Ii ABSTRACT CORRADINO, GABRIELLE. the Characterization

ABSTRACT

CORRADINO, GABRIELLE. The Characterization and Ecology of a Heterotrophic Nanoflagellate from the Coastal Water of North Carolina. (Under the direction of Dr. Astrid Schnetzer).

Nanoflagellates (2-20 µm) are key components of planktonic food webs and include single- celled eukaryotic autotrophs, heterotrophs and mixotrophs. The heterotrophic nanoflagellates

(HNANs) are the primary top-down consumers of bacteria and other picoplankton through grazing. HNANs facilitate nutrient remineralization of organic matter and act as an intermediate for energy and carbon flow to higher trophic levels. While these organisms play pivotal ecological roles and are ubiquitous in marine and freshwater systems, limited information is available on species composition change over spatiotemporal scales or on trophic interactions between individual prey and predator taxa. A major obstacle to resolving these important dynamics, and a key reason why HNAN assemblages are commonly treated as a single predatory group (‘black box’), is the paucity of easily distinguishable morphological characteristics.

Employing molecular tools has proven powerful in gaining insight into microbial community structure and functional diversity for HNANs and has, in combination with culturing and experimentation, become an essential tool. This thesis aimed to improve our general understanding of HNANs by characterizing a novel HNAN species and by describing its physiology and ecology.

Chapter 1 of this dissertation focused on the isolation, culturing and characterization of a marine

HNAN collected off the North Carolina coast. The HNAN was characterized by scanning and transmission electron microscopy, light microscopy and molecular methods. The small subunit ribosomal RNA gene (18S) was fully sequenced and supported the taxonomic placement of the

a ii

HNAN within the order Bicosoecida as a novel genus and species. On the basis of behavioral observations, morphology and molecular identification, the genus Coniuncta and species canzanellaia was proposed.

Chapter 2 explored the feeding ecology of C. canzanellaia offering various prey types at increasing prey densities. My main research objectives were to i) determine whether C. canzanellaia selects for autotrophic or heterotrophic prey, ii) examine differences in growth (µ) and grazing responses feeding on varying prey, iii) determine how HNAN stoichiometry may be impacted, and iv) estimate gross growth efficiency (GGE) in relation to diet. To answer these research questions, prey was offered in form of a mixed heterotrophic bacterial assemblage and two common picophototrophs, the cyanobacterium Synechoccocus, and the picoeukaryote

Ostreococcus. In an additional experiment, all three prey were made available as a mixed assemblage and changes in µ, ingestion rates (IR), flagellate biovolume (BV), cellular stoichiometry and GGE were examined for the HNAN. Our findings are discussed in context with previous flagellate feeding studies and the role that C. canzanellaia’s trophic interactions may play for carbon and nutrient transfer discussed.

In chapter 3, predator-prey dynamics for C. canzanellaia feeding on heterotrophic bacteria and

Ostreococcus were further explored over an ecologically pertinent temperature range (at 10°C,

15°C and 25°C). My main research objectives were to i) examine whether µ and IR rates would be prey- and/or temperature-dependent and ii) investigate if the flagellate could maintain GGEs across a temperature range characteristic for year-round ambient conditions in North Carolina coastal waters. Differences in the flagellate’s µ and grazing response and GGEs were compared to existing HNAN studies. In addition, the impact that HNANs, displayed in their feeding ecology, may have standing stocks of autotrophic and heterotrophic prey populations is discussed.

This study is one of a limited number that describe the successful isolation and characterization a novel HNAN species and genus that further led to the interrogation of trophic interactions on a species-specific level and allowed for estimates of carbon and nutrient transfer from picoplankton to a nanograzer. It is my hope that the findings of this dissertation regarding C. canzanellaia can contribute to a growing overall understanding of HNAN ecology.

The Characterization and Ecology of a Heterotrophic Nanoflagellate from the Coastal Water of North Carolina

by Gabrielle L. Corradino

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Marine, Earth, and Atmospheric Sciences

Raleigh, North Carolina 2020

APPROVED BY:

______Dr. Astrid Schnetzer Dr. Ryan Paerl Committee Chair

______Dr. Chris Osburn Dr. Adrian Marchetti

DEDICATION

This dissertation is dedicated to my parents and George who have been my support throughout my PhD.

BIOGRAPHY

Gabrielle Corradino was born in New Haven, Connecticut. She earned her Bachelor of Arts degree in Biology with a minor in Psychology from Salve Regina University. She went on to teach middle school science and work for several years before going back as a graduate student.

In 2015, Gabrielle completed her Masters of Science degree in Biology and conducted her PhD at North Carolina State University with Dr. Astrid Schnetzer. After her PhD Gabrielle will be starting a Knauss marine policy fellowship in Washington, DC with NOAA.

iii

ACKNOWLEDGMENTS

Pursuing my PhD at NC State was one of the best decisions I have made and I owe everything to my advisor Dr. Astrid Schnetzer. The support provided to me by Dr. Schnetzer is something that

I simply cannot put into words and I am very appreciative of everything that she has done for me personally and academically.

A big thank you to my committee members Dr. Chris Osburn, Dr. Ryan Paerl and Dr. Adrian

Marchetti for their support and guidance throughout all stages of my research. This dissertation has been made better with their input and help, and for that, I am grateful. Additionally, I would like to thank Dr. Carmelo Tomas from UNC-W for his kindness and guidance through each step of characterizing this plankton. I would also like to thank the Associate Dean Dr. Mike Carter, and the whole group from the Dissertation Completion Grant for editing my thesis and making it stronger.

I would like to acknowledge the Schnetzer lab members (past and current) whose laughter and support were always welcome. Also thank you to the crew of R/V Brown, R/V Pelican, RAPID and GOMECC collaborators, and their team members. This study was partially supported through funds from the National Science Foundation grant (OCE-1459406), Department of the

Interior Southeast Climate Adaptation Science Center, Sigma Xi GIAR and by the National

Geographic Society.

Over the past decade, I also owe quite a bit to Dr. Jameson Chase of Salve Regina University for his continuous support of my work, Dr. David Vasseur of Yale University and Dr. John DeLong of the University of Nebraska. Dr. Vasseur and Dr. DeLong introduced me to freshwater iv

plankton ecology in the EEB department at Yale University and I will forever be appreciative of their valuable teachings, kindness and support.

Lastly, a special thanks goes to my wonderful family, parents, friends and my significant other.

You have earned this degree with me, and I am so very thankful for each one of you.

TABLE OF CONTENTS

LIST OF TABLES ...... ix

LIST OF FIGURES ...... xii

CHAPTER 1 – Coniuncta canzanellaia gen. et sp. nov. (Bicosoecida), a Previously Uncultured

Heterotrophic Nanoflagellate from North Carolina Coastal Waters

ABSTRACT ...... 1

INTRODUCTION ...... 2

MATERIALS AND METHODS ...... 3

Collection, Isolation and Growth ...... 3

Microscopy ...... 4

Molecular Analyses ...... 6

Phylogenetic Analyses ...... 7

RESULTS ...... 8

Light- and Epifluorescence Microscopy ...... 8

Electron Microscopy ...... 9

Phylogenetic Analyses ...... 10

DISCUSSION ...... 11

Molecular Analyses ...... 11

Morphological Analyses ...... 12

Behavioral Observations ...... 13

Taxonomic Summery ...... 15

Acknowledgments...... 16

REFERENCES ...... 17

CHAPTER 2 – Grazing of the Heterotrophic Nanoflagellate Coniuncta canzanellaia on Prokaryote and Eukaryote Prey: Ingestion Rates and Gross Growth Efficiency ABSTRACT ...... 36

INTRODUCTION ...... 38

MATERIALS AND METHODS ...... 40

Prey and Predator Origins and Culture Conditions...... 40

Grazing Experiment ...... 41

Nanoflagellate Growth, Ingestion Rates and Biovolume ...... 42

Elemental Ratios and Gross Growth Efficiencies ...... 43

Statistical Analyses ...... 44

RESULTS ...... 45

Prey and Predator Biovolume and Nutrient Contents ...... 45

Flagellate Growth and Ingestion ...... 46

Gross Growth Efficiencies ...... 48

DISCUSSION ...... 48

Nanoflagellate Growth and Ingestion ...... 48

Nanoflagellate Cellular Stoichiometry, Biovolume and Gross Growth Efficiencies ... 50

Impact on Picoplankton Standing Stocks ...... 52

REFERENCES ...... 53

CHAPTER 3 – The Impact of Diet and Temperature on Growth and Grazing of the

Heterotrophic Nanoflagellate Coniuncta canzanellaia

ABSTRACT ...... 75

INTRODUCTION ...... 76

vii

MATERIALS AND METHODS ...... 77

Study Organisms ...... 77

Grazing Experiment ...... 78

Biovolumes and Cell Carbon Estimates ...... 79

Flagellate Growth and Ingestion Rates ...... 79

Gross Growth Efficiencies ...... 80

Statistical Analyses ...... 80

RESULTS ...... 81

Prey and Predator Biovolume ...... 81

Flagellate Growth and Ingestion ...... 82

Gross Growth Efficiencies ...... 83

DISCUSSION ...... 84

Nanoflagellate Growth and Ingestion Rates and Gross Growth Efficiencies ...... 86

Impact on Picoplankton Standing Stocks ...... 87

REFERENCES ...... 90

CONCLUSION ...... 108

REFERENCES ...... 111

viii

LIST OF TABLES

CHAPTER 1

Table 1: Nearest NCBI 18S BLAST matches (n = 17) to C. canzanellaia. NCBI

sequence lengths ranged from 1,455 to 1,796 bp. All E-values scored at 0.0. .... 25

Table 2: Comparison of key phenotypic characteristics for C. canzanellaia and other

common nanoflagellate genera. N = number of individuals characterized per

species; sources of each study shown in footnotes. Note: differences in

methodology such as EM protocols exist between the different studies ...... 26

CHAPTER 2

Table 1: Initial prey and predator abundances (cells mL-1) with their standard deviation

(±SD) in each of the four experiments. Prey densities in the control flasks

started at ~106 cells mL-1 (no flagellate added). HB = heterotrophic bacteria;

Syn = Synechococcus; Ost = Ostreococcus lucimarinus; Mix = mixed prey

assemblage...... 60

Table 2: Averaged prey carbon (C), nitrogen (N), phosphorus (P) content in fg cell-1

and normalized by biovolume in fg µm-3 in the control treatments. * For Ost

cell content estimates were available for T0 observations only...... 61

Table 3: Carbon (C), nitrogen (N), phosphorus (P) cell content and elemental ratios for

C. canzanellaia. Cell contents for C. canzanellaia grazing on varying prey

(HB, Syn, Ost and Mix treatments) are shown in pg cell-1 and normalized by

biovolume in fg µm-3. Flagellate nutrient concentrations are shown in

comparison to estimates reported elsewhere. Values are averages from at least

2 or more observations. a study used bacteria raised under balanced growth

conditions and under C-limitation. bC content differences linked to cell

shrinkage during preservation. HB = mixed heterotrophic bacteria; Bact Cult

= bacterial culture...... 62

Table 4: Elemental ratios for prey and C. canzanellaia fed on each prey type (n = 2) ..... 63

Table 5: Specific growth rate (µ, d-1) and ingestion rate (IR, cells flag-1d-1 and pg C

flag-1d-1) for C. canzanellaia averaged over triplicate incubations ±SD (see

exact grazer and prey abundances in Table 1) ...... 64

Table 6: A comparison table of growth (µ) and ingestion rates (IR) across various

experiments with nanoflagellate species and picoplankton prey. The average

or ranges are reported when available with standard deviation in parentheses.

Note: differences in experimental methodology exist between the studies. HB

= heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Pro =

Prochlorococcus; Bact Cult = bacterial culture; Chlor = Chlorocystis ...... 65

SUPPLEMENTAL TABLE Table S1: Prey growth (µ d-1) in control treatments shown as average ±SD ...... 67

CHAPTER 3

Table 1: Initial prey and predator abundances (cells mL-1) with their standard deviation

(±SD) in each of the experiments. Prey in Control = starting concentrations in

flasks that had no flagellate added. HB = heterotrophic bacteria; Ost =

Ostreococcus lucimarinus ...... 95

Table 2: Specific growth rate (µ, d-1) and ingestion rate (IR, cells flag-1d-1 and pg C

flag-1d-1) for C. canzanellaia averaged over triplicate incubations ±SD ...... 96

Table 3: The Q10 values across all temperatures and prey treatments with C.

canzanellaia ...... 97

Table 4: A comparison table of growth (µ) and Q10 rates across various experiments.

The average is reported at the tested temperature. Note: differences in

experimental methodology exist between the studies. HB = heterotrophic

bacteria; Ost = Ostreococcus; Syn = Synechococcus; Bact Cult = bacterial

culture; Mix = HB + Syn + Ost ...... 98

SUPPLEMENTAL TABLES Table S1: Average cell biovolumes of HB and Ost at 10, 15 and 25°C at TF (n = 40;

±SD) ...... 99

Table S2: Prey growth (µ d-1) in control treatments shown as average (n = 40; ±SD) ...... 100

LIST OF FIGURES

CHAPTER 1

Figure 1: Sampling site (star) in Bogue Sound, North Carolina (34°43’17.77”,

76º45’33.93”W) (ArcGIS Desktop, Release 10) ...... 26

Figure 2: Several nanoflagellate cells (black arrows) attached to an elongated particle

(P) with one of their flagella (60x, scale bar = 10 µm). (B) Single

nanoflagellate with ingested fluorescently-labelled bead visible inside the cell

(white arrow) (60x, scale bar denotes 5 µm) ...... 27

Figure 3: Scanning electron microscopy (SEM) image under 6,500x. Only the anterior

flimmer flagellum is covered with mastigonemes (right arrow). Flagellum

ends in a paddle-like tip (left arrow). Scale bar denotes 5 µm...... 28

Figure 4: SEM whole-mount under 20,000x showing the front of the cell and location of

food uptake (black arrow). Mastigonemes are shown surrounding the flimmer

flagellum (white arrow). Scale bar denotes 2 µm ...... 29

Figure 5: Transmission electron micrograph (TEM) of the nanoflagellate cell with a

visible microtubular flagellar root (A) and the associated basal body above the

root (B), nucleus (N) and mitochondria (M). Scale bar denotes 1 µm ...... 30

Figure 6: Maximum Likelihood (ML) phylogenetic tree created with the Tamura-Nei

model (Tamura and Nei, 1993). The tree with the highest log likelihood (-

7016.56) is shown. The percentage of trees in which the associated taxa

clustered together is shown next to the branches. The tree consists of the

closest NCBI Blast sequence matches (11) for the isolated nanoflagellate C.

xii

canzanellaia and lists the chlorophyte Oocystis solitaria as outgroup. Silva

sequences of the 18S were used to create pairwise distance estimates. The

sample collection location is listed for each match with the GenBank

accession number. Blue highlights marine and the green freshwater organisms 31

Figure 7: Maximum Likelihood (ML) phylogenetic tree created with the Tamura-Nei

model (Tamura et al. 1993). The tree with the highest log likelihood (-

17054.28) is shown. The ML tree consists of 17 previously characterized

nanoflagellate genera/species, the isolated nanoflagellate Coniuncta

canzanellaia and using Oocystis solitaria as the outgroup. Silva sequences of

the 18S were used to create pairwise distance estimates. Branch lengths

represent the number of substitutions per site. This analysis involved 18

nucleotide sequences with a total of 2,540 positions in the final dataset. The

GenBank accession numbers are listed with each species name. The

organisms’ origins are marine except indicated otherwise: F = Freshwater or S

= Soil ...... 32

SUPPLEMENTAL FIGURES Figure S1: C. canzanellaia attached and swimming near several Rhizosolenia sp. cells.

The scale bar denotes 20 µm ...... 33

Video S2: C. canzanellaia attached to a particle and creating a feeding current with the

anterior flagellum. Open access video link:

drive.google.com/open?id=1zBuAiOuHNJunSIQ_iVJoKqgy2z9G_-Kq ...... 34

xiii

CHAPTER 2

Figure 1: Average cell biovolume (µm-3; +SD) of the prey at T0 and TF (n = 40 each) .... 65

Figure 2: Average cell biovolume of C. canzanellaia fed on various prey (n = 30; +SD).

