Doctor of Environmental Sciencein Graduate School of Environmental Science
The University of Shiga Prefecture

Symbiotic microbiome with three dominant crustacean zooplankton in
Lake Biwa, Japan

Defended by Taiabur Rahman Tuhin
On 5 September 2023, in Hikone (Japan)

Supervisor: Professor Syuhei Ban
Graduate School of Environmental Science
The University of Shiga PrefectureACKNOWLEDGEMENTS

I am delighted to express my heartiest and most sincere gratitude, deep appreciation,indebtedness, and honor to my most respected teacher and thesis supervisor, Professor Dr.
Syuhei Ban, for his excellent academic guidance throughout my research. His constantinspiration, expert advice, affection, constructive criticism, exemplary editing, and continuous
meticulous encouragement throughout the study made it possible to come to this end.
I would also especially like to thank my co-supervisors, Professor Dr. Misako Urabe
and Dr. Shoko-Hosoi Tanabe, Associate Professor, for training me in using molecularlaboratory equipment and helping me get to grips with all aspects of this research. I would liketo thank Mr. Bun-ichiro Kaigai, the captain of the research vessel Hassaka II, for his kindcooperation and help during the samplings.
I owe my warm thanks to Dr. Xin Liu for his all kinds of help. I take this opportunity
to express my gratitude to all my laboratory members who have been instrumental in
successfully completing this project. And I find no words to express my sincere and heartiest
gratitude to my beloved parents, wife, and children for their continuous moral support in the
successful implementation of the research work.
At the very outset, all gratefulness to Almighty Allah, the most gracious and merciful
who has enabled me to complete this research work and manage each and everything soundly.

AuthorFUNDING

This study was supported by a Grant-in-Aid for Scientific Research (22H02425) fromthe Japan Society for the Promotion of Science (JSPS) to Professor Syuhei Ban.

Keywords
Bacterial communities, Lake Biwa, Symbiotic bacteria, Ambient bacteria, Gut, Crustacean
zooplankton, Daphnia pulicaria, Eodiaptomus japonicus, Cyclopoida spp., Flagellate, Ciliate,
Epibionts.

Host laboratory
Laboratory of Aquatic Ecosystems
Department of Ecosystem Studies
School of Environmental Science
The University of Shiga Prefecture2500 Hassaka-cho, Hikone, Shiga, Japan.

CONTENTS
GENERAL INTRODUCTION…………………………………………….…….………

1

1.1 Zooplankton in Lake Biwa……………………………………………………………

1

1.2 Microbiome of host zooplankton …………………………………………………….

3

1.3 Bacterial symbionts……………………………………………………………………

4

1.4 Flagellated and Ciliated symbionts……………………………………………………

5

1.5 Methodology for identifying symbiotic bacteria ……………………………………...

6

1.6 The scientific approach: Thesis contents……………………………………………...

7

CHAPTER1:SYMBIOTICBACTERIALCOMMUNITIESOFZOOPLANKTERS FROM LAKE BIWA, JAPAN …………………………………...

9

1. INTRODUCTION………………………………………………………...…...…...….

10

2. MATERIALS AND METHODS……………………………………………...….........

12

2.1 Sample collection and preparation…………………………………………………...

12

2.2 Metabarcoding analysis using Illumina MiSeq……………………………………...

13

2.3 Data analysis…………………………………………………………………………

14

3. RESULTS ………………………………………………………………...…...….........

15

4. DISCUSSION………………………………………………………………………….

27

CHAPTER 2: COMPARATIVE ANALYSIS OF BACTERIAL COMPOSITIONSIN THE GUT, BODY OTHER THAN GUT, AND FECES OF THE THREE
DOMINANTCRUSTACEANZOOPLANKTON
IN
LAKE
BIWA,
JAPAN…………………………….…….…….…….…….…….…….…….…….………
32
1. INTRODUCTION………………………………………………………...…...….........

33

2. MATERIALS AND METHODS……………………………………………...…...….

35

2.1 Sample collection and preparation…………………………………………………...

35

2.2 Metabarcoding analysis using Illumina MiSeq……………………………………...

38

2.3 Data analysis…………………………………………………………………………

39

3. RESULTS ………………………………………………………………...…...….........

40

4. DISCUSSION…………………………………………………………….…...…...…...

47

CHAPTER 3: COMMUNITY STRUCTURES OF SYMBIOTIC BACTERIAASSOCIATED
WITH
THE
THREE
DOMINANTCRUSTACEANZOOPLANKTON IN DIFFERENT DEPTH HABITATS OF LAKE BIWA
DURING THE STAGNATION PERIOD…….…….…….…….…….…….…….……..

52

1. INTRODUCTION……………………………………………………….…...…...…...

53

2. MATERIALS AND METHODS……………………………………….…...…...…....

55

2.1 Sample collection and preparation……………………………………………………

55

2.2 Metabarcoding analysis using Illumina MiSeq………………………………………

57

2.3 Data analysis……………………………………………………………………….....

58

3. RESULTS………………………………………………………………...…...…...…...

59

3.1 Vertical structure of the water column……………………………………………….

59

3.2 Bacterial compositions in zooplankton and ambient waters………………………….

61

4. DISCUSSION…………………………………………………………….…...…...…...

69

CHAPTER 4: TAXONOMIC AND ECOLOGICAL ASPECTS ON EPIBIOTIC
CILIATES AND FLAGELLATES ATTACHED TO CYCLOPOID COPEPODS IN
LAKE BIWA…………………………….…….…….…….…….…….…….…….……...

73

1. INTRODUCTION………………………………………………………...…...….........

74

2. MATERIALS AND METHODS……………………………………………...…...….

76

3. RESULTS………………………………………….…...…...….....................................

77

3.1 Morphological features of the epibionts………………………………………………

79

3.1.1 Flagellates………………………………………………………………………….

79

3.1.2 Ciliates …………………………………………………………………………….

80

3.2 Seasonal changes in temperature, dissolved oxygen, and chlorophyll a concentration81

3.3 Seasonal changes of host abundance and the prevalence of the epibionts …………….

83

4. DISCUSSION…………………………………………………………….…...…...…...

87

SUMMARY………………………………………………………………….…….……...

91

REFERENCES……………………………………………………………...…….……...

94

APPENDIX………………………………………………………………….…….………

105

GENERAL INTRODUCTION

1. General Introduction
1.1 Zooplankton in Lake Biwa
Lake Biwa (35.1°N, 136.1°E) is the largest and the oldest freshwater lake in Japan, witha surface area of 670.3 km2, a water volume of 27.5 km3, and a maximum depth of 104 m(Tabata et al. 2016). It is the most species-rich aquatic habitat in Japan (Rossiter 2000),
providing water for about 14.5 million people living in surrounding provinces for their daily
lives and playing an important role in fisheries and agricultural irrigation (Kawanabe et al.
2012). Lake Biwa is monomictic; the water column becomes stratified from May to September,starts mixing from October, being mixed wholly in February every year (Kagami et al. 2006),
and phytoplankton biomass in the epilimnion (0–20 m) generally increased from spring but
low in winter (Hsieh et al. 2010, Liu et al. 2020).

In Lake Biwa, seven crustacean taxa, Eodiaptomus japonicus, Cyclopoida spp.
including mainly Mesocyclops dissimilis and Cyclops vicinus, Daphnia galeata, Daphnia
pulicaria, Bosmina longirostris, and Diaphanosoma orientalis, dominated and accounted for> 90% of the total crustacean biomass in the lake (Kawabata 1989, Yoshida et al. 2001, Urabeet al. 2003, Liu et al. 2020, 2022). Among the zooplankton taxa, calanoid copepod E. japonicus
is the most dominant, followed by cladoceran Daphnia spp. and Cyclopoida spp. (Yoshida etal. 2001, Liu et al. 2020). D. pulicaria is the largest taxa among Daphnia spp. in the lake,
having distinct morphological features including a short head without any helmet or projection,with five stout teeth in the middle pectin in the post-abdominal claw, compared to D. galeata(Urabe et al. 2003). Daphnia spp. show different seasonal succession in abundance among
species (Liu et al. 2020). D. galeata usually increases after May, reaching its maximum inJune. On the other hand, D. pulicaria shows large inter-annual variability during the mainoccurrence period, that is, mainly from January to June, reaching their maximum in April to
May but declining after June toward winter (Liu et al. 2020). Cyclopoids usually increase in1

the early summer and are stable until October, and a decline during the winter months(Kawabata 1989, Liu et al. 2020). The abundance of E. japonicus becomes low during winterand early spring, starts to increase after May, reaching a maximum in June to September, anddecreases until December (Kawabata 1987, Liu et al. 2020).

