significantly different The swimming trajectories of Salpingoeca helianthica near from a random normal distribution (two-sided Kol- Actinosphaerium nucleofilum predators are shown in mogorov–Smirnov test, P = 0.0335). Large choanoflagel- Fig. 2A. The instantaneous swimming speeds of unicellu- late colonies were more likely to be transported via lar S. helianthica were not significantly different from rapid axopodial contraction than were small ones or sin- those of multicellular colonies, both when outside and gle cells (there was a significant positive association when inside the capture zone (CZ) (ANOVA–Bonferroni, between the number of cells in a choanoflagellate and P > 0.05, n = 6 A. nucleofilum predators) (Fig. 2B). Both the percent transported by rapid contraction, Kendall’s colonies and single cells moved significantly more slowly tau-b, P = 0.015, n = 30 choanoflagellates). Large helio- when inside than when outside the CZ (ANOVA–Bonfer- zoans were also more likely to use rapid axopodial con- roni, for colonies P = 0.012; for single cells, P = 0.0024, traction than were smaller predators, as there was a n = 6 predators). The straightness indices for the trajecto- significant positive association between the percent of ries of unicellular and multicellular S. helianthica were not prey transported by rapid axopodial contraction and significantly different from each other, nor did they change A. nucleofilum cell diameter (Kendall’s tau-b, P = 0.0495, when the choanoflagellates entered the CZ (Kruskal–Wal- n = 10 predators). lis, P = 0.81, n = 6 predators) (Fig. 2C). Encounters with capture zone and contacts with Mechanisms of prey capture by Actinosphaerium axopodia nucleofilum There are two steps in the feeding process of a heliozoan The process of capturing S. helianthica prey by A. nucle- for which encounter rates are important: (i) entry of the ofilum involved several steps. First, a choanoflagellate prey into the CZ and (ii) contact of prey item with an axo- swam into the CZ. Then, a prey item physically contacted pod. Because the capture zone of a passive heliozoan an axopod and became adhered to it. We observed two predator is a sphere with the length of the longest axopod

Figure 3 Examples of the two types of axopodial transport of prey by a single Actinosphaerium nucleofilum.(A) and (B) show the paths of prey (arrow indicates the location of the tip of the axopod before prey contact) and (C) and (D) show prey speeds plotted as a function of time before contact with an axopod (green), during axopodial transport (red), and during phagocytosis (blue). (A) and (C) present data for an example of axopodial flow, during which a prey item that touches an axopod is transported along the axopod toward the cell body. (B) and (D) show data for rapid axopodial contraction, in which a captured prey is rapidly moved toward the predator by the retraction of an axopod. as the radius, we can use the empirical relationship pro- dC vided by Fenchel (1982) and Shimeta and Jumars (1991) FA ¼ 2pðrc þ rpÞDA l dr for passive predators to estimate the rate (FCZ) at which motile choanoflagellates (radius rp) in still water encounter where rp is choanoflagellate radius, 2p(rc +rp) is the cir- the spherical CZ (radius rs)ofanA. nucleofilum: cumference of the site of encounter between the prey and axopod, DA is the diffusivity of the prey within the CZ, FCZ ¼ 4pCDðrs þ rpÞ and dC/dr is the concentration gradient of prey around the cylinder. Prey swimming in the water within the CZ can where C is the concentration of the prey (number/volume) approach an axopod from any direction, and our data meet and D is the diffusivity of the prey when outside the CZ. the assumptions of this model because when an axopod Diffusivity, D, is used in models to account for prey swim- is in the focal plane, we can see prey approaching it from ming when there is no quantification of the actual swim- all directions. Since both the swimming velocities and ming trajectories, and so a random walk is assumed. straightness indices of unicellular and multicellular S. he- Instead, we have measured the velocities and the straight- lianthica are the same when within the CZ of an A. nucle- ness indices of the prey trajectories and found that they ofilum, we assume that DA is the same for colonies and do not differ between single cells and colonies when out- single cells. Thus, when we calculate the ratio of the FA side the CZ. Therefore, we assume that D is the same for of colonies to the FA of single cells at a given concentra- colonies and single cells. Thus, when we calculate the tion gradient around an axopod of length l, then DA, dC/dr, ratio of the FCZ of colonies to the FCZ of single cells for a and l are constants that are canceled out. CZ of a given rs,Dis a constant that is canceled out. Colonies are more likely to encounter the CZ than are Once the prey are within the spherical CZ, they must single cells. For example, if an A. nucleofilum has a CZ also contact an axopod to be captured. We modeled an radius of 300 lm and an axopod radius of 3 lm, the ratio axopod as a cylindrical collector and calculated the contact of the FCZ for a colony of 10 cells (rp = 15.00 lm, Fig. 1B) rate (FA) of choanoflagellates in the CZ with an axopod of to the FCZ for a single cell (rp = 3.02 lm, Fig. 1B) is 1.04, length l and radius rc using the relationship given by Shi- indicating that unicellular choanoflagellates are just as meta and Jumars (1991): likely to enter the CZ as are colonies. This prediction is