# denotes a significant difference between the T0 and T1 or TF cultures (p <

0.05) ...... 66

Figure 3: C. canzanellaia C:N, N:P and C:P ratios (n = 2) fed on heterotrophic bacteria

(HB), Synechococcus (Syn), O. lucimarinus (Ost) and being offered a mix of

these three prey types (Mix) ...... 67

Figure 4: C. canzanellaia growth rates (µ) over various prey concentrations (cells mL-1)

for each of 13 sets of triplicates. Averages shown with SD...... 68

Figure 5: Ingestion rate (IR in pg C flag-1 d-1) at each of the initial prey concentrations

(n = 36) ...... 69

Figure 6: C-based ingestion rates (IR; pg C flag-1d-1) in relation to flagellate growth (µ,

d-1). Also shown is the average for preying on the mixed assemblage ±SD.

Note x-axes is log-transformed ...... 70

Figure 7: The average HNAN GGEBV and GGEC grazing on HB, Syn, Ost and Mix (n =

3). * denotes a significant difference between GGEBV and GGEC for Ost (p <

0.05) ...... 71

CHAPTER 3 Figure 1: Monthly averages for temperature data from 2015 to 2019 from the National Data Buoy (station BFTN7) for NOAA at Beaufort (34°43’17.77”, 76º45’33.93”W), North Carolina, (https://www.ndbc.noaa.gov/station_page.php?station=bftn7) are shown ±SD (n= 5) ...... 101 xiv

Figure 2: Average prey cell biovolumes (µm3 cell-1; ±SD) at T0 and TF at 10°C, 15°C

and 25°C (n = 40 each). * denotes a signficant difference in BV comparing

T0 and TF for Ost at 10°C (p <0.05) ...... 102

Figure 3: The average cell biovolume of C. canzanellaia fed on various prey at 10, 15

and 25°C (n = 30; ±SD). T0 biovolumes are averaged across temperatures (n

= 60 each) ...... 103

Figure 4: Average C. canzanellaia growth rates (µ) at 10°C, 15°C and 25°C feeding

on a mixed bacterial assemblage (HB) or Ostreococcus (Ost) ...... 104

Figure 5: Ingestion rate (IR in pg C flag-1 d-1) at each temperature (n=18) ...... 105

Figure 6: The GGEBV and GGEC for C. canzanellaia grazing on HB and Ost at all

three temperatures ...... 106

Figure 7: C-based ingestion rates (IR; pg C flag-1d-1) in relation to flagellate

growth (µ, d-1) ±SD. Note x-axes is log-transformed ...... 107

CHAPTER 1

Coniuncta canzanellaia gen. et sp. nov. (Bicosoecida), a Previously Uncultured Heterotrophic

Nanoflagellate from North Carolina Coastal Waters

Abstract

Supported through molecular and morphological analyses, we introduce a new bicosoecid genus and species, Coniuncta canzanellaia, isolated from coastal waters in North Carolina.

Comparison with existing sequence data (SSU rDNA) placed C. canzanellaia closest to the genus Bicosoeca within the order of the Bicosoecida and morphological characteristics indicated both similarities with the family Cafeteriidae and Bicosoecidae. C. canzanellaia was biflagellated with an anterior flimmer flagellum that pulls and glides the cell and the posterior flagellum frequently attaches to surfaces. The flimmer flagellum was covered with tripartite mastigonemes, whereas the shorter flagellum was acronematic. An aloricate cell body and a distinct flattened flagellum tip were demonstrated under electron microscopy. The flagellate was identified as a heterotroph based on the absence of pigment and live observations showing the flagellate ingesting bacteria-sized fluorescent beads in interception feeding mode. While isolated from the western Atlantic, environmental sequence data suggests that this organism is also found in the Aegean, Black and Caribbean seas.

Key Words: Coniuncta canzanellaia, Nanoflagellate, Bicosoecid, Electron Microscopy,

Ribosomal Genes, North Carolina

Introduction

Heterotrophic nanoflagellates (HNANs) are phylogenetically diverse protists (2-20 µm in size) found throughout the ocean (Azam et al., 1983; Logares et al., 2012; Nakayama, 2015). As primary consumers of archaea, bacteria and picoeukaryotes, HNANs play an important role in shunting carbon towards higher trophic levels or in remineralizing nutrients within the microbial loop (Azam et al., 1983; Fenchel, 1982; Sherr and Sherr, 2002; Yang et al., 2018). Discerning their biogeochemical roles requires knowledge of how HNAN assemblages are structured and reassemble in response to environmental and biological drivers and an understanding of group or species-specific physiological traits. Acquiring this information, however, has been a challenge due to their minute cell size and general lack of morphological features that allow to easily distinguish taxa using traditional microscopy (Gast et al., 2018; Jones and Lennon, 2010; Monier et al., 2013). Molecular surveys (mainly targeting SSU rDNA) have begun to provide insight into taxonomic affiliations, evolution and biogeography but these analyses also suggest that the majority of HNAN lineages in nature remain yet uncharacterized (Kühn et al., 2004; Massana et al., 2004; Nakayama, 2015; Park et al., 2006).

In addition to genetic surveys, the isolation and cultivation of HNANs has aided the study of individual taxa across several major groups including the chrysomonads, bicosoecids, bodonids and choanoflagellates (see review in Juergens and Massana, 2008). Information on morphology, genetics and basic physiology from culture trials combined with the development and employment of group/species-specific detection approaches (e.g., fluorescence in situ hybridization or quantitative polymer chain reaction assays) has allowed to better resolve in situ spatiotemporal patterns for previously unknown, but often ecologically important HNANs

(Massana et al., 2007; Massana et al., 2009). Furthermore, in combination with transcriptomic analysis, new insight is gained into the diverse nutritional diversification and functionality among nanoflagellates (Beisser et al., 2017; Caron et al., 2017).

In this study, a previously uncharacterized nanoflagellate, C. canzanellaia, is introduced that constitutes a new heterotrophic species and genus within the order Bicosoecida. The bicocoecids are a common HNAN group found in both freshwater and marine environments (del Campo and

Massana, 2011). Primarily based on molecular identification there are ~15 reported genera within the order Bicosoecida, while based on morphological analyses there are ~12 recognized genera (Guiry, 2019), however more in depth analyses of ultrastructural features had led to an estimate of 40+ morphospecies (Moestrup and Thomsen, 1976; Preisig et al., 1991). The combined information from molecular, phylogenetic, behavioral and morphological analyses supports the classification of the newly described genus and species.

Materials and Methods

Collection, Isolation and Growth

Plankton surface tows (150 µm mesh size) were conducted in eastern Bogue Sound, North

Carolina, during October 2015 (34°43’17.77”, 76º45’33.93”W; Fig 1). The nanoflagellate was initially captured together with large diatom cells of the genus Odontella. The diatoms, to which the flagellate cells were partially attached, were grown using F/20 media (Guillard and Ryther,

1962) at 22ºC under a L:D cycle of 14:10 and at 75 μE m−2 s−1 using cool white fluorescent light.

The flagellates tended to numerically dominate the diatom cultures within 2 weeks of each transfer. The <5 µm-sized cells were separated from the microalgae using a 20 µm nitex screen

and further dilution steps. Microscopical analyses conducted with brightfield and epifluorescence microscopy indicated the presence of a few pigmented cells among a majority of colorless flagellates, and hence, growth conditions were adjusted to select for the heterotrophic and/or mixotrophic type(s). Cultures were grown at the original 14:10 L:D cycle with F/20 and in the dark with a barley seed. After 2 months, general cell characteristics were reexamined using light- and epifluorescence microscopy, and genetic analyses were conducted (see below) to confirm the number of Operational Taxonomic Units (OTUs) or species present.

Microscopy

Light and Epifluorescence Microscopy

General characteristics of pigmentation and size were examined using a combination of light- and epifluorescence microscopy. Natural chlorophyll a fluorescence was observed using an

Olympus BX53 in fluorescence mode (Excitation/Emission (nm) of 440/685 for chlorophyll a).

Unstained HNAN cells were observed with differential interference contrast with both living and glutaraldehyde (2% final concentration) preserved cells (Grover and Chrzanowski, 2009). For fixation, the nanoflagellate cells were concentrated onto blackened 0.2 µm filters

(MilliporeSigma) following previously published protocols (Sherr et al., 1993). Both DIC and epifluorescence images were captured with the Olympus DP73 monochrome digital camera and analyzed with the Olympus cellSens Dimension 1.13 software.

Electron Microscopy

Scanning Electron Microscopy (SEM) was conducted for the culture that returned only colorless cells (grown in the dark) for which molecular analyses also confirmed the presence of a single

species (see details below). Nanoflagellate cells were fixed for SEM, following a previously published protocol (Tomas et al., 2012). Cells were fixed sequentially, first with 1% osmium in media (0.2 µm filtered seawater with a salinity of 40), passively filtered, rinsed in media and then fixed in 2.5% glutaraldehyde in 40% salinity of media and concentrated on a 3.0 mm

Poretic filter (Osmonics) and rinsed with DI water. Then, the cells were dehydrated through a graded ethanol series. The process ended with three washes of 100% EtOH. After dehydration, the cells were critical-point dried (Tousimis Samdri-795, Tousimis Research Corporation), mounted on aluminum stubs and gold–palladium sputter coated (Hummer 6.2 Sputter System,

Anatech). The samples were viewed in a JEOL JSM 5900LV SEM (JEOL) operated at 15 kV at the CALS Center for Electron Microscopy at North Carolina State University. SEM was followed by Transmission Electron Microscopy (TEM) to resolve internal ultrastructure and position of organelles within the nanoflagellate. Cells for TEM were fixed sequentially, first in

1% osmium in 40% salinity of media (0.2 µm filtered seawater), filtered, rinsed in media and then fixed in 2.5% glutaraldehyde in salinity of 40 for the media. The cells were concentrated via centrifugation between fixatives and after rinsing with 40% media and then pre-embedded in a

2% preparation of warm agarose, pelleted by centrifugation and solidified on ice. The pellet was removed, cut into 1mm3 blocks for dehydration and embedding in Spurr’s resin (Spurr, 1969).

Ultrathin sections were obtained using a Leica UC6rt Ultramicrotome (Leica Microsystems), and post-stained with 4% aqueous uranyl acetate for 60 minutes followed by lead citrate for 4 minutes according Reynolds (1963). All TEM observations were done using a JEOL

JEM1200EX TEM at 80 kV.

Molecular Analyses

DNA Extraction Cloning and Sequencing

Aliquots of 100 ml from nanoflagellate cultures were concentrated onto 25 mm glass fiber filters

0.7 µm pore size and stored at -20°C. As noted, these cultures included cells grown at 14:10 L:D cycle with F/20 and those kept in the dark with a barley seed. The DNA was extracted using a

DNA Isolation Kit (MoBio) and the 18S rDNA was amplified using universal eukaryotic primers

EukA (5’-AACCTGGTTGATCCTGCCAGT-3’) and EukB (5’-

GATCCTTCTGCAGGTTCACCTAC-3’) (Countway and Caron, 2006; Medlin et al., 1988).

Briefly, the PCR reaction contained 5 µl of template DNA, 1 µl 0.1 µM of each primer, 18 µl of nuclease-free water and 2X DreamTaq Green PCR Master Mix (DreamTaq DNA polymerase,

2X Green buffer, 0.4 mM of each dNTP, 4 mM MgCl2) (ThermoScientific) for a total volume of

50 µl. The PCR was run (BioRad, Thermocycler) using an initial denaturation step for 10 minutes at 95ºC, followed by 35 cycles of 30 seconds at 94ºC, 30 seconds at 55ºC, and 1 minute at 72ºC, with an extension for 8 minutes at 72ºC. The PCR products were cleaned and then ligated using a cloning kit (Qiagen) following the manufacturer’s instructions. Ligation was achieved using electro-competent cells (Thermofisher, OneShot Top 10) and electroporation

(BioRad, Gene Pulser Xcell). Both cultures were plated on LB Agarose plates with 50 μg mL-1 kanamycin (Sigma-Aldrich) and stored at 37ºC for 24 hours. Individual colonies (n = 10) were picked from each plate/culture and grown on liquid broth with 2 µl mL-1 Kanamycin for 24 hours. After plasmid purification (GeneJet, Plasmid Miniprep Kit) and DNA quantification through qubit (Invitrogen, 2.0 fluorometer), amplicon presence was confirmed through electrophoresis with 2% SYBR safe E-Gels (Invitrogen). Clones were sequenced using 575FWD

(5’-GTAATTCCAGCTCCAATAGC-3’) (Weekers et al., 1994) to compare sequence similarities

over this hypervariable region of the 18S (570 FWD). Based on these comparisons, a full SSU rDNA sequence was obtained for the culture kept in the dark. All sequencing was conducted using outside services (SimpleSeq, Eurofin). The combination of EukA, EukB, NS4 (5’-

CTTCCG TCAATTCCTTTAAG-3’), NS5 (5’-AACTTAAAGG AATTGACGGAAG-3’)

(White et al., 1990) primers ensured at least 2x coverage across the 18S gene for the four tested clones. Universal primers 1055F/1055R (Elwood et al., 1985) commonly used to target eukaryote 18S did not yield amplification. In addition, we attempted amplification of the LSU and ITS regions using varying primer sets. These included ITS1/ITS2/ITS3/ITS4 (White et al.,

1990) that had been successfully applied for a variety of stramenopile targets including HNANs belonging to the genera Meyerella and Halocafeteria (Fawley et al., 2005; Raja et al., 2017).

Primers variants for the ITS and LSU ITS1F/ITS1R which had been successfully used on other stramenopiles like Discostella pseudostelligera and Thalassiosira pseudonana (Hevia-Orube et al., 2016) and the primer set JITSF/JITSR, which was successfully used on the amoeboflagellate

Tetramitus dokdoensis, was also tested (Garstecki et al., 2005; Pin et al., 2001). Despite multiple trials, (including varied combinations of forward and reverse primers including EukA and EukB) and troubleshooting PCR settings (e.g., hot start and temperature gradients), no amplification was achieved in addition to the 18S.

Phylogenetic Analyses

All 18S sequence reads were quality-trimmed followed by manual review of the chromatogram files in BioEdit (Hall, 1999). After alignment, the consensus sequence was blasted against the

National Center for Biotechnology Information database (NCBI, http://www.ncbi.nih.gov) to provide taxonomic information for the unknown flagellate (Altschul et al., 1997). The closest

matching partial sequences (unknown eukaryotes) were retrieved from the NCBI database. Also retrieved were the closest matches to known taxa from the Silva database (https://www.arb- silva.de/) for phylogenetic analysis using MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms (Kumar et al., 2018). Phylogenetic relationships were inferred by using a maximum likelihood (ML) approach (Felsenstein, 1981) with the Tamura-Nei model

(Tamura and Nei, 1993). Initial tree(s) for the experiential search were acquired automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. ML trees were constructed with 1,000 replicates to produce bootstrap values (Kumar et al., 2018).

Results

Light- and Epifluorescence Microscopy

Flagellate isolates from the North Carolina coast (Fig. 1) were continually grown at a 14:10 light:dark (L:D) cycle plus F/20 continued to contain a mix of pigmented and colorless flagellates. Dark growth conditions, however, exclusively selected non-pigmented cells (Fig 2A) and further examination via epifluorescence microscopy confirmed that transferring the flagellate back to original growth conditions (14:10 L:D cycle plus F/20 and/or barley seed) did not result in the return of pigmented cells. Light microscopy information on pigmentation suggested that the selected species was a phagotroph sustained by feeding on heterotrophic bacteria that grew on a barley seed. The flagellate’s ability to ingest bacteria-sized prey was further confirmed in short (1 hr) feeding experiments using 0.5µm fluorescently labelled beads (Fluoresbrite YG) (Fig

2B). The HNAN cells were of spherical shape with an average size of 5.10 + 0.51 µm (+SD, n =

50). Each cell (n = 50) was equipped with one long anterior flagellum of an average length of

19.38 + 4.8 µm that pulled the cell and a trailing posterior flagellum of an average length of

10.43 + 1.69 µm.