The abundance of these zooplankton species serves as a major component of thebiological condition by affecting water quality, phytoplankton densities, fish production, andnutrient cycling. They occupy the center of the open water food web, ingest bacteria and algaethat form the base of the food web, and in turn, they are heavily preyed upon by fish, insects,
and others (Yoshida et al. 2001, Kawabata et al. 2006, Liu et al. 2020). However, the effects
of environmental disturbances can be detected through changes in zooplankton species
composition and abundance (Liu et al. 2020).

In aquatic ecosystems, metazoan zooplankton are essential elements of the trophic food
web and significant contributors to pelagic biodiversity and biochemical processes with its
symbiotic microbiome (Tang et al. 2010). Recently, microbial symbionts have been known tobe an integral part of zooplankton. Because cladoceran Daphnia and copepods depend on theirbacterial symbionts for their development and growth (Sison-Mangus et al. 2014,
Peerakietkhajorn et al. 2015, Callens et al. 2018, Akbar et al. 2020), nutrition (Macke et al.
2017, Cooper and Cressler 2020, Gorokhova et al. 2021), and pathogenic protection (Wahl etal. 2012). Interactions between the zooplankton and its symbiotic microbiome can be
obligatory or facultative, and mutualistic, commensalistic, or parasitic (Wells and Varel 2011,
Martin and Schwab 2012).
Zooplankton ecologists generally tend to focus on interactions between zooplankton
and their prey or predators, abiotic factors of surrounding environments that affect life history
traits (Kawabata 2006, Boonmak et al. 2018, Liu et al. 2014, Gao et al. 2022) while symbiotic

2

microbiomes have been overlooked by aquatic microbial ecologists. During the last two
decades, researchers have focused on host–microbiome interactions (Lim and Bordenstein2020). However, studies on interactions between zooplankton and their symbionts are limited,
especially in natural habitats, because the two are usually considered to represent different
functional units with indirect connections in nutrient cycling and food webs in aquatic
ecosystems (Azam and Malfatti 2007). It is necessary to study the interaction between thesehost-symbiont systems and their mode of interaction for a better understanding of the element
fluxes, ecosystem responses, food web architecture, carbon metabolism, and unexploredtrophic links in aquatic ecosystems (Eckert and Pernthaler 2014, Corno et al. 2013, Thébault
and Fontaine 2010, Mougi and Kondoh 2012).
In Lake Biwa, zooplankton and microbial communities in the lake have been wellstudied separately over various time scales (Kawabata 1989, Liu et al. 2014, Okazaki andNakano 2016, Okazaki et al. 2019). However, interactions between zooplankton and theirmicrobial symbionts have not been studied yet. In this study, I determined the microbialcommunities, such as bacteria and protists, associated with the three dominant zooplankton
taxa in Lake Biwa.
1.2 Microbiome of host zooplankton
In aquatic habitats, crustacean zooplankton, including copepods and cladocerans, is ahost of a wide range of microorganisms, such as viruses, bacteria, fungi, algae, and protozoans(Ho and Perkins 1985, Carman and Dobbs 1997, Bickel et al. 2012, Bass et al. 2021). These
prokaryotic and eukaryotic microorganisms live in close physical contact with a host called its
microbiome (Macke et al. 2017), and those inhabiting the host’s body surface, including
swimming appendages and filtering apparatus, are called ectosymbionts, whereas those in theinternal organs, such as digestive tracts, hemocoel, and tissue called endosymbionts (Ho andPerkins 1985). The endosymbiotic microbiota may use organic matter available in the host’s

3

gut and tissue, and ectosymbiotic ones can utilize organic matter excreted by the host (Wahl etal. 2012, Bickel and Tang 2014). By attaching to the host body surface, epibiotic
microorganisms may evade zooplankton predators (Henebry and Ridgeway 1979) and travel
to areas with available foods (Kankaala and Eloranta 1987, Wahl 1989) or use the host as a
substrate for their growth and survival (Tang 2005, Tang et al. 2010, Corre et al. 2020, Lu etal. 2020)
Previous studies reported that symbiotic fungi have parasitic effects on their calanoid
copepod hosts. Redfield and Vincent (1979) reported that a pathogenic ectosymbiotic fungus
reduced the population size of Diaptomus novamexicanus by 48.8% in an alpine lake in
California. An endosymbiotic fungus, Leptolegnia baltica, was responsible for the mortality of
Eureytemora hirundoides in the Baltic Sea (Vallin 1951, Höhnk and Vallin 1953).
Endosymbiotic parasitic fungi, Thelohania sp., affected the life history traits of Daphnia pulex
in a vernal pond in Michigan (Brambilla 1983).
1.3 Bacterial symbionts
Several previous studies reported on the bacterial symbiont of daphnids and copepods.
Bacterial components associated with their host bodies have been almost reported from
cultured daphnids (Qi et al. 2009, Sison-Mangus et al. 2014, Callens et al. 2016, Akbar et al.
2020), and calanoid and cyclopoid copepods from marine habitats (Gerdts et al. 2013, De Corteet al. 2014, 2018, Sadaiappon et al. 2021, Velasquez et al. 2022). The information on symbiotic
bacterial microbiota associated with copepods and cladocerans from natural freshwater habitats
is still limited (Grossart et al. 2009, Samad et al. 2020, Wang et al. 2021, Eckert et al. 2021).
However, all these studies described host-associated symbiotic bacteria using the whole bodywithout dissecting the gut, thus containing limited information on the specific location ofsymbionts.

4

Furthermore, Chae et al. (2021) reported the gut bacterial microbiota of Acratia tonsa,
Sinocalanus tenellus, and Pseudodiaptomus inopinus from a brackish habitat without
separating the intestine from the host’s body. Some other studies described host-associatedsurface-attached bacteria using microscopic images (Nagasawa and Nemoto 1985, Nagasawa
and Terazaki 1987, Huq et al. 1983, Skovgaard et al. 2015). Harris (1993) and Carman andDobbs (1996) described ectosymbiotic bacteria of calanoid hosts using culturing techniques.
Moreover, Hansen and Bech (1996) described bacteria in the gut, body surface, and feces of
Acratia tonsa using culture-dependent methods and without separating the gut from the hostbody. Such studies provided limited information about the species composition and diversityof bacterial microbiome attached to the host’s surface and internal organs. To elucidate thismatter, metagenomic analysis of bacterial DNA extracted separately from the gut and bodyparts without the gut allows the phylogenetic investigation of these host-associated uncultured
bacteria in a broader range. And such a study will uncover knowledge on the complex
interaction between host zooplankton and associated bacterial microbiome in the specificmicrohabitats.
1.4 Flagellated and Ciliated symbionts
Epibiosis involving a relationship between epizoic protists such as flagellates andciliates and zooplankton is a widespread phenomenon in aquatic environments (Chiavelli 2003,
Jones et al. 2016). Ciliates have free-living and symbiotic life (Mayén-Estrada et al. 2021), andabout 2600 species are symbionts to metazoan zooplankton (Corliss 2000). The peritrich
ciliates order Sessilida contains about 105 to 140 genera and more than 800 species (Foissner
et al. 2010). The genus Epistylis under the Epistylididae family belonging to Sessilida contains
over 260 nominal taxa that may adapt to diverse environmental conditions (Lu et al. 2020, Nitta
2022). Nitta (2022) reported the peritrich ciliate Epistylis wuhanensis attached to cyclopoid
copepod Lernaea cyprinacea from the Arida River in Wakayama prefecture. Several previous

5studies reported that heterotrophic peritrich ciliates attach to copepods and cladocerans(Foissner et al. 1999, Utz and Coats 2005, Clamp et al. 2016, Lu et al. 2020).
The protist Colacium and species of dinoflagellates colonize on zooplankton hosts (Hoand Perkins 1985, Rosowski and Kugrens 1973). The free-living stage of Colacium contains
flagellum but leaves when it enters a sessile life attached to a substrate (Hubber-Pestalozzi
1955). Species of Colacium are autotrophs and can be heterotrophic in a light-limiting
environment (Salmaso and Tolotti 2009). Several studies described Colacium as ectosymbionts
of copepod and cladoceran hosts (Mohlenberg and Kaas 1990, Threlkeld et al. 1993, Chiavelli
et al. 1993, Zalocar et al. 2011).
However, previous studies dealing with epibiotic ciliates and flagellates mainly focusedon describing new records using morphological and molecular data, possible effects on hosts,
and infection prevalence. But, limited information exists on the ecological aspect of theepibionts.