© 2020 International Society of Protistologists Journal of Eukaryotic Microbiology 2020, 0, 1–11 7 Predation on Uni- vs. Multicellular Choanoflagellates Kumler et al. consistent with our data showing no difference between The measured encounter frequencies of unicellular and the proportion of single cells or of colonies near an A. nu- colonial choanoflagellates with the CZ are shown in cleofilum that enter the CZ (Fig. 4A). In contrast, the ratio Fig. 4A, and the measured contact frequencies with of FA for the 10-celled colony to the FA of a unicellular axopodia are shown in Fig. 4B. The proportion of single S. helianthica is 3.0, indicating that contact rates with cells encountering the CZ (median = 0.38, n = 5 preda- axopodia are higher for large multicellular prey than for tors), was not significantly different from that of colonies small unicellular prey. This prediction is also consistent (median = 0.54, n = 6 predators) (Mann–Whitney U, with our data showing that a significantly greater propor- P = 0.463). In contrast, the proportion of colonies contact- tion of colonies in the CZ contact axopodia than do single ing axopodia (median = 0.50, n = 4 predators) appeared to cells (Fig. 4B). be greater than that of single cells (median = 0.13, n = 4 predators) (Mann–Whitney U, P = 0.059).

Feeding efficiency of Actinosphaerium nucleofilum A B 1.0 1.0 Feeding efficiency (number of choanoflagellates consumed divided by the number of choanoflagellates that entered 0.8 0.8 the CZ) was not affected by predator or prey size. There was no significant correlation between the size (cell diam- 0.6 0.6 eter) of A. nucleofilum and feeding efficiency on unicellular S. helianthica (Kendall’s tau-b, P = 0.36, n = 8 0.4 0.4 predators) or on multicellular colonies (Kendall’s tau-b, P = 0.28, n = 9 predators) (Fig. 4B). There also was no 0.2 axopod 0.2 significant difference between the feeding efficiency on colonies vs. single cells (Mann–Whitney U, P = 0.59, 0.0 0.0 n = 9 predators), nor was there any trend in feeding effi- n=5 n=6 n=4 n=4 ciency as a function of the number of cells in a prey col- Proportion contacting

Proportion entering CZ ony (Kendall’s tau-b, P = 0.41, n = 9 predators) (Fig. 4C).

Singles Singles Colonies Colonies DISCUSSION C ● Unicellular prey ● Multicellular prey Steps in the capture of Salpingoeca helianthica by Actinosphaerium nucleofilum 1.0 ● The process of capture of choanoflagellate prey by A. nu- 0.8 ● cleofilum occurs in four distinct steps: (i) entry of prey into ● 0.6 the capture zone (CZ), (ii) contact of the prey with an axo- ● pod, (iii) transport of the prey toward the cell body of the 0.4 ● ● ● ● ● 0.2 ● ●● ● ● Figure 4 Salpingoeca helianthica interactions with Actinosphaerium

Feeding efficiency Feeding ● ● ● 0.0 nucleofilum.(A) Proportion of the single-celled choanoflagellates and of colonies swimming near an A. nucleofilum that enter the CZ. 120130 140 150 160 170 180 190 A. nucleofilum µ A total of 41 single cells and 30 colonies of S. helianthica were D size ( m) tracked for 9 A. nucleofilum predator individuals. (B) Proportion of the single-celled choanoflagellates and of colonies swimming in the CZ of 1.0 A. nucleofilum that contact axopodia. A total of 22 S. helianthica colonies and a total of 35 unicellular S. helianthica were tracked for 4 indi- 0.8 vidual predators. (C) Feeding efficiency (number of prey engulfed in a 0.6 phagosome per number of prey in the CZ) of A. nucleofilum, plotted as a function of the size of the A. nucleofilum for unicellular 0.4 choanoflagellates (black circles) and for colonies (white circles). Each circle shows the efficiency for an individual predator, eight of which 0.2 fed on single cells and nine of which fed on colonies. (D) Feeding effi- 0.0 ciency plotted as a function of the size (number of cells) of S. he- n=5n=9n=5n=6n=5n=5n=4n=3n=3n=2 lianthica prey. The number of individual predators (n) for which Feeding efficiency Feeding efficiency data could be determined for a given prey size is listed on 13579246810 the graph. In (A), (B), and (D), black bars indicate medians of the val- ues from all the individual predators, boxes show the first quartiles Choanoflagellate size (# of cells) about the median, and whiskers the range.