Additional observations using light microscopy showed that, as soon as particles formed within the culture, the nanoflagellate would frequently attach to them (Fig 2A). During attachment, the shorter flagellum acted as an anchor and the second, longer flagellum acted as a whip, which created a feeding current. Direct interception feeding using the flagellum seemed to be the dominant mode of food concentration especially during attached phases; a strategy common for nanoflagellates (Fenchel, 1982). In addition to the attachment, the HNAN was observed swimming or resting on the bottom of the slide, not attached to a substrate with little to no movement. While swimming, the longer flimmer flagellum was used to pull the nanoflagellate cell forward as the other posterior flagellum trailed behind, this behavior has been reported previously for other HNANs (Fenchel, 1986).

Electron Microscopy

SEM confirmed that there were no surface coverings or scales and that the flimmer flagellum was covered with tripartite filamentous appendages called mastigonemes (hairs), whereas the shorter smooth flagellum was acronematic (Fig 4). SEM also indicated the presence of a ~900 nm flattened paddle on the end of the anterior flagellum (Fig 3). Finally, TEM allowed for the characterization of the ultrastructure of the nanoflagellate and demonstrated a subapically inserted flagellum (Fig 5). The posterior flagellum was positioned within a groove located to the side of the cell. The microtubular flagellar root and the associated basal body above the root

were also visible. The mitochondria was located near the nucleus. There were no extrusomes, lorica or surface scales observed on the nanoflagellate cell body.

Phylogenetic Analyses

Initial sequencing of the 575Fwd region consisting of read lengths from 450 to 1,000 bp resulting in sequence similarities of 100% for 9 out of the 10 clones with one showing a one-base difference (99% similarity) confirming the presence of a single species. Full-length 18S sequences (n = 4) read between 1,790 and 1,930 bp and they all matched at a 100%. BLAST results returned the closest matches to uncultured eukaryotes from marine, and subsequently, freshwater environments (Table 1 and Fig 6). While sequence similarities of 96% and higher indicated affiliations with marine organisms from the Aegean Sea (AY789790.1), Caribbean

Sea (GU823081.1) and the Black Sea HM749932.1, similarity values dropped quickly to 90% or less for freshwater taxa (Fig 6) with the highest log likelihood of -7016.56 with a total of

2,071 positions. The closest matching sequence for an unknown eukaryote (AY789790.1) returned a robust bootstrap value of 82 and a well-supported grouping (bootstrap value = 100) among the uncharacterized marine representatives (Fig 6). The first BLAST matches that could be associated with previously characterized HNANs were returned at sequence similarities of

~88 to 89% and included matches to Bicosoeca vacillans (89.6%), Bicosoecida gen. 1 sp. EK-

2010a (89.6%), Nerada mexicana (89.4%) and Pseudophyllomitus vesiculosus (88.9%); generally nesting the nanoflagellate within the order Bicosoecida (Table 1 and Fig 7).

Referring the nanoflagellate’s position using previously characterized heterotrophic stramenopiles from common clades returned the highest log likelihood of -17054.28 for a total of

2,540 positions (Fig 7).

Discussion

Molecular Analyses

Available molecular evidence suggests the bicosoecids are a basal monophyletic lineage within the stramenopiles (kingdom Heterokonta) which encompasses several important, cosmopolitan, marine HNAN groups such as uncultivated marine stramenopiles (MASTs), Cafeteria spp. and

Pirsonia spp. (Fenchel, 1982; Kühn et al., 2004; Massana et al., 2004). In this study, phylogenetic analyses (SSU rDNA) placed Coniuncta canzanellaia closest to the genus

Bicosoeca within the family Bicosoecidae. This genus currently contains 40+ documented morphospecies (Cavalier-Smith and Chao, 2006), however, only three species (B. vacillans, B. kenaiensis and B. petiolata) have been characterized based on both gene sequence and in-depth morphological analyses from SEM and/or TEM (Cavalier-Smith and Chao, 2006; Yubuki et al.,

2015). SSU rDNA comparisons grouped the three Bicosoeca species closely together (Fig 7), while Coniuncta fell outside the clade suggesting the parting of the nanoflagellate into a novel genus and species with a bootstrap value 79 (Fig 7). Its closest identified match being B. vacillans had an 89% sequence similarity. Based on comprehensive comparisons of 18S molecular data, Caron et al. (2009) suggested sequence similarities of ≤ 95% to be a conservative approach to delineate protists at the species level by the Operational Taxonomic

Unit (OTU) (Caron et al., 2009; Kim et al., 2011). Given the wide range of reported interspecies similarities that exist for well-studied protists (Caron et al., 2009; Kim et al., 2010; Okamura and

Kondo, 2015) and the lack of sequence data for the bicosoecids, approximating genus distinction based on 18S similarity is currently not feasible. The closest unidentified matches to Coniuncta that fell within a 5% sequence similarity window were associated with marine organisms from the Aegean Sea (99%), Black Sea (96%) and Caribbean Sea (98%), suggesting a more

cosmopolitan distribution for this novel genus within warm subtropical and tropical waters.

Unidentified freshwater relatives of C. canzanellaia, matched at much lower sequence similarities and originated from ponds and lake systems in Japan (89-90%), France (89%) and

Kenya (89%).

Only limited information on ITS and LSU sequence is available to support HNAN phylogeny and currently no data exists besides 18S data for the aforementioned Bicosoeca species (Raja et al., 2017). Using available ITS and LSU primers for potentially related groups did not yield any amplification for C. canzanellaia in this study. We believe that describing these attempts, albeit unsuccessful, can aid future classification efforts for the many flagellate lineages that await characterization (Nakayama, 2015; Patterson and Lee, 2000; Yubuki et al., 2015). Among the tested primers were ITS1 and ITS4, which were previously used for several ground- and surface water HNANs including Cafeteria roenbergensis, Caecitellus paraparvulus, Bodo saltans and

Paracercomonas (Wylezich et al., 2010). The ITS1, 5.8S and ITS2 region for members of the genus Halocafeteria (Raja et al., 2017) from the order Bicosoecida (Park et al., 2006) had also been successfully sequenced utilizing primers ITS1 and ITS4.

Morphological Analyses

C. canzanellaia fit within the bicosoecids based on its morphological features so far as presenting as a biflagellated cell with a forward-directed anterior flagellum with mastigonemes that pulled the cell forward and a smooth, trailing flagellum (Fenchel, 1986; Park et al., 2006;

Park and Simpson, 2010). Taxonomically important are also the presence/absence of specific ultrastructural features such as a lorica, cytopharynx and body scales (Table 2). Previous

studies (Karpov, 2000; Moestrup and Thomsen, 1976) currently support the separation of the order Bicosoecida into four families which encompass the Cafeteriidae, Bicosoecidae,

Siluaniidae, and Peudodendromonadida. Similar to members of the Bicosoecidae (B. vacillans,

B. kenaiensis and B. petiolata) and the Cafeteriidae (Cafeteria roenbergensis and Pseudobodo tremulans), C. canzanellaia was confirmed as an aloricate without body scales and carrying mastigonemes on one flagellum (Fig 3 and Table 2) (Fenchel and Patterson, 1988; Karpov, 2000;

Moestrup, 1995). While the molecular analyses placed the flagellate within the Bicosoecidae and further distance to members of the Cafeteriidae (Fig 7), ultrastructural features indicated both similarities and dissimilarities with members from both families (Table 2). For instance, a lorica was missing in C canzanellaia, but has been described for species within the genus Bicosoeca

(Picken, 1941; Yubuki et al., 2015). Additionally, C. canzanellaia lacks body scales or a cytopharynx similar to the Pseudodendromonadidae or Siluaniidae (Karpov, 2000). A feature reportedly novel for bicosoecids, which was revealed during SEM , was the presence of a small wing-like tip at the base of all intact anterior flagella (n = 8). Such a swelling at the tip of flagella has not been discussed previously for nanoflagellates. In marine bivalves and ciliates, paddle-like structures (flattened tips of cilia) have caused debate. Some refer to the structures as artifacts of

SEM sample processing (Short and Tamm, 1991) while others consider them authentic morphological features (Mohieldin et al., 2015).

Behavioral Observations

Collecting information on prey capture mechanisms in HNANs has aided taxonomic characterization. In culture, C. canzanellaia can move by gliding through the water with the longer flagellum extended outwards pulling the cell body forward and the short flagellum trailing

behind. At times, C. canzanellaia would also rest on the bottom of the glass slide with minimal movement. The observations of HNANs movement and grazing behavior has led to the categorization of three major feeding modes: filter, raptorial and interception feeding (Fenchel,

1987). C. canzanellaia attaches to available surfaces (i.e. particles, bacterial mats, the glass slide bottom) using its shorter flagellum, as it did during early isolation trials with diatom frustules

(Supplementary Material Fig S1 and V1). While attached, the flagellate used the mastigoneme flagellum to create a water current to feed on surrounding bacteria via interception feeding; a strategy commonly reported for the stramenopile genera Bodo, Cafeteria, Spumella,

Ochromonas, Monosigas and Actinomonas (Boenigk and Arndt, 2000; Christaki et al., 2005;

Ishigaki and Terazaki, 1998). Being “anchored” can aid HNANs in handling and selecting prey and food vacuole formation (Christaki et al., 2005; Davidson and Eurgain, 2001; Gonzalez et al.,

1993; Yang et al., 2018). Attachment of nanoflagellates (e.g. Salpingoeca sp., Cryothecomonas longipes) to diatoms has been reported numerous times (Galvao, 1990; Schnepf and Kühn, 2000;

Simek et al., 2004; Tillmann et al., 1999) and, in case of a bloom, resulted in up to 65% - 90% of the diatoms having cells attached (Drebes and Schnepf, 1982, 1998; Schnepf et al., 1990;

Tillmann et al., 1999). Less commonly, HNAN attachment has also been associated with parasitic behavior where the flagellates of the genera Pirsona and Cryothecomonas proceed to penetrate the diatom frustule to perform phagocytosis (Drebes et al., 1996; Schweikert, 2015).

Such parasitic behavior was never observed for C. canzanellaia. Still very little is known about the conditions that trigger these protist interactions or their overall impact on biogeochemical cycling (Schweikert, 2015). Cultured HNAN representative such as C. canzanellaia, will facilitate the characterization of these microbial interactions and their importance for energy flux within ocean environments.

Taxonomic Summary

Coniuncta is being proposed as a new genus and belongs to a heterotrophic stramenopile lineage of nanoflagellates and is has been described under the International Code of Zoological

Nomenclature (ICZI).

Coniuncta n. gen.

Cells have two unequal flagella and move by gliding, one anterior flagellum with tubular mastigonemes (tripartite flagellar hairs), with cytostome supported by a curving flagellar microtubular root. No cytopharynx, extrusomes, or lorica. Growth has been observed in water of

30-35 salinity at temperatures ranging from 10-28°C.

The name Coniuncta is Latin for attached, which is in recognition to the original isolation of this organism while it was attached to a diatom.

Type Species canzanellaia n. sp.

Description

Canzanellaia is a proposed new species with ovoid colorless cell shape ~5 μm in diameter.

Flagella of unequal length with the anterior flagellum ~19-23 µm and posterior flagellum at ~10

µm. The posterior flagellum is used to attach to particle surfaces.

A slide of preserved cells from a monoprotistan culture of Coniuncta canzanellaia is deposited in the Protist Type Specimen Slide Collection, US Natural History Museum, Smithsonian

Institution, Washington, DC

The species name canzanellaia is based off of the surname Canzanella.

Type locality

Morehead City, North Carolina, collected from surface waters in October 2015.

Assignation

Eukaryota; Stramenopiles; Bicosoecida incertae sedis

Acknowledgments

This work was partially supported by National Science Foundation grant OCE-1459406 and a

Sigma Xi Grant in Aid of Research. Support was also provided through a Department of the

Interior Southeast Climate Adaptation Science Center graduate fellowship awarded to Gabrielle

Corradino. Thank you to the Center for Electron Microscopy at North Carolina State University for their help with the processing of the SEM and TEM. Special thanks to Dr. Øjvind Moestrup for his help expertise with nanoflagellates and interpreting the SEM and TEM images.

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Yubuki N, Pánek T, Yabuki A, Čepička I, Takishita K, Inagaki Y, Leander BS (2015) Morphological Identities of Two Different Marine Stramenopile Environmental Sequence Clades: Bicosoeca kenaiensis (Hilliard, 1971) and Cantina marsupialis (Larsen and Patterson, 1990) gen. nov., comb. nov. Journal of Eukaryotic Microbiology 62: 532-542.

Table 1. Nearest NCBI 18S BLAST matches (n = 17) to C. canzanellaia. NCBI sequence lengths ranged from 1,455 to 1,796 bp. All E-values scored at 0.0.

Sample Location % Year Accession # Reference Match Submitted Environmental Aegean Sea 99.5 2008 AY789790.1 (Dalby et al., sequence 2008) Environmental Cariaco Basin, 98.0 - 2011 GU823081.1, (Edgcomb et sequence Caribbean 97.9 GU823193.1 al., 2011)

Environmental Black Sea 96.3 2011 HM749932.1 (Wylezich and sequence Jürgens, 2011) Environmental Lake Kusaki 90.1 - 2012 AB771824.1, Unpublished sequence 89.4 AB622309.1, AB771883.1, AB771814.1 Environmental Motojuku 90.1 - 2012 AB721079.1, Unpublished sequence Water 89.6 AB721078.1, Purification KX465213.1, Plant AB721078.1 Environmental Ponds in Paris, 89.9 2005 AY821965.1 (Šlapeta et al., sequence France 2005) Bicosoeca Narragansett 89.6 2006 AY520445.1 (Cavalier- vacillans Bay, Rhode Smith and Island Chao, 2006) Filos agilis Jyme and 89.4 2010 FJ971856.1 (Kim et al., Hook Lake, 2010) Wisconsin Adrimonas Unpublished 89.3 2003 AF243501.1 Unpublished peritocrescens Environmental Lake Oloidien, 89.1 2016 KX465212.1 (Luo et al., sequence Kenya 2017)

Table 2. Comparison of key phenotypic characteristics for C. canzanellaia and other common nanoflagellate genera. N = number of individuals characterized per species; Sources of each study shown in footnotes. Note: differences in methodology such as EM protocols exist between the different studies.

Family Bicosoecaceae Siluaniaceae Cafeteriaceae None assigned

Genus and Coniuncta Bicosoeca Adriamonas Cafeteria Pseudobodo Halocafeteria Species canzanellaia kenaiensis1 peritocrescen2 roenbergensis3 tremulans4 seosinensis5

Habitat Marine & Marine & Marine Soil Marine Marine Fresh Brackish Size 4-5 µm 2-6 µm 8-9 µm 3.5-6 µm 5-8 µm 4-8 µm N=50 N=30 N=38 N=n/a N=n/a N=n/a Flagellum 2 2 2 2 2 2

Mastigonemes + + - + + -

Body Scales ------

Lorica - + - - - -

Extrusomes n/a n/a + + + -

Paddle Flagella Tip + - - - - -

1. Yubuki et al., 2015 2. Verhagen et al., 1994 3. O'Kelly and Patterson, 1996 4. Karpov, 2000 5. Park et al., 2006

81°39’00 76°54’00 W W

Virginia 00

N36°15’ North Carolina

0 N34°42’ South Carolina

Figure 1. Sampling site (star) in Bogue Sound, North Carolina (34°43’17.77”, 76º45’33.93”W)

(ArcGIS Desktop, Release 10).

A B

Figure 2. (A) Several nanoflagellate cells (black arrows) attached to an elongated particle (P) with one of their flagella (60x, scale bar = 10 µm). (B) Single nanoflagellate with ingested fluorescently-labelled bead visible inside the cell (white arrow) (60x, scale bar denotes 5 µm).

Figure 3. Scanning electron microscopy (SEM) image under 6,500x. Only the anterior flimmer flagellum is covered with mastigonemes (right arrow). Flagellum ends in a paddle-like tip (left arrow). Scale bar denotes 5 µm.

Figure 4. SEM whole-mount under 20,000x showing the front of the cell and location of food uptake (black arrow). Mastigonemes are shown surrounding the flimmer flagellum (white arrow). Scale bar denotes 2 µm.

A/B

Figure 5. Transmission electron micrograph (TEM) of the nanoflagellate cell with a visible microtubular flagellar root (A) and the associated basal body above the root (B), nucleus (N) and mitochondria (M). Scale bar denotes 1 µm.