1.5 Methodology for identifying symbiotic bacteria

In the past, physical characteristics were the only basis for identifying and categorizing
the bacteria, which proved to be challenging due to their wide range of variations. However,
the emergence of DNA sequencing has transformed the field of biology, enabling more precise
analysis of microorganisms. In 1990, Carl Woese and his team first began analyzing andsequencing the 16S rDNA genes of various bacteria using state-of-the-art DNA sequencing
technology and used these sequences for phylogenetic studies. With the advent of PCR and
automated DNA sequencing in the past two decades, as well as subsequent investigations on
the 16S rDNA sequencing of bacteria and 18S rDNA sequencing of eukaryotes, vast amountsof sequence data on the rDNA genes of the smaller subunit of ribosomes in numerous livingorganisms were collected. Comparing these sequences showed that rDNA gene sequences are6highly conserved within living organisms of the same genus and species but differ between
organisms of other genera and species (Woo et al. 2008).

Recently, researchers have conducted extensive research on zooplankton-associatedbacteria, specifically focusing on sequencing the 16S rDNA gene (Shoemaker and Moisander2015, 2017, Eckert et al. 2014, 2021). By using 16S rDNA sequences, researchers have beenable to rename and reclassify bacterial genera and species, as well as identify and categorize
new ones with possible functions (Qi et al. 2009, Sadaiappon et al. 2021). Moreover, using 16S
rDNA sequences has greatly aided in classifying bacteria and their relationships, even for thosethat cannot be cultivated (Staley et al. 2013, Lentini et al. 2014). In this study, I utilized thisadvanced 16S rDNA gene sequencing technique and the Illumina Mi-seq platform, which
proved to be highly effective in identifying different bacterial species.

1.6 The scientific approach: Thesis contents

In this research, I aimed to identify symbiotic bacteria associated with the bodies of thethree most dominant crustacean zooplankton taxa, E. japonicus, Cyclopoida spp., and Daphnia
pulicaria having different feeding habits from Lake Biwa, Japan. Secondly, I established anovel technique to separate the whole intact gut from these three crustacean zooplankton,
which allowed me to identify resident symbiotic bacteria in the gut and body parts without thegut of these three zooplankton taxa for the first time.

The thesis, after a general introduction, is structured as follows:

In Chapter 1: Symbiotic bacterial communities of zooplankters from Lake Biwa,
Japan

7

Chapter 2: Comparative analysis of bacterial compositions in the gut, body otherthan gut, and feces of the three dominant crustacean zooplankton in Lake Biwa, Japan.

Chapter 3: Community structures of symbiotic bacteria associated with the threedominant crustacean zooplankton in different depth habitats of Lake Biwa during thestagnation period.

Chapter 4: Taxonomic and ecological aspects on epibiotic ciliates and flagellates
attached to cyclopoid copepods in Lake Biwa.

8

CHAPTER 1:“Symbiotic bacterial communities of zooplankters from Lake Biwa, Japan.

This chapter is mainly based on the manuscript:“Symbiotic bacterial communities of zooplankters from Lake Biwa, Japan” by Taiabur
Rahman Tuhin, M.F.A. Anny, Xin Liu, Shoko Hosoi-Tanabe, and Syuhei Ban, accepted in
Plankton and Benthos Research.

91. Introduction

Ecto- and endosymbiotic bacteria on zooplankton can form functional assemblages that,through diverse interactions, significantly impact surrounding environments (Green 1974,
Ebert 2005). Interactions between the host and its symbiotic bacteria can be obligatory or
facultative, and mutualistic, commensalistic, or parasitic (Wells and Varel 2011, Martin
and Schwab 2012). Generally, the symbiotic bacteria promote host fitness by contributing
to nutrition (Mangus et al. 2015, Cooper and Cressler 2020), detoxification (Macke et al.
2017a, Manakul et al. 2017), pathogen protection (Verschuere et al. 2000, Cooper andCressler 2020), development (Mangus et al. 2015), and reproduction and growth(Peerakietkhajorn et al. 2015, 2016, Callens et al. 2018). However, studies on interactions
between zooplankton and their bacterial symbionts are few, especially in natural habitats,because the two are usually considered to represent different functional units with indirectconnections in nutrient cycling and food webs in aquatic ecosystems (Azam and Malfatti2007).
Zooplankton ecologists generally stress interactions between zooplankton and theirprey or abiotic factors of surrounding environments that affect life history traits (Boonmak
et al. 2018, Liu et al. 2014, Gao et al. 2022) and overlook interactions with symbiotic
bacteria. Study of interactions in host–symbiont systems will improve understanding ofelement flow, ecosystem dynamics, food web structure, and unknown trophic links between
them in aquatic ecosystems (Eckert and Pernthaler 2014, Corno et al. 2013, Thébault andFontaine 2010, Mougi and Kondoh 2012).
Although previous studies have reported microbial symbionts associated with thebodies of crustacean zooplankton from freshwater lakes (Grossart et al. 2009, Wang et al.
2021, Eckert et al. 2021), information on bacterial compositions in the feces released fromthe zooplankton is scarce. This is also important to understand carbon flux throughout the10water column because fecal material is an essential component of vertical flux throughout
the water column (Poulsen and Kiørboe 2006). Previous studies suggested that zooplankton
feces may contain bacteria that either originate from their digestive system (Gowing andSilver 1983, Tang 2005) or are acquired from the surrounding environment through theconsumption of foods (Hansen and Bech 1996). Callens et al. (2020) found that both thehost genotype and environmental microbes influenced the gut bacterial composition ofDaphnia magna Straus, 1820. Similarly, Grossart et al. (2009) observed that the bacterial
composition of a freshwater cyclopoid varies with the environmental bacterial assemblages
and differs from that of its cladoceran counterpart. There are several other explanations for
how these symbiotic bacterial communities could be constructed; bacterial compositions
associated with zooplankton body rely on the feeding habits and behavior of their hosts(Datta et al. 2018, Chae et al. 2021, Velasquez et al. 2022), host specificity of microbial
symbionts (Wang et al. 2021), or ambient environmental factors (Huq et al. 1984, Eckert
et al. 2021, Chae et al. 2021).
It has been shown that major symbiotic bacterial classes in cultured daphnids, Daphnia
pulicaria Forbes 1893, Daphnia pulex Leydig, 1860, and D. magna, include
Betaproteobacteria and Gammaproteobacteria, and to a lesser degree Bacteroidetes andActinobacteria (Qi et al. 2009). Callens et al. (2016) reported Burkholderiales,
Aeromonadales, Cytophagales, and Flavobacteriales to be the most abundant order incultured D. magna and Aeromonas sp. to be abundant in its gut. Species of Limnohabitans,ubiquitous in freshwater lakes, were dominant bacteria in the intestine of laboratory-reared
D. magna (Freese and Schink 2011). Although this intensive knowledge of key bacterial
components associated with their host bodies has been almost reported from cultured
daphnids, the information from natural habitats is still limited for both copepods andcladocerans (Samad et al. 2020, Wang et al. 2021, Eckert et al. 2021).

11

Lake Biwa is the largest lake in Japan, and zooplankton and microbial communities in
the lake have been well-studied separately over various time scales (Kawabata 1989, Liuet al. 2014, Okazaki and Nakano 2016, Okazaki et al. 2019). However, interactions between
zooplankton and their bacterial symbionts have not been studied yet. In this study, we
reported the bacterial communities associated with bodies and feces of three different
zooplankton taxa with different feeding habits in Lake Biwa using a metagenomic analysisof 16S rDNA. We can enhance the information on the relationship between microbial
symbionts and their zooplankton hosts with different feeding habits in situ.