© 2020 International Society of Protistologists 8 Journal of Eukaryotic Microbiology 2020, 0, 1–11 Kumler et al. Predation on Uni- vs. Multicellular Choanoflagellates predator, either by axopodial flow or by rapid axopodial predator, and thus no axopodial transport could be contraction, and (iv) phagosome formation around prey tracked. Prior studies of various species of Acti- and movement of the phagosome into the cell body of the nosphaerium have noted both successful (Greenwood predator. Steps 2, 3, and 4 have also been documented 1886; Suzaki et al. 1980) and failed (Bovee and Cordell for A. nucleofilum feeding on protozoan prey other than 1971; Greenwood 1886; Tilney and Porter 1967) prey cap- choanoflagellates (Suzaki et al. 1980). ture, but did not report the number of successful captures The swimming speeds and the straightness indices of relative to the number of prey encountered. the trajectories of unicellular and multicellular S. helianth- We found that multicellularity does not affect the sus- ica are the same, both outside and within the CZ (Fig. 2). ceptibility of S. helianthica to capture by a heliozoan pas- The choanoflagellates swim more slowly when in the CZ sive predator, A. nucleofilum. Our result suggests that (Fig. 2B). Viscous forces determine the hydrodynamic per- colony formation is not an effective defense against such formance of microscopic swimmers, so the viscous resis- heliozoan predators, but other passive predators such as tance of the water to being sheared between swimming foraminiferans and radiolarians should also be tested. If choanoflagellates and nearby axopod surfaces is most the mechanisms we documented in this study also oper- likely the mechanism responsible for the reduction in ate for other passive predators, this would suggest that swimming speed in the CZ (Vogel 1994). predation on the protozoan ancestors of animals by such We found that S. helianthica that contacted axopodia passive protozoan predators might not have been an were transported by A. nucleofilum using either rapid important selective factor in the evolution of multicellular- axopodial contraction, (~75% of the transports) or axopo- ity. However, colony formation by choanoflagellates dial flow. In contrast, Suzaki et al. (1980) found that A. nu- increases susceptibility to capture by raptorial predators cleofilum used only axopodial flow to transport the and decreases vulnerability to ingestion by suspension- flagellated cryptophyte Chilomonas and the ciliates feeding ciliates (reviewed by Koehl 2020). Many active Paramecium and Stentor, while both rapid contraction and heterotrophic eukaryotes show size-selective feeding, with axopodial flow were used to transport the flagellated uni- preferences for large prey in some cases and smaller prey cellular alga Chlamydomonas and the ciliate Tetrahymena. in others (e.g. Fenchel 1980, 1986; Hansen et al. 1996; Jonsson 1986; Koehl 2020; Montagnes et al. 2008; Strom and Loukos 1998; Verity 1991; Weisse et al. 2016), so col- Susceptibility of unicellular vs. multicellular ony formation may well increase or decrease susceptibility choanoflagellates to predation by passive protozoan to being eaten in ecosystems where such predators are predators abundant. Models of passive protozoan predators indicate that large, motile prey are more likely than small ones to contact a predator (Fenchel 1982; Shimeta and Jumars 1991), as ACKNOWLEDGMENTS long as the prey item is not significantly larger than the This research was supported by NSF grant #IOS-1655318 predator (Jabbarzadeh and Fu 2018). This suggests that (to M. A. R. Koehl), by a SURF Rose Hills Fellowship (to W. multicellular choanoflagellates might be more susceptible Kumler), and by the Undergraduate Research Apprentice- than unicellular choanoflagellates to predation. In contrast, ship Program at the University of California, Berkeley. We it has been suggested that the larger size of choanoflagel- thank K. Waxman and T. Cooper for technical assistance. late colonies makes them less vulnerable than unicellular choanoflagellates to protozoan predators and hence that predation might have been an important selective factor in LITERATURE CITED the evolution of multicellularity in the ancestors of animals (Boraas et al. 1998; Fenchel 1986; Jonsson 1986; Richter Armstrong, H. A. & Brasier, M. D. 2005. Microfossils, 2nd ed. Blackwell Publishing, Malden, MA. and King 2013; Stanley 1973). Our study addresses these Arndt, H., Dietrich, D., Auer, B., Cleven, E. J., Grafenhan, T., contradictory suggestions. Weitere, M. & Mylnikov, A. P. 2000. Functional diversity of het- We found that the feeding efficiency (ratio of the num- erotrophic flagellates in aquatic ecosystems. In: Leadbeater, B. ber of prey taken into a phagosome in the cell body of the S. C. & Green, J. C. (ed.), The Flagellates. 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