Kenya, Lake Oloidien (KX465213.1)

France, Ponds in Paris (AY821965.1)

Japan, Lake Kuaski (AB622309.1) Japan, Lake Kuaski (AB771824.1) Japan, Lake Kuaski (AB771883.1)

Japan, Motojuku Water Purification Plant (AB721079.1) Japan, Motojuku Water Purification Plant (AB721078.1) Aegean Sea (AY789790.1)

Coniuncta canzanellaia Black Sea (HM749932.1) Caribbean Sea (GU823193.1)

Caribbean Sea (GU823081.1) Oocystis solitara (AF228686.1)

Figure 6. Maximum Likelihood (ML) phylogenetic tree created with the Tamura-Nei model

(Tamura and Nei, 1993). The tree with the highest log likelihood (-7016.56) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches.

The tree consists of the closest NCBI Blast sequence matches (11) for the isolated nanoflagellate

C. canzanellaia and lists the chlorophyte Oocystis solitaria as outgroup. Silva sequences of the

18S were used to create pairwise distance estimates. The sample collection location is listed for each match with the GenBank accession number. Blue highlights marine and the green freshwater organisms.

Caecitellus parvulus (AF174367.1) Caecitellus paraparvulus (DQ220713.1) Cafeteria roebergensis (KY886365.1) F Halocafeteria seosinensis (DQ269470.1) Siluania monomastiga (AF072883.1)

Adriamonas peritocrescens (AF243501.1) S Paramonas globosa (AY520452.1) F Nerada mexicana (AY520453.1) S Bicosoeca kenaiensis (KM816648.1) Bicosoeca vacillans (AY520445.1)

Bicosoeca petiolata (AY520444.1) Coniuncta canzanellaia Oblongichytrium sp. PBS05 (FJ799794.1) Pseudobodo tremulans (AF315604.1) Rictus lutensis (GQ223284.1)

Oocystis solitara (AF228686.1) Symbiomonas scintillan (AF185053.1) Bodo saltans (AY490229.1) F

Figure 7. Maximum Likelihood (ML) phylogenetic tree created with the Tamura-Nei model

(Tamura et al. 1993). The tree with the highest log likelihood (-17054.28) is shown. The ML tree consists of 17 previously characterized nanoflagellate genera/species, the isolated nanoflagellate

Coniuncta canzanellaia and using Oocystis solitaria as the outgroup. Silva sequences of the 18S were used to create pairwise distance estimates. Branch lengths represent the number of substitutions per site. This analysis involved 18 nucleotide sequences with a total of 2,540 positions in the final dataset. The GenBank accession numbers are listed with each species name.

The organisms’ origins are marine except indicated otherwise: F = Freshwater or S = Soil

Supplemental Figures

Figure S1. C. canzanellaia attached and swimming near several Rhizosolenia sp. cells. The scale bar denotes 20 µm.

Video S2. C. canzanellaia attached to a particle and creating a feeding current with the anterior flagellum. Open access video link: https://drive.google.com/open?id=1zBuAiOuHNJunSIQ_iVJoKqgy2z9G_-Kq

CHAPTER 2

Grazing of the Heterotrophic Nanoflagellate Coniuncta canzanellaia on Prokaryote and Eukaryote Prey: Ingestion Rates and Gross Growth Efficiency

Abstract

Heterotrophic nanoflagellates are important grazers of prokaryotic and eukaryotic picoplankton in marine ecosystems. A growing number of studies provide insight on how the ability of flagellates to control prey populations may depend on grazer-specific feeding responses, but only limited information is currently available. In the present study, the growth and feeding response of the heterotrophic nanoflagellate Coniuncta canzanellaia was examined when grazing on a mixed heterotrophic bacterial assemblage (HB), the cyanobacterium Synechococcus spp. (Syn,

WH8102) and the picoeukaryote Ostreococcus lucimarinus (Ost) over varying prey densities.

Additionally, the flagellate was offered the three prey types as a mixed assemblage. The highest average growth rates were detected with ~2 d-1 feeding on HB at densities of ~107 cells mL-1 and maximum ingestion rates on Ost with 4,980 pg C d-1 at prey densities of ~106 cells mL-1.

Flagellate growth generally increased with ingestion rates up to ~200 pg C d-1 but overall growth seemed constrained to ~2 d-1 when the flagellate fed on either HB or Ost. Despite the flagellate reaching ingestion rates of up to 1,586 pg C flag-1 d-1 feeding on Syn, its growth response remained the lowest (1.2 d-1). Examining the elemental stoichiometry of the nanoflagellate compared to its prey indicated that a relatively low P-content in Ost drove a shift in C:N:P ratios of C. canzanellaia in the single-prey experiment and also when Ost was part of the mixed assemblage. When given a choice, the highest contribution to daily carbon intake for the flagellate came from the Ost with 51%, followed by HB with 46% and Syn with only 3%. The benefits of a more balanced diet were indicated as C. canzanellaia yielded the highest gross

growth efficiencies (GGEs) feeding on a mix of prey, followed by HB being offered alone and then, at much lower GGEs the picophototrophs. Applying daily ingestion rates from the mixed prey trial to an entire nanoflagellate community could have resulted in the removal of 23% of the

HB, 69% of the Ost and 17% of the Syn standing stocks. The findings presented in this study further corroborate the importance of a species-approach when investigating the biogeochemical role of heterotrophic nanoflagellates and are unique in signifying the importance of Ost in contributing to the diet of a heterotrophic flagellate.

KEY WORDS: Coniuncta canzanellaia, Heterotrophic Nanoflagellates, Microbial Food Web, Ingestion Rates, Growth, Gross Growth Efficiency

Introduction

Heterotrophic nanoflagellates (HNANs; 2 – 20 µm in size) are important grazers of picoplankton

(0.2 – 2 µm) throughout the oceans and play pivotal roles in biogeochemical cycling within microbial food webs (Pomeroy 1974, Azam et al. 1983, Pernthaler 2005). HNAN grazing may account for the daily removal of as little as ~5% but up to 100% of bacterial standing stocks, typically constituting the primary cause of bacterial mortality (vis-à-vis viral lysis) (Caron et al.

1999, Christaki et al. 2001, Tsai et al. 2013). Similarly, the extent to which HNANs exert top- down control over picophytoplankton (e.g., Synechococcus spp.) may range markedly with 1 -

93% of biomass consumed on a daily basis (Safi & Hall 1999, Worden et al. 2004, Karayanni et al. 2005, Worden 2006). Both laboratory and field studies have demonstrated how prey quantity and quality may affect rates of flagellate community grazing (Gonzalez et al. 1990, Dolan &

Šimek 1999, Christaki et al. 2001). Fewer studies, however, have focused on how feeding ecologies and preferences differ among individual nanoflagellate species (Boenigk & Arndt

2000, Schnepf & Kühn 2000, Christaki et al. 2005). For instance, ingestion rates for Cafeteria and Ochromonas were found to differ depending on prey type and, moreover, ingestion rates for

Ochromonas exceeded those for Cafeteria by orders of magnitude when feeding on the same prey (Boenigk et al. 2001). A major obstacle to studying species-specific trophic interactions, and a reason why HNAN assemblages have been commonly treated as a single predatory group

(‘black box’), is the paucity of easily distinguishable morphological characteristics (Jürgens et al.

2008, Caron et al. 2012).

Variability in HNAN bacterivory has been attributed to prey size and mobility with larger, mobile cells typically being ingested at higher rates (Gonzalez et al. 1990, Boenigk et al. 2001).

Next to prey size as a potential indicator of nutritional value, some studies have also examined nutrient composition (C, N and P budgets) for prey and predator to aid in interpreting HNAN- prey dynamics (Eccleston-Parry & Leadbeater 1995, Grover & Chrzanowski 2009). Previous work has shown that the typical C:N:P range for HNANs fall within 6:1.5:1 to 66:10:1 and can shift based on the nutrient composition of ingested prey (Eccleston-Parry & Leadbeater 1995,

Chrzanowski & Foster 2014). A growing number of studies emphasize the role that phototrophic prey, mainly cyanobacteria Synechococcus and Prochlorococcus, play in flagellate diet next to feeding on heterotrophic bacteria (Dolan & Šimek 1998, 1999, Guillou et al. 2001, Christaki et al. 2002) and a few have examined HNAN feeding on picoeukaryotes, mainly Ostreococcus and

Choricystis spp. (Christaki et al. 2005, Bręk-Laitinen & Ojala 2011). Generally, HNANs are able to ingest varying phototrophs, however, their ability to sustain population growth on such diets seems to differ from flagellate to flagellate (Dolan & Šimek 1998, Christaki et al. 2001, Bec et al. 2006).

Prey quantity and quality can impact flagellate growth and may also affect gross growth efficiency (GGE), the amount of prey carbon converted into HNAN biomass (Straile 1997,

Dahlgren et al. 2010). While carbon-based GGEs for HNANs feeding on natural prey communities typically fall between 23-54% (Fenchel 1982a, Børsheim & Bratbak 1987, Rose et al. 2009), lab studies suggest variations dependent on prey and grazer “matches.” For instance, in experiments where the same prey (E.coli) was offered to differing flagellates, maximum GGE for Pteridomonas sp. reached 22%, while values were twice as high (~43%) for Ochromonas sp.

(Wikner et al. 1986, Pelegri et al. 1999). In another example, grazing on various strains of

Synechoccocus, the HNAN Goniomonas pacifica yielded GGEs from 13% to 45% (Apple et al.

2011). Christaki et al. (2005) also reported decreases in biovolumes of certain flagellates feeding on prey like the picoeukaryote Ostreococcus compared to heterotrophic bacteria. A growing number of studies continues to provide better insight into how the biogeochemical roles of

HNANs link to grazer-specific and prey-dependent responses.

The objective of this study was to examine grazing and growth of a newly characterized HNAN,

C. canzanellaia (Chapter 1). The flagellate, a member of the bicosoesids, was originally isolated and characterized from the North Carolina coast but, based on public gene databases (NCBI

BLAST matches), may have a wider geographical distribution encompassing varying subtropic and tropic regions including the Aegean Sea, Caribbean Sea and the Black Sea (Chapter 1).

Grazing was examined for three common prey types/groups including a mixed heterotrophic bacterial assemblage, the cyanobacterium Synechococcus sp. and the picoeukaryote

Ostreococcus lucimarinus. Prey was offered over increasing concentrations (~104 to 107cells mL-

1) in separate short-term incubations (24 hours), and then, all prey types were combined to examine HNAN feeding on the mixed assemblage. The effects of prey concentration and type on

C. canzanellaia growth, ingestion, predator biovolume and GGE are discussed.

Materials and Methods

Prey and Predator Origins and Culture Conditions

Cultures of the cyanobacteria Synechococcus sp. (Syn; NCMA 2370, WH8102), the eukaryote

Osterococcus lucimarinus (Ost; NCMA 3430, Mamiellophyceae) and a freshly collected mixed assemblage of heterotrophic bacteria (HB) were used as prey. The motile Syn. (NCMA 2370) had been originally collected from the Sargasso Sea and Ost (NCMA 3430) was isolated in the

North Pacific. The HB assemblage was collected from the same location as C. canzanellaia was originally isolated along the North Carolina coast, in January 2019 (34.721791N, -76.759982W;

Chapter 1). The bacteria were grown in the dark at 16ºC with 0.2 µm-aged seawater with F/2 and a single barley seed (Guillard & Ryther 1962), transferred every 6 days and used in the feeding experiments within 60 days of collection. Syn and Ost were grown under a 14:10 light:dark cycle

(75 μE m−2 s−1) with F/2 as was the nanoflagellate with the addition of a baked barley seed.

Grazing Experiment

A series of feeding experiments (24 hours) were conducted at 16°C to examine HNAN growth rates (µ; d-1), ingestion rates (IR; cells flag-1 d-1 and fg C-1 flag-1 day-1), shifts in HNAN biovolume (BV; µm3 cell-1) and cell stoichiometry (C, N and P). The incubation temperature was chosen based on the original isolation temperature for C. canzanellaia. Exponentially growing

HB, Syn and Ost were each inoculated with the flagellate and, on one occasion, combined to provide a mixed prey assemblage. Prey-predator and control treatments were set up in triplicates where each flask contained a total volume of 170 mL of artificial seawater (ASW; ASTM

D1141-98 Lake Products Company), spiked initially with F/2 to account for prey growth due to the availability of remineralized nutrients in bottles with the flagellate compared to the control treatments (Selph et al. 2003). Initial prey concentrations for each of the three prey types ranged from ~104 to 107 cells mL-1 (Table 1). The control treatments were run at ~106 cells mL-1 for each of the prey items (no flagellate added). For the mixed prey experiment, starting concentrations were ~106 cells mL-1 for the mixed HB assemblage and ~104 cells mL-1 for both

Syn and Ost cultures (Table 1); organismal concentrations typical for coastal waters (Dolan &

Šimek 1999, Countway & Caron 2006). The flagellate was gently concentrated using a 3 µm

filter by passive filtration and then transferred into 0.2 µm-filtered ASW ~2 hours prior to all experiments to allow for emptying of food vacuoles under decreased concentrations of background bacteria at ~102 to 103 mL-1 (Šimek & Chrzanowski 1992, Bratvold et al. 2000,

Zwirglmaier et al. 2009). Samples to determine cell abundances for the HNAN and the prey were obtained at the beginning (T0), after 12 hours (T1) and after 24 hours (TF). Triplicate subsamples (10 µL) from each flask were used to enumerate HNAN abundances live using light microscopy (Olympus BX53) under differential interference contrast. Picoplankton were enumerated using 3 mL from each triplicate flask after preservation with ice-cold 2% glutaraldehyde and, in the case of the HB, staining with DAPI (Slowfade gold antifade,

Thermofisher) (Porter & Feig 1980). The picoplankton prey were quantified under 60x and 100x using epifluorescence microscopy (Table 1). Samples to determine HNAN biovolumes (30 µL) and elemental composition (150 mL combined from each of the triplicate bottles) were collected at T0 and at TF for the control bottles (prey only) and in grazer treatments was measured within the first hour of feeding and then after 24 hours at TF.

Flagellate Growth, Ingestion Rates and Biovolumes

Growth (µ) and ingestion rate (IR) were calculated using the Frost equations (Frost 1972) modified by Heinbokel (Heinbokel 1978),

μ = ln (PTF/PT0) /(TF-T0)

IR= BT0 – BTF ((PTF – PT0) /(lnPTF – lnPT0))(TF-T0) where B are the prey and P the flagellate concentrations (both in cells mL-1) at the onset (T0) and the final time point (TF).

Cell size estimates for the nanoflagellate and prey were obtained by measuring cells preserved in glutaraldehyde and capturing images (n = 30 for C. canzanellaia and n = 40 for the prey in each treatment and at each time point) with an Olympus DP73 monochrome digital camera plus

Olympus cellSens Dimension 1.13 software. Comparison with live flagellate cells indicated 24-

30% cell shrinkage with preservation, which is within the range of previously reported values

(Hondeveld et al. 1992). The HNAN biovolumes (BV) were calculated using the ellipsoid equation of Hillebrand et al. (1999). The HB were categorized as filamentous, rod or ellipsoid and shapes for Syn, Ost were approximated as ellipsoids.

Elemental Ratios and GGE

Particulate nutrient samples were collected at T0 and TF from flasks that contained HB, Syn, Ost, and the mixed prey assemblage. To accumulate sufficient biomass, subsamples from three treatment bottles were combined (total of 150 mL) and filtered onto precombusted 0.2 µm glass fiber filters. For the single prey experiments, these samples were collected from treatment bottles containing ~106 cells mL-1. Each of the glass fiber filters was individually stored in a petri dish at

-20°C for ~5 weeks prior to elemental analysis. Briefly, particulate carbon (PC) and particulate nitrogen (PN) concentrations were determined following modified methods of Froelich (1980).

Each filter was wrapped in methanol cleaned tin boats and combusted at 1000°C in a Perkin

Elmer 2400 elemental analyzer. Total particulate phosphorus (TPP) was determined using a modification of the Aspila method. Filters were combusted at 550°C to convert all organic P present into inorganic P forms and extracted using a weak hydrochloric acid (Aspila et al. 1976,

Benitez-Nelson et al. 2007). A standard reference material (NIST #1573a, tomato leaves) was analyzed with each run to evaluate analytical accuracy and monitor run to run variability. HNAN

cell nutrient contents were calculated by correcting total estimates for C, N and P values for prey.

For this, prey cell content values for each prey type (control bottles) were multiplied by prey cell abundance in the varying grazer bottles and subtracted. Finally, changes in C:N, N:P and C:P ratios were examined for each prey type and the flagellate.