2. Materials and Methods
2.1 Sample collection and preparation
Zooplankton were collected by a NORPAC net (mesh size, 200 µm; mouth diameter,
45 cm) hauled vertically from 20 m to the surface at a pelagic site in the north basin of Lake
Biwa, Japan (351832.6ʺN, 136838.9ʺE, 70 m deep) on 25 September 2020, which enable tocompare the bacterial community among multiple taxa because the biomass of dominantcrustacean zooplankton increase from July to September above 20 m in the north basin of Lake
Biwa (Liu et al. 2020). Live zooplankton were transferred in an insulated container to thelaboratory within 1 h.
Three dominant zooplankton taxa were selected: Daphnia pulicaria Forbes, 1893, asuspension feeder that non-selectively ingests suspended materials, including phytoplankton(Ebert 2005, Rothhaupt 1997); Eodiaptomus japonicus Kiefer, 1932, a suspension feeder that
selectively ingests phytoplankton and microzooplankton (Liu et al. 2021); and Cyclopoida spp.,
especially late copepodites and adults, which are raptorial feeders that ingest smaller
zooplankton such as larval E. japonicus (Kawabata 1991). Two species in the cyclopoid
copepods, i.e., Mesocyclops dissimilis Defaye and Kawabata, 1993 and Cyclops vicinus Uljanin,

121875, occurred throughout the study period. We did not discriminate the species because of thesimilar morphology and combined the two species as one taxon for the following analysis.
Both species are at the same trophic level, i.e., raptorial feeders.
The number of animals, i.e., 100 and 120 adults of each taxon, were sorted from live
samples under a dissecting microscope (Olympus, SZX12, Japan), washed twice with aged tap
water filtered through a glass-fiber filter (Whatman, GF/F) (ATW), autoclaved at 120 °C for
20 min, and incubated into 1-L glass jars filled with ATW. Animals were incubated in a growthchamber (Sanyo, MLR-350, Japan) at ambient temperature (20 °C) for 24 h to evacuate gut
contents, then removed from the jar and washed twice with Milli-Q water (better for DNA
extraction than ATW to avoid contamination). Feces in jars were filtered through and collected
upon a 0.2-µm pore membrane filter (Advantec, K020A047A, Japan) after extraneousmaterials such as broken body parts, carcasses, or newly hatched individuals had been removed
from the sample (beneath a microscope).
Separate samples of 15–25 specimens (without eggs or embryos for females) from eachtaxon (15 individuals of D. pulicaria, 20 individuals of E. japonicus, and 25 individuals of
Cyclopoida spp.) were transferred into sterile 1.5-mL micro-tubes (Eppendorf, Germany) filledwith sodium phosphate (978 µL) and MT buffer (122 µL) in a Fast DNA® Spin Kit for Soil
(MP Biomedicals, LLC, USA). In preliminary experiments, we confirmed that 12–15
individuals of D. pulicaria, 18–20 individuals of E. japonicus, and 22–25 individuals of
Cyclopoida spp. were required for extracting a sufficient amount of DNA for metabarcoding
analysis. The filters with feces were transferred into a 5-mL tube (Labcon, U.S.A.) andsimilarly treated. All samples were frozen at −80 °C until the following analysis.
2.2 Metabarcoding analysis using Illumina MiSeq
Samples were thawed and homogenized using a sterile disposable plastic pellet pestle.
DNA was directly extracted from each sample using a Fast DNA ® Spin Kit for Soil.

13

Metabarcoding targeted the V4 region of the 16S rRNA gene with primers 515F (5ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGTGCCAGCMGCCGCGGT
AA-3)

and

806R

(5-

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGGACTACHVGGGTWTCTAAT-3) (Glenn et al. 2019). All PCR reactions were performed for each sample in a totalof 10 L reaction mixture, mixes with Ex TaqHS mix (TaKaRa Bio Inc., Japan), 10 M ofeach forward and reverse primer, and about 5 ng of extracted DNA. Initial denaturation
proceeded at 94 C for 2 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing
at 50 C for 30 s, elongation at 72 C for 30 s, followed by a final extension at 72 C for 5 min.
Before the second PCR was performed, the first PCR product was purified by Beckman Coulter
AMPure XP (Beckman Coulter, Inc., USA). To attach the index sequence to the products ofthe first PCR, products of the second PCR were processed with the primer set F:
AATGATACGGCGACCACCGAGATCTACAC-Index-2(8bp)ACACTCTTTCCCTACACGACGC

and

R:

CAAGCAGAAGACGGCATACGAGAT-

Index1(8bp)-GTGACTGGAGTTCAGACGTGTG (Tanaka et al. 2020). Initial denaturation
was at 94 C for 2 min, followed by 10 cycles of denaturation at 94 C for 30 s, annealing at
60 C for 30 s, elongation at 72 C for 30 s, and a final extension at 72 C for 5 min. After
purification using the Beckman Coulter AMPure XP, library quality was assessed using an
Agilent fragment analyzer system and dsDNA 915 reagent kit (Advanced Analytical
Technologies). Libraries were sequenced on an Illumina MiSeq platform, where 2  300 bp
paired-end reads were generated. A total of 318,657 raw reads were obtained.
2.3 Data analysis
Raw data collected from the Illumina MiSeq sequencing platform were considered raw
tags. Basic tags were prepared after removing the barcode and PCR primer sequences using
the fastq_barcode_splitter from the Fastx Toolkit software (ver. 0.0.14). After removing the14primer sequence (50 bp at the 3 end), chimera sequence, and noise sequence using the dada2
plugin of Qiime2 (ver. 2020.8), effective tags with 97% similarity were classified into one
operational taxonomic unit (OTU). OTUs containing “unidentified” taxonomic annotations
and singleton (only a single sequence across the entire dataset) were removed from data
analysis. All OTUs with best Blast matches to non-bacterial organisms were also discarded
from the analysis. OTU sequences belonging to bacteria were deposited to the DNA data bankof Japan (DDBJ) under the accession numbers DRR493548–493553.
Before analyzing OTUs, a rarefied OTU table was generated using the QIIME
rarefaction analysis script with the rarefied depth in the minimum sequence number. The
Shannon diversity index (H) was calculated for the bacterial community of each sample. We
discarded nonbacterial OTUs such as chloroplasts (Eckert et al. 2021, Wang et al. 2021) andarchaea (Wäge et al. 2019) from all samples because we used bacterial 16S rRNA gene primers.
DNA sequences belonging to cyanobacteria had a closest match as a “partial chloroplast
containing genome” of the eukaryotic euglenoid alga Colacium Ehrenberg, 1834 whencompared against the NCBInt database using BLAST search. We, therefore, discarded the
cyanobacterial OTUs as well.
We calculated H to examine the diversity of OTUs in each sample and performed a
non-metric multidimensional scaling (NMDS) analysis to visualize dissimilarities in bacterial
composition among samples using all OTUs. Heatmap analysis was performed using the mostabundant bacterial families ( 100 reads for an OTU) in all samples. Analyses were performed
using the R software ver. 4.0.3 (2020) ‘vegan’ package.
3. Results
A total of 293,018 sequences comprising 669 bacterial OTUs were generated, including
three zooplankton bodies, in which 54 (D. pulicaria), 88 (E. japonicus), and 84 (Cyclopoidaspp.) OTUs were identified, and in their feces, 132, 156, and 155 OTUs, respectively. Among15

the identified OTUs, 7 (D. pulicaria), 48 (E. japonicus), and 51 (Cyclopoida spp.) OTUs wereunique in the host bodies, and 13, 36, and 31 OTUs were in their feces, respectively (Table 11). H was lower in bodies (2.15–2.38) than in feces (2.44–2.83) and was highest for E.
japonicus.
Table 1-1. Total numbers of sequences and identified OTUs, and Shannon’s H index ofbacteria associated with bodies and feces of Daphnia pulicaria, Eodiaptomus japonicus, andCyclopoida spp. from Lake Biwa.
Taxa

Body orfeces

Total no. ofsequences

No. of OTUs

No. of unique
OTUs

Shannon’s H

D. pulicaria

Body

41529

54

7

2.157

E. japonicus

Body

46603

88

48

2.379

Cyclopoida spp.

Body

36561

84

51

2.153

D. pulicaria

Feces

56441

132

13

2.442

E. japonicus

Feces

50781

156

36

2.827

Cyclopoida spp.

Feces

61103

155

31

2.645

Bacteria were attributed to 15 identified phyla and 6 unknown phyla (termed asunidentified), with Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes predominant
in host bodies and feces (Fig. 1-1). Proteobacteria accounted for > 80% of all bacterial
sequences for D. pulicaria and E. japonicus bodies, and Bacteroidetes ~9%. In Cyclopoidaspp., Proteobacteria accounted for 55% and Bacteroidetes 35% of all bacterial sequences.
Proteobacteria (52%–66%) were also abundant in the feces of all zooplankter taxa, followedby Actinobacteria (11.6%–34%), which was also more in feces. Proteobacteria andBacteroidetes were less abundant in feces.