Gross growth efficiency (GGE) of C. canzanellaia was calculated based on abundance and C biomass change in the cultures in relation to changes for the nanoflagellate (GGEC) (Fenchel

1982b). The GGE was also calculated comparing the change in BVs between prey and predator

(GGEBV). Following equation from Choi and Peters was used (1992):

GGE= ((PTF- PT0) x Cpred) / ((BT0- BTF) x Cprey)) x 100 where B is the prey concentration at the beginning (T0) and the end of the experiments (TF)

(cells mL-1), P is the HNAN concentration at the beginning (T0) and the end of the experiments

-1 (TF) (cells mL ) and Cpred and for Cprey are C-based predator (pred) and prey (prey) biomass. For the computation of GGEBV the same equation was used but instead of Cpred and Cprey values for

BVpred and BVprey were substituted.

Statistical Analyses

Statistical analyses were performed using the JMP Pro 14 software package (SAS Institute, Cary,

NC). Differences in growth, ingestion, GGEBV, and GGEC among treatments were examined using a one-way ANOVA test (Zar 1984). Means were compared using a Tukey test (p < 0.05).

Results

Prey and Predator Biovolume and Nutrient Contents

No significant changes were detected for cell BV over time for any of the prey organisms in the control treatments (p > 0.05). At T0 Syn cell size averaged 0.9 ±0.9 µm3 (n = 40) and Ost 3.0

±1.1 µm3 (n = 40) and at TF 1.3 ±0.9 and 3.3 ±1.0 µm3 cell-1, respectively (Fig. 1). For HB a decrease in BV from 1.4 ±1.7 to 0.4 ±0.8 µm3 cell-1 was indicated, however, this overall change did not prove statistically significant (p > 0.05) (Fig. 1). The initial BV for C. canzanellaia averaged 36 ±10 µm3 cell-1 and changed when the flagellate was fed Syn (Fig. 2). In this treatment, BVs increased to 64 ±17 µm3 cell-1 by TF (n = 30 for each time point; p < 0.05; Fig.

2). Grazer BVs also increased feeding on the mixed assemblage averaging 68 ±26 µm3 cell-1 (n =

30). These shifts in predator size were already observable after the first 12 hours of each experiment (T1 in Fig. 2).

For the computation of C-based IR and GGE, prey nutrient contents were computed per cell and per BV by averaging T0 and TF values (Table 2). Estimates for the HB were determined with 55 fg C µm-3, 9 fg N µm-3 and 4 fg P µm-3 (Table 2). Estimates for Syn were 181 fg C µm-3, 37 fg N

µm-3 and 6 fg P µm-3 and for Ost 656 fg C µm-3, 37 fg N µm-3 and 6 fg P µm-3 (Table 2). These values compared well to previously published C estimates with 5 - 80.5 fg C cell-1 for HB (Lee

& Fuhrman 1987, Eccleston-Parry & Leadbeater 1995, Theil-Nielsen & Søndergaard 1998) and

82 - 250 fg C µm-3 for Syn (Kana & Glibert 1987, Worden et al. 2004). Cellular C concentrations for Ost in this study were somewhat higher than previously published values with 233 - 247 fg C

µm-3 (Worden et al. 2004) but fell well within the wide range reported for picoeukaryotes

(Zubkov et al. 1998, Garcia et al 2018). C. canzanellaia nutrient contents were averaged using

values collected approximately ~1 hour into the experiments and at TF (Table 3). Feeding on Ost yielded nutrient concentrations of 495 fg C µm-3, followed by the flagellate fed on HB with 403 fg C µm-3, 330 fg C µm-3 for Syn and 214 fg C µm-3 for flagellates fed on the mixed assemblage

(Table 3). Molar elemental ratios (C:N:P) for each prey organism were similar with 14:2:1 and

31:7:1 for HB and Syn but deviated from Ost with 119:7:1, indicating a higher C content for Ost in relation to N and especially P. In the single prey treatments, C. canzanellaia feeding on Ost yielded similar C:N values (overall range = 7 - 9) but N:P and C:P ratios were relatively high with 10 and 64 compared to the other treatments reflecting differences in prey nutrient composition (i.e., low P content for Ost; Table 4). Moreover, C:N:P results for C. canzanellaia feeding on the mixed prey treatments (including Ost) yielded ratios that fell in between single- prey estimates for HB and Syn compared to the Ost single prey treatments (Table 4).

Nanoflagellate Growth and Ingestion

Abundance estimates in the control bottles indicated little to no prey growth over the 24-hour incubations (Table S1). Average µ rates for C. canzanellaia increased from 0.7 to 1.9 d-1 with increasing HB abundances from 104 to 107 cells mL-1 (Fig. 4 and Table 5). Feeding on Syn and

Ost over the same prey densities yielded average growth rates from 0.7 to 1.2 d-1 and from 1.1 to

1.4 d-1, respectively (Fig. 4 and Table 5). HNANs feeding on picophototrophs reached maxima at lower prey densities around 105 cells mL-1 partly decreasing thereafter (Fig. 4). Provided with mixed prey, HNAN growth averaged 0.9 d-1 (range = 0.6 to 1.3 d-1). Comparison across all treatments showed that HNAN growth was significantly different (lower) only when comparing the Syn and HB treatments at 106 cells mL-1 (p <0.05) (Fig. 4 and Table 5).

Ingestion rates (IRs), both computed using cell abundance and C estimates, generally increased with prey concentrations for all prey types with the exception for the Ost treatment at ~107 cells mL-1, where IRs had started to decline (Table 5 and Fig. 5). The HNAN reached its highest individual IRs with 3,856 ±707 cells d-1 for HB, 6,898 ±2,980 cells d-1 for Syn and 3,703 ±449 d-1 cells for Ost (Table 5). Applying prey C conversion factors, maximal ingestion rates corresponded to 296 ±54 pg C flag-1 d-1 for HB 1,586 ±685 pg C flag-1 d-1 for Syn and 4,980

±477 pg C flag-1 d-1 for Ost (Table 5, Fig. 5). There was a significant difference in C-based IRs between Syn and Ost treatments at 105 and 106 cells mL-1 (p <0.05). Additionally, there was a significant difference in C-based IRs between HB and Ost at 105 and 106 cells mL-1 (p <0.05).

Overall, IRs reached their highest for Ost at prey densities ~106 cells mL-1. In the mixed assemblage, Ost was grazed at the highest rates (16 ±6 pg C flag-1 d-1), followed by Syn (0.93

±0.39 pg C flag-1 d-1) and HB (14 ±4 pg C flag-1 d-1).

Comparing the single prey treatments showed that flagellate µ rates increased with IRs up to

~200 pg C d-1 but overall growth seemed constrained to ~2 d-1, rates that were reached when the flagellate fed on either HB or Ost (Fig. 6). Notably, IRs of ~3 pg C flag-1 d-1 for HB prey already facilitated µ rates at 1.6 d-1. Despite C. canzanellaia reaching average C- based IRs as high as

4,980 and 1,586 pg C flag-1 d-1 feeding on Ost and Syn, respectively, µ had levelled off under the experimental conditions. The flagellate’s overall µ response remained the lowest with 1.2 d-1 which was its maximum for a Syn diet compared to HB and Ost treatments (Fig. 6). Growth for mixed prey conditions fell into the lower range of observations with 0.9 d-1 with 10.7 pg C flag-1 d-1 (Fig. 6).

Gross Growth Efficiencies

Average gross growth efficiencies based on BV (GGEBV) and C estimates (GGEC) ranged from

<1 to 50% and <2 to 37% GGEC, respectively (Fig. 7). C. canzanellaia had maximum GGEs feeding on the mixed prey assemblage (HB + Syn + Ost) (50 ±29% for GGEBV and 37 ±11% for

GGEC) exceeding all other prey treatments, followed by GGE estimates feeding on HB (GGEBV of 15 ±4% and GGEC of 50 ±14%) and those for picophototrophic prey (Syn and Ost, Fig. 7).

Both C- and BV-based GGEs were significantly higher for the Mix treatment compared to the

Syn and Ost treatment and for BV-based GGEs a difference was also detected to the HB treatment (p <0.05). Comparing between BV and C-based GGEs only rendered a significant difference for C. canzanellaia grazing on Ost (p <0.05).

Discussion

Nanoflagellate Growth and Ingestion

C. canzanellaia demonstrated differential growth and ingestion dependent on the prey that the flagellate was offered. Prey abundances for each of the prey types that fell below their “typical” densities in coastal waters yielded low IRs indicating minimum prey thresholds that are required to prompt a sound feeding response. For instance, C. canzanellaia IRs increased at prey abundances > 105 cells mL-1 for HB and 104 cells mL-1 for the picophytoplankton Syn and Ost.

While µ generally increased for C. canzanellaia with increased IRs of up to ~200 pg C d-1, maximum µ seemed constraint to ~2 d-1 when the flagellate fed on HB and, in some bottles, on

Ost (Fig. 6). Despite the flagellate reaching its highest IRs with > 1.500 pg C flag-1 d-1 feeding on

Syn, its µ response was the lowest overall, leveling at ~1.2 d-1. When all three prey organisms were offered together with the mixed assemblage, the HNAN selected for HB (14 ±4 pg C flag-1

d-1) and Ost (16 ±6 Ost) over Syn (0.94 ±0.39 pg C flag-1 d-1, Table 6). In a study by Guillou et al.

(2001), single-prey experiments with Syn and with mixed prey for the HNAN Picophagus flagellatus showed similar findings when Syn was offered alone and resulted in some of the lowest µ recorded for P. flagellatus (0.6 d-1 compared to ~1.1 d-1 for C. canzanellaia). However, unlike our results, Syn was selected for by the HNAN over other organisms when it was offered a mixed assemblage with Prochlorococcus (Guillou et al. 2001).

Relatively little information is available for individual HNAN species feeding on multiple prey types including phototrophic prey and picoeukaryotes and the majority of studies base IRs on cell abundance rather than C intake (Table 6). IRs for Syn in this study ranged from ~100 to >

6,900 cells-1 flag d-1 and exceeded previously reported rates of <1 - 57 cells-1 flag d-1 over similar prey densities (Table 6). For Ost, IRs ranged from ~5 to >2.300 cells-1 flag d-1 and also exceeded most of the available published values ~5 - 15 cells-1 flag d-1 (Table 6). While comparison across these studies is somewhat problematic due to the differences in experimental methodology, future incorporation of information on prey quality (C content, size and elemental composition) will allow for wider comparisons and better resolution of individual, grazer-specific feeding responses.

Nanoflagellate Cellular Stoichiometry, Biovolume and GGE

C. canzanellaia varied in its nutrient composition dependent on the prey quality that was offered.

Overall C:N:P ratios from 12:1:1 to 64:10:1 fell well within the range seen in a limited number of studies that incorporated direct measurements of cell stoichiometry (Table 3). Ost ratios of

17.6 for C:N and 119 for C:P(Table 4), fell within reported ranges for O. lucimarinus that found

the C:N to be 5.25 and 119.5 for the C:P (Garcia et al. 2018). In this study, the relatively low P- content in Ost (C:P ratios of 119:1 for Ost compared to 14:1 and 31:1 for HB and Syn, Table 4) drove a shift in C:N:P ratios of C. canzanellaia in both the single-prey experiment and when offered a mixed prey assemblage (Table 3). Similarly, a study by Chrzanowski (2010) reported a shift in cellular composition using a single HNAN species grazing on the heterotrophic bacterium Pseudomonas fluorescens. Fed on P. fluorescens raised under balanced nutrient conditions, Ochromonas danica yielded C:N:P ratios of 161:10:1 compared to 80:12:1 when the bacterium grew under C-limited conditions before being offered to the flagellate (Chrzanowski et al. 2010). Variations in prey C:N:P drive HNAN stoichiometry but there are also differences in incorporation efficiency among varying grazer species that require consideration (Grover 2004,

Chrzanowski & Foster 2014). For instance, Eccleston-Parry et al. (1995) showed cellular C:N:P ratios of 71:13:1 for Jakoba libera in contrast to 6:2:1 for Bodo designis feeding on the same HB assemblage. A major factor that has hindered the resolution of stoichiometric relationships is that most HNAN feeding studies have used common, fixed conversion factors for prey cell nutrient concentrations and these can vary significantly dependent on an organism’s physiological state

(Choi & Peters 1992, Pelegri et al. 1999, Selph et al. 2003). Moreover, only a very small number of investigations, similar to this study, extended nutrient measurement to include N and P (Caron et al. 1990, Eccleston-Parry & Leadbeater 1995, Chrzanowski et al. 2010).

Our study demonstrated that HNAN growth on varying prey can be linked to changes in flagellate BV. In the single prey treatments, C. canzanellaia had the highest increase in cell BV

(79%) by the end of the experiment while grazing on Syn. This increase, however, was also associated with low µ rates. A limited ability to divide was likely due to C. canzanellaia not fully

digesting Syn, leading to low assimilation and noticeably low GGEs (< 5%). Inefficient prey processing and subsequent egestion (Dolan & Šimek 1998, Shannon et al. 2007) have been previously linked to low GGEc or GGEBV (Pelegri et al. 1999) suggesting that Syn is of low prey quality and adversely impacts HNAN population growth and fitness (Shannon et al. 2007).

Furthermore, there might be additional gradients in the palatability and digestibility dependent on the Syn species or strain (Gorsky et al. 1999, Guillou et al. 2001, Apple et al. 2011). For instance, grazing experiments using Syn strain WH8102 (Sargasso Sea, isolated 1981) confirmed that cell surface proteins can play a role in the defense against predation from heterotrophic protists such as dinoflagellates (Strom et al, 2012). Based on this study, C. canzanellaia did poorly on a sole Syn diet compared to the other offered prey. Grazing on the mixed assemblage,

Syn contributed only 3% to daily C intake by the flagellate with 46% and 51% coming from the

HB and Ost, respectively. Our findings did indicate that C. canzanellaia seemed to benefit from being offered a mixed prey assemblage, however, since the flagellate reached thehighest GGEs

(50% and 37% based on BV and C, respectively; Fig. 7). The significant increase in C. canzanellaia BV in the mixed treatment paired with modest µ could have been a result of Syn being ingested as part of the mix. A study by Samuelsson and Andersson (2003) showed that in the absence of grazers (removal via size-fractionation) both µ rates and average BV increased within a natural HNAN community. Hence, BV shifts in flagellates based on diet choices may have important consequences for the flagellate’s own susceptibility to grazers. Future studies can address the impact that HNAN diet may have on prey-predator dynamics and C transfer with C. canzanellaia being prey to larger grazers.

Impact on Picoplankton Standing Stocks

Daily C removal rates were calculated employing average HNAN IRs from the single prey treatment and assuming natural abundances of HB (106 cells mL-1), of Syn (104 cells mL-1) of Ost

(104 cells mL-1) and HNANs (103 cells mL-1). Under these specific conditions HNAN grazing would have accounted for a high loss of over 100% of HB standing stocks and 8% and 48% for

Syn and Ost biomass, respectively. These ranges shifted when IRs were pulled from the mixed prey trial at compatible prey densities, which was considered a somewhat better representation of natural feeding conditions. Here, C. canzanellaia IRs extrapolated to a daily removal of 23% and

17% of HB and Syn biomass with Ost being affected the strongest with 69% of carbon biomass being grazed. Previous studies have reported average removal rates of incident heterotrophic bacterial populations with 45% to 87% and maxima of >100% in coastal waters (Solic &

Krstulovic 1994, Christaki et al. 2001). Similar to our estimate, previous laboratory and field studies suggested lower removal with 1% to 20% of C standing stocks and a maximum of 45% for Syn (Safi & Hall 1999, Christaki et al. 2001). Currently there are no comparative studies on the grazing impact of flagellates on in situ Ost populations, but this study suggested that HNAN grazing could account for high losses (~50%). The indicated potential of HNANs, with feeding ecologies like C. canzanellaia, to exert top-down control on Ost populations warrants further investigation given that the cosmopolitan Ost taxa are major contributors to primary production

(Leconte et al. 2020).

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Table 1. Initial prey and predator abundances (cells mL-1) with their standard deviation (±SD) in each of the four experiments. Prey densities in the control flasks started at ~106 cells mL-1 (no flagellate added). HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

Treatment HNAN Prey Prey in Control HB 4.5 ±0.5 x 102 5.1 ±3.0 x 104 5.3 ±0.6 x 102 2.2 ±5.7 x 105 3.8 ±0.3 x 102 2.4 ±3.3 x 106 2.0 ±3.6 x 106 4.3 ±0.6 x 102 1.4 ±2.6 x 107 Syn 2.8 ±2.8 x 102 8.1 ±1.5 x 104 2.0 ±0.0 x 102 1.7 ±9.4 x 105 2.8 ±2.8 x 102 2.5 ±4.0 x 106 6.8 ±5.4 x 106 3.0 ±0.0 x 102 3.4 ±2.3 x 107 Ost 6.4 ±3.1 x 102 3.4 ±3.0 x 104 4.3 ±1.1 x 102 5.8 ±8.8 x 105 5.3 ±3.0 x 102 8.7 ±2.0 x 106 1.1 ±8.3 x 106 5.6 ±2.0 x 102 1.1 ±8.2 x 107 HB 1.0 ±0.94 x 106 1.1 ±0.47 x 106 Mix 1.9 ±0.53 x 102 Syn 3.5 ±1.1 x 104 5.5 ±2.2 x 10 4 Ost 4.0 ±1.7 x 104 4.5 ±2.5 x 104

Table 2. Averaged prey carbon (C), nitrogen (N), phosphorus (P) content in fg cell-1 and normalized by biovolume in fg µm-3 in the control treatments. * For Ost cell content estimates were available for T0 observations only. HB = heterotrophic bacteria; Syn = Synechococcus; Ost

= Ostreococcus

Prey C N P C N P fg cell-1 fg µm -3

HB 77 12 5 55 9 4

Syn 230 48 7 181 37 6

Ost* 2,144 122 18 656 37 6

Mix 222 37 11 202 34 10

Table 3. Carbon (C), nitrogen (N), phosphorus (P) cell content and elemental ratios for C. canzanellaia. Cell contents for C. canzanellaia grazing on varying prey (HB, Syn, Ost and Mix treatments) are shown in pg cell-1 and normalized by biovolume in fg

µm-3. Flagellate nutrient concentrations are shown in comparison to estimates reported elsewhere. Values are averages from at least 2 or more observations. a study used bacteria raised under balanced growth conditions and under C-limitation. bC content differences for the same organisms linked to cell shrinkage during preservation. Syn = Synechococcus; Ost = Ostreococcus; HB = mixed heterotrophic bacteria; Bact Cult = bacterial culture

Nanoflagellate(s) Prey C N P C:N C:N:P C N P Source pg cell-1 fg µm-3 C. canzanellaia HB 15 2 0.9 8.5 17:2:1 406 47 27 This Study Syn 15 2 1.3 8.3 12:1:1 330 30 16 Ost 19 3 0.3 6.6 64:10:1 495 272 38 Mix 9 0.9 0.2 10.4 39:4:1 214 14 3 Paraphysomonas Eccleston-Parry (1995) imperforate HB 32.5 7.4 1.2 4.5 27:6:1 149 35 6 Bodo designis HB 12.6 3.1 2.2 4.1 6:1.5:1 233 57 41 Stephanoeca diplocostata HB 5.4 1.1 0.1 4.9 45:9:1 156 31 3 Jakoba libera HB 14.2 2.6 0.2 5.5 71:13:1 189 34 3 Ochromonas danicaa Bact Cult 32.8 2.6 0.6 18.2 161:10:1 386 31 7 Chrzanowski (2010) Bact Cult 46.6 7.9 1.5 6.9 80:12:1 885 150 28 Bact Cult - - - - 66:10:1 - - - Chrzanowski (2014) Paraphysomonas spp. Bact Cult - - - 5.0 - 466 93 - Sin (1998) Bact Cult - - - 10.6 - 181 17 - Paraphysomonas Selph (2003) bandaiensis HB - - - 5.2 - 130 - - Monas sp.b Bact Cult 100 Børsheim & Bratbak (1987) - - - 4.6 - - - Bact Cult 220

Table 4. Elemental ratios for prey and C. canzanellaia fed on each prey type (n = 2). HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

Prey (Control) C:N:P C:N N:P C:P HB 14:2:1 6.5 2.2 14

Syn 31:7:1 4.8 6.5 31 Ost 119:7:1 17.6 6.8 119

Mix 21:4:1 5.9 3.5 21

HNAN fed on C:N:P C:N N:P C:P HB 17:2:1 8.5 1.9 16.6

Syn 12:1:1 8.3 1.4 11.9

Ost 64:10:1 6.6 9.6 63.6 Mix 39:4:1 10.4 3.8 39.2

Table 5. Specific growth rate (µ, d-1) and ingestion rate (IR, cells flag-1d-1 and pg C flag-1d-1) for

C. canzanellaia averaged over triplicate incubations ±SD (see exact grazer and prey abundances in Table 1). HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

Prey Prey Abund Flag µ IR IR cells mL-1 d-1 cells flag-1 d-1 pg C flag-1 d-1 HB 104 0.8 ±0.2 0-11 0 - 1.3 105 1.4 ±0.1 43 ±26 3 ±2 106 1.7 ±0.1 1,106 ±71 85 ±2 107 1.9 ±0.1 3,856 ±707 296 ±54 Syn 104 0.7 ±0.9 120 ±146 27 ±33 105 1.1 ±0.2 98 ±52 22 ±12 106 1.2 ±0.7 2,352 ±1,056 540 ±243 107 0.9 ±0.7 6,897 ±2,980 1,586 ±685 Ost 104 1.1 ±0.1 4 ±3 9 ±6 105 1.4 ±0.01 179 ±54 384 ±118 106 1.2 ±0.8 2,322 ±222 4,980 ±477 107 1.4 ±0.1 370 ±49 794 ±962 HB 106 HB 191 ±60 HB 14 ±4 Mix Syn 104 0.9 ±0.3 Syn 4 ±1 Syn 0.94 ±0.39 Ost 104 Ost 7 ±3 Ost 16 ±6

Table 6. A comparison table of growth (µ) and ingestion rates (IR) across various experiments with nanoflagellate species and picoplankton prey. The average or ranges are reported when available with standard deviation in parentheses. Note: differences in experimental methodology exist between the studies. HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Pro =

Prochlorococcus; Bact Cult = bacterial culture; Chlor = Chlorocystis. # Marks flagellates that are known mixotrophs

Prey Nanoflagellate(s) Prey Flag Abund µ IR Temp Duration Reference Abund cells mL-1 cells mL-1 d-1 cells flag-1 d-1 °C hrs C. canzanellaia HB 5.3 x102 2.2 x105 1.4 (0.1) 3 (2) 16 24 This study HB 3.8 x102 2.4 x106 1.7 (0.1) 1,106 (71) 16 24 HB 4.3 x102 1.4 x107 1.9 (0.1) 3,856 (707) 16 24 Syn 2.8 x102 8.1 x104 0.7 (0.9) 120 (146) 16 24 Syn 2.0 x102 1.7 x105 1.1 (0.2) 98 (52) 16 24 Syn 2.8 x102 2.5 x106 1.2 (0.7) 2,352 (1,056) 16 24 Syn 3.0 x102 3.4 x107 0.9 (0.7) 6,897 (2,980) 16 24 Ost 6.4 x102 3.4 x104 1.1 (0.1) 4 (3) 16 24 2 5 Ost 4.3 x10 5.8 x10 1.4 (0.1) 179 (54) 16 24 Ost 5.3 x102 8.7 x106 1.2 (0.8) 2,322 (222) 16 24 Ost 5.6 x102 1.1 x107 1.4 (0.1) 370 (49) 16 24 Pseudobodo sp. Syn WH8103 3.9 x104 1.3 x106 -- 0.012-64 18 12 Christaki et al. Pro MED4 2.8 x106 -- 0.024-160 18 12 (2002) Picophagus Pro SS120 2.3 x104 5.2 x105 1.6 28 19 10 Guillou et al. flagellates# Syn WH8103 2.4 x103 2.7 x105 0.6 16 19 10 (2001) Pro SS120 2.6 x105 9 2.8 x104 1.5 19 10 Syn WH8103 1.2 x104 9 Jakoba libera Ost 4.5 x102 0.5 x106 1.2 15 18 60-114 Christaki et al. Cafeteria and (2005) Ost 2.3 x102 1.9 x106 3.1 19 18 60-114 Monosiga sp. Paraphysomonas 9.4 x104- 2.5 Choi and Peters Bact Cult 3- 6.7 x108 2.7 (0.08) 1,488 (117) 15 18-36 imperforata x105 (1992)

Table 6 cont’. A comparison table of growth (µ) and ingestion rates (IR) across various experiments with nanoflagellate species and picoplankton prey. The average or ranges are reported when available with standard deviation in parentheses. Note: differences in experimental methodology exist between the studies. HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Pro =

Prochlorococcus; Bact Cult = bacterial culture; Chlor = Chlorocystis. # Marks flagellates that are known mixotrophs

Prey Nanoflagellate(s) Prey Flag Abund µ IR Temp Duration Reference Abund cells mL-1 cells mL-1 d-1 cells flag-1 d-1 °C hrs 9.4 x104- 2.5 Choi and Peters P. imperforata Bact Cult 3- 6.7 x108 2.3 (0.12) 2,376 (237) 15 18-36 x105 (1992) Eccleston-Parry 8 x105 - 2.24 P. imperforata Bact Cult 2 x103 0.21 -- 20 48 and Leadbeater x108 (1994) Boenigk et al. Spumella sp. Bact Cult 1-2 x103 2 x108 0.3 (0.17) -- 16 15 (2006) Bact Cult 1-2 x103 2 x108 2.8 (0.06) -- 16 15 Christaki et al. Mixed Syn WH8103 3.4 x 04 1.4 x106 -- 57 18 12 (2002) Pro MED4 2.7 x 06 -- 153 18 12 Christaki et al. Mixed Ost 4.2 x104 0.32 x106 -- 6 18 0-144 (2005)

Supplemental Table

S1. Table 1. Prey growth (µ d-1) in control treatments shown as average ±SD. HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

Prey µ

HB 0.67 ±0.70

Syn -0.24 ±0.20

Ost 0.05 ±0.02 Mix (HB) 0.50 ±0.72

Mix (Syn) 0.14 ±0.19 Mix (Ost) -0.07 ±0.08

) 1 - TF

cell 3 3

2 Prey Biovolume (µm Prey 1

0 HB SynSyn Ost

Prey

Figure 1. Average cell biovolume (µm-3; +SD) of the prey at T0 and TF (n = 40 each). HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

90 T0

80 T1

)

1 -

70 # TF

cell 3 60

50 #

20 Flagellate Biovolume (µm Flagellate 10

0 HB SynSyn OstOst Mix Prey

Figure 2. Average cell biovolume of C. canzanellaia fed on various prey (n = 30; +SD).

# denotes a significant difference between the T0 and T1 or TF cultures (p < 0.05). HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

70 HB

60 Syn

Ost 50 Mix

30 Ratio Nutrent 20

0 C:N N:P C:P Prey

Figure 3. C. canzanellaia C:N, N:P and C:P ratios (n = 2) fed on varying prey. HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage

2.5 104 105 106 2 107

) 1

- 1.5 µ(d

0.5

0 HB SynSyn Ost Mix

Prey

Figure 4. C. canzanellaia growth rates (µ) over various prey concentrations (cells mL-1) for each of 13 sets of triplicates. Averages shown with SD. HB = heterotrophic bacteria; Syn =

Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

HB Ost Syn 10000 Mix (HB) Mix (S) Mix (O)

1000

)

1 -

100 IR Cd (pg IR

1 0.1 1 10 100 1000 Prey Concentration 104

Figure 5. Ingestion rate (IR in pg C flag-1 d-1) at each of the initial prey concentrations (n=36).

HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

2.5 HB Ost

Syn 2.0

Mix (all)

) 1

- 1.5

(d µ

1.0

0.5

0.0 0 1 10 100 1,000 10,000 IR (pg C flag-1 d-1)

Figure 6. C-based ingestion rates (IR; pg C flag-1d-1) in relation to flagellate growth (µ, d-1).

Also shown is the average for preying on the mixed assemblage ±SD. Note x-axes is log- transformed. HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

70 BV

Carbon 60

40 GGE (%) GGE 30

20 *

0 HB Syn OstreoOst Mix Prey

Figure 7. The average HNAN GGEBV and GGEC grazing on HB, Syn, Ost and Mix (n = 3). * denotes a significant difference between GGEBV and GGEC for Ost (p < 0.05). HB = heterotrophic bacteria; Syn = Synechococcus; Ost = Ostreococcus; Mix = mixed prey assemblage.

CHAPTER 3

The Impact of Diet and Temperature on Growth and Grazing of the Heterotrophic Nanoflagellate

Coniuncta canzanellaia

Abstract

The relationship of temperature with growth, grazing and gross growth efficiency was investigated for the heterotrophic nanoflagellate C. canzanellaia feeding on heterotrophic bacteria and the phototrophic picoeukayrote Ostreococcus lucimarinus over an ecologically pertinent temperature range (10 – 25°C). Flagellate growth rates (maximum with 1.1d-1) seemed to show little impact from changed temperature or with prey type with only one exception, when the flagellate fed on bacteria at 10°C. Both temperature and prey type impacted carbon-based ingestion rates over the same temperature range and ingestion rates were consistently higher when C. canzanellaia fed as a herbivore. Feeding on the bacterial assemblage, however, the flagellate was able to achieve high growth rates at much lower ingestions rates. Additionally, gross growth efficiencies for the flagellate (based on biovolume changes in prey in relation to the flagellate over the duration of the feeding experiments) were highest at 10°C on a heterotrophic bacterial diet (60%) while the highest values feeding on O. lucimarinus were reached at 25°C (52%). These findings indicated that the flagellate was better adapted to utilize bacterial prey at low temperatures (<15°C) while feeding as a herbivore played a more important role at higher temperatures.

Key Words: C. canzanellaia, Nanoflagellate, Temperature, Grazing Rate, Herbivory, Bacterivory

Introduction

Heterotrophic nanoflagellates (HNANs; 2 - 20 µm) are ubiquitous grazers of picoplankton in marine systems (Fenchel 1982, Azam et al. 1983, Pernthaler 2005). The impact that HNAN grazing has on the standing stocks of picophytoplankton and heterotrophic bacterial populations make them drivers of carbon flow and remineralization within microbial food webs (Pelegri et al.

1999, Laybourn-Parry & Parry 2000, Selph et al. 2003). The amount of energy that is transferred to higher trophic levels changes with the composition of prey and predator assemblages (prey- predator “match”) and will depend on physical variables, such as temperature (Caron et al. 1986,

Goldman et al. 1987, Yang et al. 2018).

Studies that have examined the responses of individual protistan species (including ciliates and dinoflagellates) have shown that growth may increase with temperature until reaching an upper limit or can be maintained over a relatively wide temperature range (e.g., 5-10°; Caron et al.

1986, Choi & Peters 1992, Rose & Caron 2007). Numerous studies have also reported that temperature-dependent growth responses and changes in gross growth efficiencies will vary dependent on prey quantity and quality (Dolan & Šimek 1999, Rose & Caron 2007, Vázquez-

Domínguez et al. 2012). Temperature increases during summer months have been linked to increased grazing rates, lower biovolumes and lower carbon content for several HNAN genera

(Choi & Peters 1992, Grover & Chrzanowski 2009, Vázquez-Domínguez et al. 2012). For instance, Ishigaki et al. (2001) showed that Paraphysomonas and Pteridomonas showed higher growth with decreased biovolumes and gross growth efficiencies at 25°C compared to 10°C. A decrease of flagellate size with higher temperatures has been seen in several field studies and has been attributed to increases in respiration and higher mortality due to predation on larger cells

(Rivkin & Legendre 2001, Vázquez-Domínguez et al. 2007, 2012). Temperature-dependent changes in growth and grazing of HNANs impact flagellate competition for limited resources as well as prey-predator relationships over temporal (i.e., seasons) and spatial scales (Caron et al.

1986, Boenigk et al. 2007). In some species, like Paraphysomonas, even a moderate temperature shift of 2-4°C may modify the gross growth efficiencies, and thereby, the amount of carbon transferred to upper trophic levels (Caron et al. 1986, Marrasé et al. 1992, Rose & Caron 2007,

Rose et al. 2009a).