16

Fig. 1-1 Bacterial community (all OTUs) phyla associated with bodies and feces of Daphnia
pulicaria (Dp), Eodiaptomus japonicus (Ej), and Cyclopoida spp. (Cy), from Lake Biwa.
At the order level, dominant bacterial OTUs in host bodies (based on numbers of sequences)were Aeromonadales (37%) and Burkholderiales (25%), while in feces, these wereActinomycetales (20%) and Rickettsiales (41%) (Fig. 1-2). Rhodobacterales was dominant
only in cyclopoid bodies (4%). For D. pulicaria and E. japonicus bodies, Flavobacteriales(9.3% and 3.4%, respectively) and Pseudomonadales (2% and 7.5%, respectively), wererelatively abundant, but not so (<0.2%) in Cyclopoida spp. Both Holophagales andMethylophilales occurred in bodies of E. japonicus but not D. pulicaria or Cyclopoida spp.
Saprospirales occurred in bodies of E. japonicus and Cyclopoida spp., but not D. pulicaria.

17Fig. 1-2 Bacterial community ( 100 OTU reads) orders associated with bodies and feces ofDaphnia pulicaria (Dp), Eodiaptomus japonicus (Ej), and Cyclopoida spp. (Cy), from Lake
Biwa.
The orders Spirobacillales, Legionellales, Chlamydiales, and Cerasicoccales occurred
only in bodies of D. pulicaria (Fig. 1-3); Procabacteriales, Deinococcales, Gemmatales, andHolophagales occurred only in bodies of E. japonicus (Fig. 1-4); and Rhizobiales,
Caulobacterales, Rhodospirillales, Bdellovibrionales, Xanthomonadales, Pasteurellales,
Solirubrobacterales, Lactobacillales, Fimbriimonadales, Armatimonadales, Fusobacteriales,
and Gemmatimonadales occurred only in cyclopoid bodies (Fig. 1-5).

18

Bacteroidetes(3918)

Flavobacteriia(3873)

Flavobacteriales (3873)

Sphingobacteriia(45)

Sphingobacteriales (45)Alphaproteobacteria(62)

Rickettsiales (62)Burkholderiales (7878)

Proteobacteria(36657)

Betaproteobacteria(16556)

Unclassified (8655)
Neisseriales (23)Deltaproteobacteria(40)

Spirobacillales (40)Aeromonadales (18486)

Gammaproteobacteria(19999)Bacteria(41529)Pseudomonadales (828)Enterobacteriales (351)
Alteromonadales (289)
Legionellales (32)Firmicutes(427)

Bacilli(427)

Bacillales (427)

Chlamydiae(49)

Chlamydiia(49)

Chlamydiales (49)Actinobacteria(364)

Chloroflexi(11)

Verrucomicrobia(36)

Actinobacteria(257)

Actinomycetales (257)

Acidimicrobiia(107)

Acidimicrobiales (107)

Chloroflexi(11)

Roseiflexales (11)

Opitutae(30)

Cerasicoccales (30)

Methylacidiphilae(6)

Methylacidiphilales (6)Unidentified (67)Fig. 1-3 Taxonomic tree of the bacteria associated with Daphnia pulicaria, collected from apelagic site in the north basin of Lake Biwa on 25 September 2020 from phylum to order leveltaxonomic assignment using all OTUs. Figures in parentheses represent the cumulative numberof OTUs assigned.

19

Bacteroidetes(4376)

Saprospirae (2721)

Saprospirales (2721)

Cytophagia (67)

Cytophagales (67)

Bacteroidia (19)Bacteroidales (19)

Flavobacteria (1569)

Flavobacteriales (1569)

Alphaproteobacteria(707)

Proteobacteria(38389)Betaproteobacteria(18175)
Deltaproteobacteria(35)

Sphingomonadales (250)
Rickettsiales (388)Unclassified (69)
Neisseriales (1269)
Burkholderiales (16864)
Procabacteriales (27)Methylophilales (15)Myxococcales (10)Unclassified (25)
Aeromonadales (15656)

Gammaproteobacteria(19472)Bacteria(46603)
Actinobacteria(414)

Verrucomicrobia(34)
Thermi (70)

Pseudomonadales (3504)
Enterobacteriales (44)Alteromonadales (268)Actinobacteria(372)

Actinomycetales (372)

Acidimicrobiia(42)

Acidimicrobiales (42)

Methylacidiphilae(27)

Methylacidiphilales (27)

Unclassified (7)Deinococci (70)

Deinococcales (70)

Chlorobi (9)Chloroflexi (21)

Chloroflexi (10)Anaerolineae (11)Unclassified (11)Planctomycetes(61)
Acidobacteria(29)
Firmicutes(395)

Planctomycetia (15)Gemmatales (15)

Chloracidobacteria (8)Holophagales (21)

Holophagae (21)Clostridia (34)

Clostridiales (34)

Bacilli (361)

Bacillales (361)

Unidentified (2805)

Fig. 1-4 Taxonomic tree of the bacteria associated with Eodiaptomus japonicus, collected froma pelagic site in the north basin of Lake Biwa on 25 September 2020 from phylum to orderlevel taxonomic assignment using all OTUs. Figures in parentheses represent the cumulative
number of OTUs assigned.

20

Saprospirae (266)

Saprospirales (266)

Cytophagia (25)

Cytophagales (25)Bacteroidia (10)Bacteroidales (10)Bacteroidetes(12828)

Flavobacteriia (68)

Flavobacteriales (68)

Sphingobacteriia (2994)

Sphingobacteriales (2994)

Unclassified (9465)

Caulobacterales (2)Sphingomonadales (42)Rhodobacterales (1374)
Rickettsiales (25)Rhizobiales (10)Rhodospirillales (16)
Unclassified (4)Alphaproteobacteria(1473)

Proteobacteria(20188)

Betaproteobacteria(6730)
Deltaproteobacteria(23)
Gammaproteobacteria(11956)
Unclassified (6)Bacteria(36561)
Actinobacteria(231)
Acidobacteria

Actinobacteria(209)
Thermoleophilia(10)
Acidimicrobiia(12)

Burkholderiales (6730)
Myxococcales (8)Bdellovibrionales (15)Aeromonadales (11835)
Pseudomonadales (90)Enterobacteriales (17)Xanthomonadales (5)Pasteurellales (9)Actinomycetales (209)
Solirubrobacterales (10)Acidimicrobiales (12)(17)

Verrucomicrobia(2)
Gemmatimonadetes(8)
Fusobacteria (14)Armatimonadetes(22)
Planctomycetes(26)
Firmicutes(125)

Methylacidiphilae(2)Methylacidiphilales (2)Gemmatimonadetes(8)

Gemmatimonadales (8)Fusobacteriia (14)Fusobacteriales (14)Fimbriimonadia (5)Fimbriimonadales (5)Armatimonadia (17)Armatimonadales (17)Unclassified (7)Planctomycetia (19)Plactomycetetales (19)

Clostridia (72)

Clostridiales (72)

Bacilli (53)Lactobacillales (6)Bacillales (47)

Unidentified (3100)

Fig. 1-5 Taxonomic tree of the bacteria associated with Cyclopoida spp., collected from apelagic site in the north basin of Lake Biwa on 25 September 2020 from phylum to order leveltaxonomic assignment using all OTUs. Figures in parentheses represent the cumulative numberof OTUs assigned.

21

Excluding the dominant Actinomycetales and Rickettsiales, zooplankton feces of all
three taxa contained Methylophilales, Acidimicrobiales, Burkholderiales, Flavobacteriales andPseudomonadales, and the bacterial composition differed from that of their bodies. Proportionsof Methylophilales and Acidimicrobiales OTUs in feces of D. pulicaria (5.6% and 4%,
respectively) and E. japonicus (6% and 7%, respectively) were slightly higher, while those of
Pseudomonadales (8.3%), Aeromonadales (5.8%), and Flavobacteriales (5%) were relatively
more abundant in feces of Cyclopoida spp. NMDS using all OTU sequences revealed thatbacteria in feces differed from those in host bodies and that variation in the body was greater

0.6(Fig. 1-6).

0.4

stress < 0.001

++

+

+

0.2

Ej++

+

Dp−F

+

+

+
+
+
+
+
+
+
+
+
+
+
+++
++ + ++ ++ ++++++ + ++++ +++++++++++++ + ++
+
+
++
+
+ +
+
+
+
+ +
++
+ +++
+
+
++

0.0

+ ++

+

+
+

+ +
+ + + +++
+ + + ++++

+

+
+

−0.2

NMDS2

+

+

Dp
+

+

+
+

Ej−F

Cy−F

Cy
++ +

+

+

−0.6

−0.4

+ ++ ++

+

−1.0

−0.5

0.0

0.5

NMDS1

Fig. 1-6 Non-metric multidimensional scaling (NMDS) analysis (all OTUs) of dissimilarities
among zooplankter bodies and feces. Eodiaptomus japonicus (Ej), Daphnia pulicaria (Dp),and Cyclopoida spp. (Cy); Ej-F, Dp-F, and Cy-F denote feces of each taxon. Encircled areas
indicate confidence 95% intervals. Open circles represent zooplankter taxa; red crosses
represent bacterial OTUs.

22

Heatmap analysis reveals a large difference in dominant bacterial families between
zooplankter bodies and feces (Fig. 1-7). Bacterial families associated with D. pulicaria andCyclopoida spp. bodies were most similar, whereas those of feces of D. pulicaria and E.
japonicus were more similar.

Fig. 1-7 Heatmap of abundant bacterial families ( 100 OTU reads) associated withzooplankter bodies and feces. Eodiaptomus japonicus (Ej), Daphnia pulicaria (Dp), andCyclopoida spp. (Cy); Ej-F, Dp-F, and Cy-F denote feces of each taxon. Individual lines
correspond to bacterial families; each column corresponds to a different sample. The upper
dendrogram represents similarities in bacterial composition among zooplankter bodies andfeces; left dendrogram represents similarities in patterns of occurrence of a bacterial family ina zooplankter body and feces. ACK-M1 and C111 are candidate Actinobacteria families.

For each zooplankton taxon, Aeromonadaceae was the dominant family in bodies, thenfor D. pulicaria and Cyclopoida spp., Comamonadaceae (17% of all bacterial OTUs), and forE. japonicus, Oxalobacteraceae (28%). In feces, the proportional contributions ofComamonadaceae and Oxalobacteraceae are lower thanin zooplankton bodies,

Pelagibacteraceae (40% of all sequences) was dominant, followed by ACK-M1 belonging to23

Actinobacteria, Methylophilaceae, and C111 for D. pulicaria (5.6%) and E. japonicus (6%),
and Moraxellaceae for Cyclopoida spp. (8%, below the percentage in bodies). Proportions of
Chitinophagaceae, Cryomorphaceae, and Rhabdochlamydiaceae were higher in feces than inbodies of each zooplankton. Neither the Legionellaceae nor Rhabdochlamydiaceae occurred ineither copepod body; Peptostreptococcaceae, Intrasporangiaceae, and Sphingomonadaceae
occurred only in the cyclopoid body; and Trueperaceae occurred only in the E. japonicus body.
Other notable families associated with the bodies of D. pulicaria included the
Flavobacteriaceae (8.6%) and Moraxellaceae (2%), E. japonicus the Moraxellaceae (7%),
Weeksellaceae (3%), and Neisseriaceae (3%), and Cyclopoida spp., the Rhodobacteraceae(4%) (Table 1-2).
Bacterial genera Aeromonas, Ralstonia, and Acinetobacter were commonly detected in
the bodies of each zooplankton taxon. Limnohabitans, Rhodoferax, Perlucidibaka, andBrevibacillus, occurred only in the body of D. pulicaria. Polaromonas, Hydrogenophaga,
Comamonas, Corynebacterium, Truepera, Spirosoma, Staphylococcus, and Sphingomonas
occurred only in the body of E. japonicus. Delftia, Rhodobacter, Segetibacter, Rodocytophaga,and Pontibacter occurred only in the cyclopoid body (Table 1-2). Flavisolibacter andCandidatus Rhabdochlamydia occurred only in zooplankton feces, while Flavobacterium,
Polynucleobacter, Fluviicola, and Chryseobacterium occurred in both bodies and feces (Table
1-3). Flavobacterium, which can digest polysaccharides (Gavriilidou et al. 2020), wasrelatively abundant in the body of D. pulicaria, less abundant in that of E. japonicus, andrelatively scarce in the body of cyclopoids.

24

25

26

4. Discussion
The heatmap analysis revealed that the bacterial communities of zooplankton bodies
mostly differ from those of their feces. Bacteria from zooplankton bodies can be directly
attached to the body surface (Carman and Dobbs 1997, Qi et al. 2009, Skovgaard et al. 2015)
and/or colonize the gut (Tang et al. 2010). Some free-living environmental bacteria may also
attach to potential zooplankter foods (Simon et al. 1992) and enter the gut through ingestion.
Bacteria attached to the food ingested may be both intestinal residents, persisting in the gut(Gowing and Silver 1983), and transients, passing through it (Olafsen 1984, King et al. 1991,
Nagasawa 1992). Transient bacteria might only remain in the gut for a short period of time
(Harris 1993, Tang et al. 2010), and because some ingested material may remain undigested ategestion (Parsons et al. 1977), some may remain intact following egestion (Hansen and Bech
1996).
In the present study, the most abundant bacterial family in the feces of all threezooplankton hosts was Pelagibacteraceae, but it was quite low in abundance in the bodies.
Actinobacterial ACK-M1 and C111 were also abundant in the feces but quite scarce in thebodies of the three hosts. In our other campaigns conducted in 2020 and 2021,
Pelagibacteraceae, ACK-M1, and C111 were dominant in the epilimnetic waters of Lake Biwa
in September (authors’ unpublished data). This implies that the dominant bacterial families in
the feces were similar to those in the water column. Okazaki and Nakano (2016) showed that
Actinomycetales was predominant order in the epilimnion of Lake Biwa from April to
December 2010, when strict stratification caused major nutrient concentrations to be reduced(Kim et al. 2006) and dissolved organic carbon (DOC) to be generally abundant (Maki et al.
2010). Pelagibacteraceae (Rickettsiales) can grow in nutrient-limited conditions (Ortmann andSantos 2016) using DOC (Giovannoni et al. 2005). This implies that the bacteria found in the27

feces may be mostly transient and therefore differ from the residential bacteria associated withthe host bodies.
Since our three zooplankton taxa were collected from the same depth and had different
bacterial compositions in the feces, some fecal bacteria may be related to host feeding habits.
The fecal bacteria of D. pulicaria and E. japonicus are similar and differ slightly from those of
Cyclopoida spp. Both D. pulicaria and E. japonicus are suspension feeders that ingest
particulate organic matter (POM), including bacterial aggregates, while Cyclopoida spp.,
especially adults, are fundamentally carnivorous and mainly ingest other small zooplankton.
Adult M. dissimilis, a typical dominant cyclopoid in Lake Biwa, prey on the late nauplii andearly copepodites of E. japonicus (Kawabata 1991).
Of the three zooplankton taxa, H indices revealed the fecal bacterial community of E.
japonicus to be the most diverse. Potential foods for this species are more variable than eitherof the other zooplankton taxon, including phytoplankton and microzooplankton (Yoshida et al.
2001, Wang et al. 2021). This omnivorous habit may lead to the bacterial community of its
feces being more diverse than that of the herbivorous D. pulicaria and carnivorous Cyclopoida
spp.
Flavobacterium, capable of degrading polysaccharides (Gavriilidou et al. 2020, Inoueet al. 2016), was more abundant in the body of herbivorous D. pulicaria than in the body ofomnivorous E. japonicus, and it was only present at low abundances in carnivorous adult
cyclopoids. Phytoplankton cell walls are mainly composed of polysaccharides (Qi et al. 2009)
such as cellulose, hemicellulose, and pectins, which are preferred substrates for
Flavobacterium bacteria. The association between Flavobacterium spp. and the bodies of D.
pulicaria and E. japonicus might indicate that this bacterium is an intestinal symbiont that
contributes to phytoplankton digestion. Furthermore, species of Aeromonadaceae show highchitinase activity (Tran et al. 2018). A plausible explanation for the prevalence of28