In this study, the marine nanoflagellate Coniuncta canzanellaia was investigated under varying pertinent temperature regimes (10, 15, 25°C) using either a mixed assemblage of heterotrophic bacteria or the phototroph Ostreococcus lucimarinus as prey. In a previous study, we focused on the flagellate’s growth and grazing on a variety of prey types at 16°C, which corresponded to the original temperature that C. canzanellaia had been isolated at from the North Carolina coast

(Chapter 2). The flagellate was shown to thrive both as a bacterivore and herbivore but prey abundances and type had a significant impact on C. canzanellaia rates and efficiencies (Chapter

2). Here we examined the combined effect of temperature and prey type on the growth, ingestion rates, gross growth efficiency and biovolume of C. canzanellaia and discuss them in context with earlier findings.

Materials and Methods

Study Organisms

Grazing experiments were conducted either using a mixed community of heterotrophic bacteria

(HB) or Ostreococcus lucimarinus (Ost, NCMA3430) as prey. HB were collected from coastal waters near Morehead City, North Carolina, near where the nanoflagellate C. canzanellaia was

initially isolated in 2015 (Corradino et al., in preparation). Prey and the nanoflagellate were grown in 0.2 µm-filtered artificial sea water (ASW; ASTM D1141-98 Lake Products Company) with added F/2 medium (Guillard & Ryther 1962) at 16ºC and at a 14:10 light:dark cycle (75 μE m−2 s−1). Cultures of C. canzanellaia also had a baked barley seed added which allowed for sustained growth via bacterivory.

Grazing Experiments

HNAN µ and feeding were determined over 24 hours at 10°C, 15°C and 25°C. These temperatures are typical for seasonal averages during winter, spring/fall and summer in the coastal waters from which C. canzanellaia originated https://www.ndbc.noaa.gov/station_page.php?station=bftn7; Fig 1). Exponentially growing HB at abundances of ~106 cells mL-1 and Ost at densities of ~104 cells mL-1 were each provided to the flagellate which was added at ~103 cells mL-1 (Table 1). Prey-predator treatments and controls (HB and Ost without the flagellate) were set up in triplicates (60 mL) using ASW spiked with F/2 medium to account for prey growth due to the availability of remineralized nutrients in bottles containing HNAN grazers compared to the control treatments (Selph et al. 2003). Both prey and the HNAN cultures were acclimated to the experimental temperatures, with a change of

~2°C every 24 hours until 10°C or 25°C were reached. The organisms were then maintained at these same conditions for ~3 weeks. At the start of the experiment the HNAN was gently concentrated using a 3 µm filter by passive filtration. Then, it was transferred into 0.2 µm- filtered ASW ~2 hours prior to all experiments to allow for emptying of food vacuoles under decreased concentrations of background bacteria at ~102 to 103 mL-1 (Šimek & Chrzanowski

1992, Bratvold et al. 2000, Zwirglmaier et al. 2009). Abundance counts for the HNAN and the

prey were obtained at the beginning (T0) and after 24 hours (TF). Triplicate subsamples (10 µL) from each flask were used to enumerate C. canzanellaia abundances live via light microscopy

(Olympus BX53) and using differential interference contrast. The picoplankton prey were enumerated using 3 mL from each triplicate flask after preservation with ice-cold 2% glutaraldehyde and, in the case of the HB, staining with DAPI (Slowfade antifade, Thermofisher)

(Porter & Feig 1980) at 60x and 100x using epifluorescence microscopy (Table 1).

Biovolumes and Cell Carbon Estimates

Cell size estimates for the nanoflagellate and prey were obtained by measuring cells preserved in glutaraldehyde and capturing images (n = 30 for the HNAN and n = 40 for the prey in each treatment and for each time point) with an Olympus DP73 monochrome digital camera plus

Olympus cellSens Dimension 1.13 software. Comparison with live HNAN cells indicated 24-

30% cell shrinkage with preservation, which is within the range of published values for HNAN

(Hondeveld et al. 1992) and was previously reported for C. canzanellaia (Corradino et al., in preparation). BVs were calculated using the cellular ellipsoid equation of Hillebrand et al.

(1999). The HB and Ost cells were also measured assuming an ellipsoid shape. Previously published carbon (C) conversion factors for C. canzanellaia, the HB and Ost were used with 403 fg C µm-3 for the HNAN, 55 fg C µm-3 for the HB and 656 fg C µm-3 for Ost (Corradino et al., in preparation).

Flagellate Growth and Ingestion Rate

The HNAN growth (µ, d-1) and ingestion rate (IR, cells flag-1 d-1) were calculated using the Frost equations (Frost 1972) modified by Heinbokel (Heinbokel 1978),

μ = ln (PTF/PT0) /(TF-T0)

IR= BT0 – BTF ((PTF – PT0) /(lnPTF – lnPT0))(TF-T0) where B are the prey and P the HNAN concentrations (both in cells mL-1) at the onset (T0) and the final time point (TF). The IRs based on abundances (cells flag-1 d-1) were further converted

C- based estimates (pg C flag-1 d-1) using available conversion factors (see earlier).

Gross Growth Efficiencies

The gross growth efficiency (GGE) of C. canzanellaia was calculated based on abundance and prey C change in the treatments in relation to changes for the nanoflagellate (GGEC) (Fenchel

1982b). The GGE was also calculated comparing the change in BVs in prey and predator

(GGEBV). Following equation from Choi and Peters was used (1992),

GGE= ((PTF- PT0) x Cpred) / ((BT0- BTF) x Cprey)) x 100 where B is the prey concentration at the beginning (T0) and the end of the experiments (TF)

(cells mL-1), P is the HNAN concentration at the beginning (T0) and the end of the experiments

-1 (TF) (cells mL ) and Cpred and for Cprey are C-based predator (pred) and prey (prey) biomass. For the computation of GGEBV the same equation was used but instead of Cpred and Cprey values for

-1 -1 BVpred and BVprey were substituted. The Q10 was calculated for µ rates (d ) and for IR (C flag day-1) over the tested temperature range using the following equation from Caron et al. (1986),

-1 (10 x (t1 – t2)) Q10 = (R0 x RF ) where R0 and RF are the µ or IR at T0 or TF and t1 and t2 are the corresponding temperatures at the respective time point.

Statistical Analyses

Statistical analyses were performed using the JMP Pro 14 software package (SAS Institute, Cary,

North Carolina). To assess the differences in HNAN growth and grazing among treatments, a one-way ANOVA was used (Zar 1984). Comparisons of means were examined using a Tukey test (p < 0.05). Multivariate analyses were performed using the PRIMER v7 statistics software package with the permutational analysis of variance/multivariate analysis of variance

(PERMANOVA) (Anderson et al. 2008). Square root-transformed µ rates, IRs, BV and GGEs were compared based on Euclidean distance measures and using ANOSIM routines (one-way and two-way crossed design) For the two-way crossed design this allowed for the testing of the average effect on µ (or other factors) separately of temperature removing differences in prey type, and the average effect of prey type removing differences in temperature (Clarke &

Warwick 2001). ANOSIM tests compute R values and significance levels (P) where R = 0 implies no difference among groups, and R = 1 suggests that group separation is so large that all dissimilarities among groups are larger than any dissimilarity within them.

Results

Prey and Predator Biovolumes

Ost cells were significantly larger than HB cells (one-way ANOSIM; P = 0.0001, R = 0.542 -

0.66, Fig 2). Besides prey type, temperature seemed to also have a small effect on prey size within each prey group but no consistent trend was apparent (one-way ANOSIM; P <0.004; R =

0.028 – 0.062). Comparisons between BVs at T0 and TF for each of the temperatures and prey types revealed only a difference for Ost at 10°C with a ~18% decrease (p < 0.05; Fig. 2). Initial

BVs for C. canzanellaia did not differ significantly across the varying temperatures (Fig. 3) and

its overall average was 42.7 ±14.4 µm3 cell-1 (n = 180). Feeding on the mixed HB assemblage, C. canzanellaia BVs showed a decrease with temperature by the end of the experiments with the strongest change in the 25°C compared to the 10°C treatments (p < 0.05; average of 52.4 µm3 compared to 38.0 µm3 cell-1, respectively). Grazing on Ost resulted in lower flagellate BVs only comparing the 25°C to the 15°C treatment (p < 0.05; average of 42.4 µm3 compared to 51.0 µm3 cell-1, respectively; Fig. 3). There were no detectable differences between BV for the flagellate feeding on the different prey types comparing across the same temperatures. ANOSIM testing indicated that overall changes in HNAN BVs were not related to prey type and only weakly associated with temperature (one-way, P = 0.0007, R = 0.051).

Flagellate Growth and Ingestion

Prey abundance estimates in the control bottles indicated low growth over the 24-hour incubations at all temperatures (Table S2). C. canzanellaia µ ranged from 0.51 to 1.13 d-1 grazing on HB and ranged from 0.94 to 1.13 d-1 when offered Ost as prey (Fig. 4 and Table 2).

The differing temperatures did not result in a changed µ rate when the flagellate fed on the picoeukaryote and there was a difference in µ in the HB treatments comparing 10ºC and 15ºC with lower µ at 10ºC (p < 0.05). Overall, C. canzanellaia achieved the highest µ rate with 1.13

±0.35 d-1 feeding on Ost at 25°C and the lowest with 0.51 ±0.09 d-1 feeding on HB at 10°C.

Comparison across all treatments using a two-way ANOSIM approach indicated that neither temperature nor prey type affected µ. The Q10 values derived from flagellate µ in the HB treatments were 4.1 from 10°C to 15°C and 1.2 from 15°C to 25°C (Table 3). Feeding on Ost, the µ Q10 values remained relatively consistent with 1.3 at 10°C to 15°C and 1.1 at 15°C to 25°C

(Table 3).

Ingestion rates (IR) based on prey abundance changes increased from 10°C to 15°C and then decreased from 15°C to 25°C for HB. In the Ost treatment the IR were highest at 25°C and decreased at 15°C (Table 2). Maximum IRs were significantly higher feeding on HB (239±91 cells-1 flag-1 d-1) compared to Ost (193 ±23 cells-1 flag-1 d-1, p < 0.05; Table 2). C-based IRs, which take differences in prey cell size and prey C-content into account, showed the same trends

(Fig. 5). Here, maxima were reached with 18 ±7 pg C flag-1 d-1 for the HB treatment at 15°C and

414 ±49 pg C flag-1 d-1 for Ost at 25°C (Table 2, Fig. 5). HNAN IRs, both derived from abundance and prey C, differed for the HB treatment between 10 and 15°C with IRs being higher at 15ºC (p <0.05). IR-C on Ost consistently exceeded those for HB (p <0.05). ANOSIM testing confirmed that prey type had a strong overall effect on HNAN IRs (one-way, P = 0.0001, R =

0.792) while temperature showed no significant relationship with IRs. The Q10 values derived from flagellate IRs in the HB treatments were relatively consistent with 1.5 from 10°C to 15°C and 1.4 from 15°C to 25°C, while feeding on Ost yielded lower values of 0.5 at 10°C to 15°C compared to 3.1 at 15°C to 25°C (Table 3).

Gross Growth Efficiencies

GGE estimates were computed based on changes in BV (GGEBV) and based on C prey content

(GGEC). GGEs for the flagellate feeding at the HB were significantly higher at 10°C with no difference between the higher temperatures (p <0.05; Fig. 6). At 10°C GGE based on BV and C contents averaged 60 ±35 % and 134 ±58%, respectively. Conversely, while grazing on Ost the flagellate’s GGE showed an overall increase with temperature from 31% to 52% GGEBV and from 6% to 26% GGEC (Fig. 6). Fed with Ost, GGEC for C. canzanellaia was significantly higher at 25ºC with a GGEC of 26 ± 11% compared to 6 ± 6% at 10ºC (p < 0.05). ANOSIM

testing across all temperatures and for the differing prey types could not explain overall variability in GGEs. However, basing estimates on C prey contents indicated that both temperature and prey type were drivers of GGE variability (two-way ANOSIM, P < 0.008, R =

0.387 and 0.617, respectively).

Discussion

Nanoflagellate Growth, Ingestion Rates and Gross Growth Efficiencies

Many studies have examined physiological responses to temperature change in HNAN communities but a smaller number has contrasted shifts in rates and efficiencies for the same flagellate species feeding on varying prey (Ishigaki & Sleigh 2001, Rose et al. 2009b). Rates for biological processes are expected to increase consistent with temperature until an optimum range is exceeded, but this response is not unified for HNAN µ or IR as they depend on other factors such as prey quantity or quality (Caron et al. 1986, Choi & Peters 1992, Savage et al. 2004). For instance, the HNAN Paraphysomonas maintained high µ rates (~6 d-1 at 20°C) feeding on heterotrophic bacteria even as temperatures decreased below 15°C while µ rates dropped (~2 d-1 at 14°C) when the flagellate fed on picophytoplankton (Caron et al. 1991, Rose et al. 2009b). A comprehensive review by Rose et al. (2009) demonstrated that such a diet-based difference in µ is observed for several heterotrophic protists (including ciliates and dinoflagellates). Based on their review, it was proposed that a diet-driven shift in µ contributed to mismatches in the µ of phytoplankton prey compared to their herbivorous grazers, allowing for the development of picophytoplankton blooms in high-latitude (low-temperature) environments (Rose & Caron

2007, Rose et al. 2009b).

When C. canzanellaia was exposed to three temperature regimes, the flagellate seemed able to maintain a consistent overall µ rate of ~1 d-1 across the entire temperature range from 10 - 25°C with one exception (Fig. 3). Growth seemed impeded at the lowest temperature when the flagellate grazed as a bacterivore followed by a dramatic increase at 15°C. This was reflected in a relatively high Q10 value of 4.1 at 10 - 15°C compared to 1.2 at 15 - 25°C and compared to Q10 values on an Ost diet which remained virtually unchanged (1.1 - 1.3, Table 3). The Q10 approach has been used frequently to describe the relationship between temperature change and basic physiological rates for HNANs and to allow for comparison among observations (Table 4).

Previous research has shown that Q10 values that are higher than 2 - 3 may indicate the removal of constraining factors (i.e., low temperature) on HNAN physiology as indicated in our HB treatment at 10°C (Hochachka & Somero 2002, Montagnes et al. 2003). However, the relationship between biological processes and temperature can be varied as rates reflect an integration of several processes (i.e. growth, respiration, digestion) that may be limited at different temperatures (Montagnes & Franklin 2001, Berges et al. 2002, Clarke & Fraser 2004).

This may partially explain why changes in µ did not “match” those in IRs for the flagellate on either prey in this study (Fig. 7). As mentioned before, on an Ost diet, no change was seen in µ rate but an overall decrease in average IRs from 10 to 15°C was followed by a strong increase from 15 to 25 °C (Q10 values of 0.5 and 3.1, respectively; Fig. 7, Table 3). Overall, neither temperature nor prey type could explain the variability in µ rate for C. canzanellaia. However, for IRs, statistical comparison across all experimental treatments did identify temperature as an influencing factor and, even more strongly, prey type.

Field and laboratory studies have reported significant differences in µ and IRs with prey type linked to prey size, motility or cell surface characteristics (Nagata 1988, Weisse 1997,

Šestanović et al. 2004, Shannon et al. 2007). Comparing C-based IRs in the present study showed the flagellate consume Ost at significantly higher rates across all temperatures (10°C,

15°C and 25°C, Fig. 7) which agreed with findings in our previous study (Corradino et al., in preparation). In the earlier study, IRs were examined for the same two prey organisms (plus

Synechocccus and a mix of all three prey) over increasing prey densities. Comparing results for

C. canzanellaia at 16°C feeding on Ost prey (Corradino et al., in preparation), with rates at 15°C in the present study at the same prey densities (104 cells mL-1), showed similar µ rates with ~1 d-

1 but lower IRs with 9 ±6 pg C flag-1 d-1 compared to 105 ±11 pg C flag-1 d-1 in the present study.

The HNAN feeding on the mixed HB assemblage at the same prey densities (106 cells mL-1) resulted in µ rates of 1.7 d-1 compared to 1 d-1 in the present study and in IRs of 85 ±2 compared to 18 ±7 pg C flag-1 d-1 (Corradino et al., in preparation). We speculate that part of the reason why the flagellate did not achieve higher µ rates in the present study, might be attributed to bacterial community structure changes due to prolonged growth of the HB assemblage in the lab.