Aeromonadaceae in the bodies of the three hosts could be the plentiful suspended chitin-basedorganic particulate aggregates within their ambient environments, which serve as an available
food source for zooplankton. These particles would be derived from the remains of crustacean
zooplankton, such as carcasses and molts, in freshwater systems (Grossart and Simon 1998,
Grossart et al. 1997). In Lake Biwa, the absence of small phytoplankton during summer toautumn (Yoshida et al. 2001) may imply that herbivorous suspension-feeding hosts, like D.
pulicaria and E. japonicus, consume more of these organic particles, which contain a highamount of chitin (Tang et al. 2010). Contrastingly, the carnivorous cyclopoid primarily feeds
on other zooplankton that have chitin-based exoskeletons.
Bacterial communities associated with host bodies differed among the threezooplankton taxa. Bacterial OTUs referable to Trueperaceae occurred only with E. japonicus;
those

of

Peptostreptococcaceae,

Intrasporangiaceae,

Rhodobacteraceae,andSphingomonadaceae occurred only with Cyclopoida spp.; and Legionellaceae andRhabdochlamydiaceae occurred only with D. pulicaria. Additionally, Oxalobacteraceae,
Moraxellaceae, Neisseriaceae, and Weeksellaceae were abundant with E. japonicus but scarcewith Cyclopoida spp.; and the family Rhodobacteraceae was abundant in Cyclopoida spp., but
was not detected in E. japonicus. Some bacteria associated with aquatic organisms are hostspecific and play protective roles (Wahl et al. 2012), protecting a host from biological andphysical stresses (Erken et al. 2015). Attached benign bacteria may also contribute to the
probiotic effect of the host Artemia body against pathogenic bacteria (Verschuere et al. 2000).
Therefore, the specific bacterial components of host zooplankton may play important roles inmaintaining host health.
Heatmap analysis reveals bacterial families associated with host cyclopoid and D.
pulicaria bodies to be similar but differ from those of E. japonicus. During the stagnation
period in Lake Biwa, E. japonicus usually occurs shallower than 20 m depth within the29epilimnion (Kawabata 1987). Because Mesocyclops thermocyclopoides Harada, 1931 (close
relative of M. dissimilis) and D. pulicaria can occur in deeper layers during the day and migrate
into shallow waters at night (Liu and Hu 2001, Stich and Lampert 1981), their environments
may be more similar to each other than to that of E. japonicus. Habitat and behavioral
differences may affect bacterial assemblages in the bodies of our three zooplankton taxa.
Migrating D. magna can transport bacteria across depth layers and release them during
migration (Grossart et al. 2010). In Lake Biwa’s summer months, E. japonicus is the mostabundant species and does not exhibit diel vertical migration (Kawabata 1987, Yoshida et al.
2001). A higher prevalence of Oxalobacteraceae associated with this host during September
could be attributed to the mesophilic characteristics of this bacteria, thriving at temperatures of
20 °C or higher (Baldani et al. 2014).
The dominant families of symbiotic bacteria associated with D. pulicaria bodies in thepresent study were very similar to those found in the bodies of Daphnia obtusa Kurz, 1874 andDaphnia galeata Sars, 1864 from freshwater lake/pond in Italy (Eckert et al. 2021), and thosefound from the whole-bodies of the cultured daphnids, D. magna, D. pulicaria and Daphnia
pulex (Leydig, 1860) (Freeze and Schink 2011, Qi et al. 2009, Peerakietkhajorn et al. 2015,
Callens et al. 2016) (see Appendix 1)). This implies that this symbiotic bacterial family
associated with daphnid species may follow a pattern termed phylosymbiosis, that is, a groupof taxonomically similar zooplankton species shows similarity in their microbial symbionts(Lim and Bordenstein 2020). Symbiotic Aeromonas sp., which was dominant in the present
study and a lot of previous studies (see Appendix 1), has been shown to support the growth ofits daphnid host in laboratory experiments (Mangus et al. 2015).
In the previous studies, the dominant bacterial families reported in the bodies offreshwater calanoids, Sinocalanus doerrii (Brehm, 1909) and Sinodiaptomus sarsi (Rylov,
1923), were Pseudomonadaceae, Bacillaceae, and Moraxellaceae (Wang et al. 2021), while30

those in Eudiaptomus padanus (Burckhardt, 1900) and Calanoida spp. were Comamonadaceae,
Pseudomonadaceae, Flavobacteriaceae, Neisseriaceae, and Oxalobacteraceae (Eckert et al.
2021). These families were different from those found in E. japonicus in the present study (seeAppendix 1). The prevalent bacterial families associated with the body of Cyclopoida spp. in
the present study also differed from those found in other cyclopoid copepods, e.g.,
Thermocyclops oithonoides (Sars, 1863) and Mesocyclops leuckarti (Claus, 1857) from
freshwater lakes in Europe (see Appendix 1). These facts suggest that copepod-associatedbacteria may be host-specific and/or depend on the ambient environmental conditions,
including available food types and bacterial communities in the surrounding waters (Grossart
et al. 2009, Velasquez et al. 2022).
Bacterial communities associated with the bodies of E. japonicus and Cyclopoida spp.
were more diverse than in D. pulicaria. The cephalothorax of copepods has many sensory
organs and excretory pores (Koomen and von Vaupel Klein 1998), whereas daphniids do not,and they also have reduced carapace segmentation (e.g., Sars 1993). This implies that thesurface of copepods is structurally more complex than the outer shield of daphniids. Bacteria
on zooplankter body surfaces may attach to and/or colonize the fine pits and holes of sensory
organs.
In this study, since the sampling was conducted during the period when the biomass ofthe zooplankton hosts increased in Lake Biwa, a comparative analysis of bacterial communities
among the three hosts could be done. Moreover, analyzing the bacterial compositions
associated with not only whole bodies of the hosts but also feces, which has not been previously
done in zooplankton (see Appendix 1), has allowed us to discuss the relationship between thesymbiotic bacterial communities and feeding habits of the hosts. On the other hand, seasonal
succession and generality of the bacterial community is still an open question. An additional
analysis, e.g., in the period of lower zooplankton biomass (e.g., winter), is necessary.

31CHAPTER 2:“Comparative analysis of bacterial compositions in the gut, body other thangut, and feces of the three dominant crustacean zooplankton in Lake Biwa,
Japan.”

32

1. Introduction
Symbiotic bacteria can be associated with their hosts’ external surfaces and internalorgans through various adherence and penetration mechanisms (De Corte et al. 2014, 2018).
Zooplankton may acquire bacterial symbionts from their mother during embryonicdevelopment (Shoemaker and Moisander 2017) and from their surrounding environments(Tang et al. 2010. Wahl et al. 2012). The digestive tracts, especially the intestine ofzooplankton, harbors a diverse symbiotic microbiota (Nagasawa and Nemoto 1988, Harris
1993, Peter and Sommaruga 2008) that may digest the food materials ingested by theirhosts and convert them into essential metabolites accessible for their hosts (Flint et al. 2008,
Wu et al. 2012). Resident bacteria in the intestine may also take part in the degradation of
egested fecal materials and play a profound role in nutrient cycling in an aquatic ecosystem(Gowing and Silver 1983, Tang et al. 2011, Yeh et al. 2020). In addition, the intestinal
environment may contain an acidic and hypoxic to anoxic condition, which may influence
the colonization of anaerobic bacteria (Tang et al. 2011, Shoemaker and Moisander 2015,2017).

The microbial composition in the digestive tract of zooplankton can be changed by thediet of the hosts (Tang 2005, Tang et al. 2009, Sadaiappon et al. 2021), species-specificfeeding behaviors (Chae et al. 2021, Velasquez et al. 2022) and surrounding environmental
factors including bacterial community structures (Grossart et al., 2009, Callens et al. 2020).
The remaining parts of the zooplankton body, excluding the digestive tract, also provide
suitable environments for colonizing diverse bacterial microflora (Tang et al. 2010, Wanget al. 2021). Bacteria may directly attach to the surface of the chitinous exoskeleton(Hansen and Bech 1996, Carman and Dobbs 1997, Qi et al. 2009), filtering apparatus of afilter feeder (Qi et al. 2009), oral area and the feeding appendages (Huq et al. 1983,
Nagasawa 1988, Skovgaard et al. 2015) and anal region (Bickel and Tang 2014), which33may attract bacteria as microhabitats enriched with organic matters. Bacteria attached tothe exoskeleton of zooplankton may protect their hosts from pollutants in the surrounding
environment and smoothen mobility (Manakul et al. 2017, Nagasawa 1989). However,knowledge of the community composition and functions of the bacterial community in/on
the exterior of the zooplankton body is scarce to date.