Gross Growth Efficiencies

Differences between GGEBV and GGEC values are typically attributed to the fact that GGEBV take changes in the grazer into account while GGEC are based mainly on prey changes (Caron et al. 1986, Pelegri et al. 1999, Ishigaki & Sleigh 2001). For C. canzanellaia both GGEs showed the same patterns with the highest values at 10ºC feeding on HB while maximum GGEs on the

Ost diet peaked at 25ºC (Fig. 6). At these respective temperatures, GGEs based on BV were rather comparable at 60 and 52% for feeding on HB and Ost, respectively (Fig. 5). Notably, one

of the triplicate bottles for the 10°C treatment holding HB prey had a much lower GGE compared to the other two replicates however, removing the value did not have an effect on treatment comparisons or differences being significant. Based on our previous and present study,

C. canzanellaia achieved high µ at much lower IRs fed with HB compared to an Ost diet (Fig 6 in Chapter 2 and Fig. 6). These differences may be due to reported differences in prey quality

(e.g., nutritional composition, Chapter 2) or other cell characteristics (similar to Synechococcus strains) that make Ost less easy to digest or assimilate (Boenigk et al. 2001, Zwirglmaier et al.

2009). Higher GGEBV on HB prey at 10°C, combined with the ability to promptly increase µ at

-1 -1 15°C (Q10 of 4.1) at yet relatively low IRs (6 pg C flag d ), indicated that the flagellate can take advantage of modest temperature changes at 10°C and above when grazing on HB.

Comparatively, grazing on Ost, C. canzanellaia yielded high GGEs and µ rates at the higher temperatures only at markedly higher IRs (Fig. 7). These results suggest that the HNAN is better adapted to utilize bacterial prey at temperatures below 15°C, while feeding as a herbivore comes into play at higher ambient temperatures when picoeukaryote prey might also be more readily available.

Impact on Prey Carbon Stocks

Daily C removal rates were calculated at each temperature by applying average IRs from this study and assuming natural prey and flagellate abundances (106, 104 and 103 cells mL-1 for HB,

Ost and flagellates, respectively). C. canzanellaia consumption of HB was estimated highest at

15°C accounting for the potential removal of ~76% of bacterial standing stocks and the lowest at

10°C with 8%. Herbivory, based on Ost as sole prey, was estimated highest at 25°C with >100% and the lowest at 15°C with 40%. These estimates are oversimplified approximations that do not

take into account the diverse and complex nature of picoplankton and HNAN assemblages that come with an assortment of species-specific prey-HNAN trophic interactions, or the fact that nanoflagellates are subject to competition and grazing themselves (González 1996, Christaki et al. 1999, Tsai et al. 2018, Yang et al. 2018, Corradino et al., in preparation).

Despite Ost being a key member of picoplankton communities around the globe (Tragin and

Vaulot 2019), there is little to no information on its population dynamics along the East Coast. A multi-year study along the California coast showed that Ost can form short-lived high-abundance blooms multiple times throughout the year reaching cell abundances of >105 cells mL-1

(Fouilland et al. 2004, Countway & Caron 2006). One study conducted in a Mediterranean

Lagoon environment investigated Ost µ and grazing losses under nutrient-amended conditions showing that Ost µ rates of 2 to 8 d-1 were paired with high grazing losses of up to 6.5 d-1 possibly explaining the rapid population fluctuations reported for the smallest known eukaryote

(Fouilland et al. 2004, Countway & Caron 2006, Worden 2006).

When C. canzanellaia was offered a mixed prey assemblage containing HB, Ost and

Synechococcus at an experimental temperature of 16°C, it derived as much as 51% of its C intake from Ost, followed by HB with 46% and Syn with 3% (Corradino et al., in preparation).

Together with findings presented here, this may suggest that HNANs with feeding ecologies similar to C. canzanellaia could account for measurable losses in Ost standing stocks in East coast waters during spring and summer months, when water temperatures are at ~15°C and above. C. canzanellaia’s ability to sustain itself on both heterotrophic bacteria and

picophototrophic prey under the temperature regimes it was exposed to in this study seemed to indicate its suitability for temperate waters off the North Carolina coast.

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two heterotrophic nanoflagellates on marine Synechococcus strains. Environmental

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Table 1. Initial prey and predator abundances (cells mL-1) with their standard deviation (±SD) in each of the experiments. Prey in Control = starting concentrations in flasks that had no flagellate added. HB = heterotrophic bacteria; Ost = Ostreococcus lucimarinus

Treatment Temp °C HNAN Prey Prey in Control cells ml-1 cells ml-1 cells ml-1

HB 10 1.7 ±0.18 x 103 2.7 ±0.06 x 106 5.5 ±0.19 x 105 15 1.0 ±0.05 x 103 4.5 ±0.38 x 106 3.2 ±0.08 x 105 25 1.5 ±0.42 x 103 3.8 ±0.31 x 106 3.8 ±0.31 x 105 Ost 10 1.3 ±0.44 x 103 5.4 ±0.18 x 104 5.4 ±0.19 x 105 15 1.8 ±0.15 x 103 4.8 ±0.10 x 104 4.4 ±0.11 x 105 25 1.5 ±0.40 x 103 4.7 ±0.27 x 104 3.4 ±0.06 x 105

Table 2. Specific growth rate (µ, d-1) and ingestion rate (IR, cells flag-1d-1 and pg C flag-1d-1) for

C. canzanellaia averaged over triplicate incubations ±SD.

Prey Temp Flag Abund Prey Abund Flag µ IR IR cells flag-1 °C cells mL-1 cells mL-1 d-1 pg C flag-1 d-1 d-1

HB 10 1.7 ±0.2 x 103 2.7 ±0.1 x 105 0.5 ±0.1 75 ±28 6 ±2

15 1.0 ±0.1 x 103 4.5 ±0.4 x 105 1.1 ±0.1 239 ±91 18 ±7

25 1.5 ±0.4 x 103 3.8 ±0.3 x 105 1.0 ±0.5 103 ±30 8 ±2

Ost 10 1.3 ±0.4 x 103 5.4 ±0.2 x 105 0.9 ±0.7 85 ±64 180 ±137

15 1.8 ±0.2 x 103 4.8 ±0.1 x 105 1.0 ±0.2 49 ±6 101 ±11

25 1.5 ±0.4 x 103 4.7 ±0.3 x 105 1.1 ±0.4 193 ±23 414 ±49

Table 3. The Q10 values across all temperatures and prey treatments with C. canzanellaia.

Temp (°C) Prey Q10 (µ) Q10 (IR)

10-15 HB 4.1 1.5

15-25 HB 1.2 1.4 10-25 HB 1.8 1.4

10-15 Ost 1.3 0.5

15-25 Ost 1.1 3.1

10-25 Ost 1.1 1.7

Table 4. A comparison table of growth (µ) and Q10 rates across various experiments. The average is reported at the tested temperature. Note: differences in experimental methodology exist between the studies. HB = heterotrophic bacteria; Ost = Ostreococcus; Syn =

Synechococcus; Bact Cult = bacterial culture; Mix = HB + Syn + Ost

Q10 µ HNAN -1 IR Temp HNAN Prey Temp µ (d ) Q10 µ Q10 IR Source Size cells flag-1 d-1 Range (°C) (°C) C. canzanellaia 4-5 µm HB 10 0.51 75 10-15 4.1 1.1 This Study HB 15 1.09 239 15-25 1.1 1.4 HB 25 0.77 103 10-25 1.7 1.3 Ost 10 0.90 85 10-15 1.1 0.5 Ost 15 0.96 49 15-25 1.1 3.1 Ost 25 1.03 193 10-25 1.1 1.7 Corradino 4-5 µm Mix 16 0.90 67 ------in prep HB 16 1.65 1,106 ------Syn 16 0.72 120 ------Ost 16 1.06 4 ------Natural HNAN Mixed HB -- n/a 96-2,448 17-28 -- 1.6 Park and Assemblage Cho (2002) Mixed HB -- n/a 96-2,448 17-28 -- 1.8 Mixed HB -- n/a 96-2,448 17-28 -- 3.2 -- -- Rivkin Mixed HB + Syn -- 6-13 -- 2.4-2.5 (1999) Syn ------6-13 -- 1.2 -- Sherr 3-5 µm HB n/a 124-658 12-20 n/a 2.8 (1988a) 3-5 µm HB -- -- 494-658 16-20 -- 2.0 Paraphysomonas 7-12 Caron Cult Bact 14 1.37 43 14-18 2.5 1.8 imperforata µm (1986) Cult Bact 18 1.92 54 18-22 2.0 1.6 Cult Bact 22 2.52 65 22-26 3.2 18.5 Cult Bact 26 4.04 209 14-26 2.5 3.7

7-12 Delaney Cult Bact 10 0.61 187 10-15 0.20 0.80 µm (2003) Cult Bact 15 1.29 209 10-15 0.20 0.80 18- 1.2- -- 18- Sherr Monas sp. 3-5 µm HB 1.1 n/a 25.5 4.8 25.5 (1988b)

Supplemental Tables

Table S1. Average cell biovolumes of HB and Ost at 10, 15 and 25°C at TF (n = 40; ±SD).

Prey °C µm3 cell-1

HB 10 0.57 ±0.58

HB 15 0.64 ±0.33

HB 25 0.47 ±0.36

Ost 10 1.46 ±0.28

Ost 15 1.38 ±0.36

Ost 25 1.87 ±0.53

Table S2. Prey growth (µ d-1) in control treatments shown as average (n = 40; ±SD).

Prey °C µ

HB 10 -0.30 ±0.05

HB 15 0.40 ±0.11 HB 25 0.09 ±0.02

Ost 10 0.02 ±0.03

Ost 15 0.11 ±0.08

Ost 25 0.23 ±0.10

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Figure 1. Monthly averages for temperature data from 2015 to 2019 from the National Data Buoy (station BFTN7) for NOAA at Beaufort (34°43’17.77”, 76º45’33.93”W), North Carolina, (https://www.ndbc.noaa.gov/station_page.php?station=bftn7) are shown ±SD (n= 5).

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10 15 3.5 25

)

1 -

cell 2.5 3 3 2

1.5

Biovolume (µm Biovolume 1

0.5

0 Hetero OstreoOst Hetero OstreoOst T0 TF

Prey Type

Figure 2. Average prey cell biovolumes (µm3 cell-1; ±SD) at T0 and TF at 10°C, 15°C and 25°C

(n = 40 each).

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10 15 70

) 1

- 60 cell 3 50

Flagellate Biovolume (µm Flagellate 10

0 HB Ost T0 TF

Figure 3. The average cell biovolume of C. canzanellaia fed on various prey at 10, 15 and 25°C

(n = 30; ±SD). T0 biovolumes are averaged across temperatures (n = 60 each).

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HB 1.8

1.6 Ost

1.4

1.2

)

1 -

(d 1.0 µ 0.8

0.6

0.4

0.2

0.0 10 15 20 Temp (°C)

Figure 4. Average C. canzanellaia growth rates (µ) at 10°C, 15°C and 25°C feeding on a mixed bacterial assemblage (HB) or Ostreococcus (Ost).

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1,000 HB

Ost

) 1

- 100

1 -

IR Cflag (pg IR 10

1 5 10 15 20 25 30 Temp (°C)

Figure 5. Ingestion rate (IR in pg C flag-1 d-1) at each temperature (n=18).

105

200 10°C

180 15°C

160 25°C

140

120

100 GGE % GGE 80

0 BV C BV C

HB OstOst

Figure 6. The GGEBV and GGEC for C. canzanellaia grazing on HB and Ost at all three temperatures.

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HB 1.6 Ost 1.4

1.2 15°C

) 1 - 25°C 25°C

(d 1.0 15°C µ 0.8 10°C

0.6 10°C 0.4

0.2 1 10 100 1,000 10,000 IR (pg flag-1 d-1)

Figure 7. C-based ingestion rates (IR; pg C flag-1d-1) in relation to flagellate growth (µ, d-1).

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Conclusion

This study allowed for the characterization of a previously unidentified HNAN affiliated with the

Bicosoecids and led to the characterization of a new genus and species. At present, only a relatively small number of HNANs have successfully been cultured to study their functional roles while the majority of cultured protists remain to be phototrophic algae (Keeling et al.,

2014). Cultivation of HNANs is an important step in understanding functional diversity in natural assemblages as they do not form a homogenous trophic guild, but show succession

(Massana et al. 2009). For instance, in the Mediterranean Sea, the dominance of bacterivores and colloidal matter feeders was reported before a phytoplankton bloom with a shift to omnivorous feeders that prey upon phytoplankton and heterotrophic prey during and after the bloom (Maria

Moustaka-Gouni et al. 2016). Successful cultivation and identification also create the basis to employ whole genome sequencing or transcriptomics approaches which have begun to provide new and unexpected insights into microbial processes (e.g., Armbrust et al. 2004; Worden et al.

2009). This study, on the feeding ecology of the novel HNAN C. canzanellaia, is one of a limited number that explored not only growth and grazing but also nutrient and carbon flow associated with the selection of heterotrophic versus phototrophic prey.

In Chapter 2, I was able to document that C. canzanellaia can sustain growth on a variety of prey types but prefers HB to Syn and Ost. The HNAN seemed unable to digest the Syn strain used in this study as efficiently as the other prey resulting in decreased GGEs. Since the genetic and biogeographic diversity of Syn is wide-ranging and their digestibility or ability to resist grazing was shown to depend on the strain (Gorsky et al. 1999, Guillou et al. 2001, Apple et al. 2011), C. canzanellaia can be used as a model to further explore varying prey-predator matches.

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Understanding how Syn species composition ties to grazing mortality is of interest since this picophototroph contributes significantly to global primary production (Partensky et al. 1999,

Flombaum et al. 2003). Additionally, the HNAN BV increase dependent on diet may have fundamental consequences for the HNANs own susceptibility to grazers and further impact carbon transfer to upper trophic levels. Extrapolating daily C-based IRs from the mixed prey trial

(i.e., HB, Syn and Ost) to an entire nanoflagellate community, resulted in the estimated removal of ~17% of the Syn standing stocks compared to 23% of the HB, and 69% of the Ost populations.

The high contributions from Ost to C. canzanellaia’s diet was another significant finding of this study and hinted towards the potential of HNANs, that display similar feeding ecology to C. canzanellaia, to exert strong top-down control on incident Ost populations.

Chapter 3 demonstrated that C. canzanellaia was able to sustain consistent µ rates across a temperature range that would be typical for temperate coastal waters independent of prey type

(i.e., HB versus Ost). However, IRs varied strongly with both prey type and temperature.

Feeding on the HB, the flagellate was able to achieve higher µ rates at much lower IRs indicating that the HNAN was better adapted to utilize bacterial prey at lower temperatures (<15°C) while feeding on Ost led to increased µ at 15 and 25°C; warmer temperatures being associated with spring bloom conditions in North Carolina coastal waters. Similar to the findings in Chapter 2, the results indicated that HNANs such as C. canzanellaia can have a measurable impact on standing stocks of picoplankton. Herbivory, based on Ost as sole prey, was estimated highest at

25°C with >100% and the lowest at 15°C with 40%. Further investigations are warranted into how Ost populations, which have been seen to fluctuate (Countway and Caron, 2006) and may be controlled in part by HNAN grazing. Again, C. canzanellaia can be used as a model to

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explore grazing mortality in key primary producers (e.g. Prochlorococcus, Synechococcus, and

Ostreococcus) and estimate how much energy (C) is shunted up the food web via this picophytoplankton-HNAN trophic link.

An important, but yet unexplored, component of this study is that the attachment of C. canzanellaia to particles and diatoms is a key feature to its behavioral ecology. The HNAN was originally isolated attached to several diatoms and the role that organismal interactions within the phycosphere play for carbon turnover or export from ocean waters has become a major focus point in context with the biological pump. Questions on how attachment to bloom-forming diatoms may impact bloom duration and/or the formation and sinking rates of marine snow are of key importance to understand carbon export rates from surface waters (Kühn et al. 1997,

Arndt et al. 2000). Further studies that explore these questions using C. canzanellaia can help decipher these processes. In conclusion, further investment in HNAN taxonomic and ecological research will greatly aid to break apart the trophic “black box” that we currently use to represent the most abundant group of oceanic predators. This research serves to provide new information on a novel HNAN and hopes to contribute to our understanding of complexities within the microbial food web.

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