In most studies dealing with the bacterial community associated with crustacean
zooplankton, whole body of the hosts without dissecting their body parts were used foranalyzing the microbial symbionts (Grossart et al. 2009, Chae et al. 2021, Eckert et al. 2021,
Valesquez et al. 2022). Therefore, the information on the locations of residential bacteria,
e.g., in the gut and body surface of the hosts, was limited. Previously, symbiotic bacteriaon the external surface of the hosts’ body were detected by culturing them (Harris 1993,
Carman and Dobbs 1997, Huq et al. 1983) or using microscopic analysis (Nagasawa andNemoto 1985, Qi et al. 2009, Skovgaard et al. 2015). However, such methods may notprovide sufficient information about species composition and diversity of the microbiome
attached to the hosts’ exterior. Metagenomic analysis allows us to know sufficient
information on the diversity and phylogeny of the symbiotic microbiota, including
uncultured ones.

It has been shown that zooplankton suffer from reduced fitness and negatively impact
host life-history traits without symbiotic microbiota (Mangus et al. 2015, Akbar et al. 2020).
However, there is limited information on community structure and diversity of hostassociated bacteria in the specific sites of the exterior and interior of the host zooplankton
collected from freshwater lakes. In addition, because of the difficulty of separating a gut
from a small-sized zooplankton body (<1mm of body length), the studies dealing withbacterial communities associated with the body surface and gut separately were limited34

(Tang et al. 2010). There were no studies dealing with host-associated bacterial community
compositions separately in the gut and body parts other than the gut of zooplankton fromboth culture and natural habitats.

In this chapter, I focused on identifying andcharacterizing the bacteria in the gut and body parts excluding the gut (BP) of the threedominant zooplankton with different feeding habits in Lake Biwa, Japan, and comparedwith bacterial compositions in their feces and surrounding environment.

2. Materials and methods
2.1 Sample collection and preparation
Zooplankton were collected with a vertical net haul from 20 m to the surface using a
NORPAC net (mesh size, 200 µm; mouth diameter, 45 cm) at a pelagic site (3518.793ʺN,
1368.854ʺE, 70 m deep) in the north basin of Lake Biwa, Japan on 4 March 2021. A verticalprofile of water temperature was obtained with a conductivity-temperature-depth (CTD)
profiler (AAQ-Rinko, JFE advantec). The lake waters for analyzing ambient bacterial
compositions were also collected from 0, 10, and 20 m with a Van-Dorn bottle (volume, 6 L)
and then combined with each other at the same volume. The collected live zooplankton andlake water were transferred in an insulated container to the laboratory within 1 h.

I selected three dominant zooplankton taxa, i.e., Daphnia pulicaria which is a non-selective
suspension feeder and mainly ingests phytoplankton (Ebert 2005, Rothhaupt 1997),
Eodiaptomus japonicus, which is an omnivorous suspension feeder and ingests both
phytoplankton and microzooplankton (Yoshida et al. 2001), and Cyclopoida spp. which are
raptorial feeders and ingest smaller zooplankton such as larval E. japonicus (Kawabata 1991).
A sufficient number, i.e., 100 –120 adult individuals, of each taxon were sorted from live
samples under a dissecting microscope (Olympus, SZX12, Japan), washed twice with aged tap

35

water filtered through a glass-fiber filter (Whatman, GF/F) (ATW), autoclaved at 120 °C for
20 min, and incubated separately into 1-L glass jars filled with ATW. The animals wereincubated in a growth chamber (Sanyo, MLR-350, Japan) at ambient temperature (8 °C) for 24
h to evacuate gut contents, then removed from the jar and washed twice with Milli-Q water.
Feces in the jars were filtered through and collected upon a 0.2-µm pore membrane filter(Advantec, K020A047A, Japan) after extraneous materials such as broken body parts,
carcasses, or newly hatched individuals had been removed from the jar under a microscope.

For the following DNA extraction experiments, 15 individuals of > 2 mm in D. pulicaria,
30 individuals of adult males and females in E. japonicus, and 35 individuals of adult males
and females in cyclopoids were sorted from the zooplankton evacuated the gut contents. In
each individual, the gut was removed from the body with a sterile needle under a dissecting
microscope (Olympus, SZX9) at a magnification of 40x−50x (Fig. 2-1). Each of the removed
gut and all other body parts excluding the gut in each taxon was separately transferred into asterile 1.5-mL micro-tube (Eppendorf, Germany) filled with sodium phosphate (978 µL) andMT buffer (122 µL) in a Fast DNA® Spin Kit for Soil (MP Biomedicals, LLC, USA) for
following DNA extraction. A batch of the feces collected on a filter in each taxon wastransferred into a 5-mL tube (Labcon, U.S.A.) filled with the same reagents as treating the body.

36

Fig. 2-1 Microscopic images in outer shapes of three zooplankton taxa tested (a, d, g), theirbody parts (b, e, h), and digestive tract (c, f, i) dissected from the body of Daphnia pulicaria(a-c), Eodiaptomus japonicus (d-f) and Cyclopoida spp. (g-i) collected at a pelagic site in thenorth basin of Lake Biwa on 4 March 2021.

37

To analyze bacterial compositions in the ambient lake waters, 1 L of the lake waters
combined from the three depths was firstly sieved with a 20-µm mesh netting. A 300 ml of the
pre-sieved water was filtered with a 0.2-µm pore polycarbonate membrane filter (Advantec,
K020A047A, Japan). The filter was transferred into a 5-mL tube (Labcon, U.S.A.) filled withsodium phosphate (978 µL) and MT buffer (122 µL) in a Fast DNA® Spin Kit for Soil (MPBiomedicals, LLC, USA) for DNA extraction.
I prepared another zooplankton, i.e., laboratory-reared D. magna fed on axenic
Chlamydomonas reinhardtii (IMA C−9). Healthy 1–2 days old animals were randomly selectedfrom the stock culture and reared in a 1 L glass jar filled with autoclaved ATW for 21 days
with excess food of axenic C. reinhardtii (>105 cells mL-1). When the animals grew up adult,
ten animals were sorted, washed twice with ATW to remove loosely attached materials on acarapace surface, and transferred to a new jar filled with ATW to evacuate gut contents for 24hours at 20˚C. Then, 8 individuals were sorted from the jar, washed twice with MilliQ water,and dissected gut from the body in an aseptic condition. The guts and all other body parts
excluding the gut, were separately transferred to a sterile 1.5-mL micro-tube (Eppendorf,
Germany) for DNA extraction with the same method for the field samples. All samples werefrozen at −80 °C until analysis.
2.2 Metabarcoding analysis using Illumina MiSeq
The frozen samples were thawed and homogenized using a sterile disposable plasticpellet pestle. DNA was directly extracted from each sample using a Fast DNA ® Spin Kit forSoil. Metabarcoding targeted the V4 region of the 16S rRNA gene with primers 515F (5ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGTGCCAGCMGCCGCGGT
AA-3)

and

806R

(5-

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGGACTACHVGGGTWTCTAAT-3) (Glenn et al. 2019). All PCR reactions were performed in each sample with a 1038

L mixture of an Ex TaqHS-mix (TaKaRa Bio Inc., Japan), 10 M of each forward and reverse
primer, and about 5 ng of extracted DNA. Initial denaturation proceeded at 94C for 2 min,followed by 30 cycles of denaturation at 94C for 30 s, annealing at 50C for 30 s, elongation
at 72C for 30 s, followed by a final extension at 72C for 5 min. Before the second PCR was
performed, the first PCR product was purified by Beckman Coulter AMPure XP (Beckman
Coulter, Inc., USA). To attach the index sequence to the products of the first PCR, products ofthesecondPCR

wereprocessed

with

the

primerset

F:

AATGATACGGCGACCACCGAGATCTACAC-Index-2(8bp)ACACTCTTTCCCTACACGACGC

and

R:

CAAGCAGAAGACGGCATACGAGAT-

Index1(8bp)-GTGACTGGAGTTCAGACGTGTG (Tanaka et al. 2020). Initial denaturation
was at 94 C for 2 min, followed by 10 cycles of denaturation at 94 C for 30 s, annealing at
60 C for 30 s, elongation at 72 C for 30 s, and a final extension at 72 C for 5 min. After
purification using the Beckman Coulter AMPure XP, library quality was assessed using an
Agilent fragment analyzer system and dsDNA 915 reagent kit (Advanced Analytical
Technologies). Libraries were sequenced on an Illumina MiSeq platform, generating 2300 bp
paired end reads. A total of 501,356 raw reads were obtained.
2.3 Data analysis
Raw data collected from the Illumina MiSeq sequencing platform were considered raw
tags. Basic tags were prepared after removing the barcode and PCR primer sequences using
the fastq_barcode_splitter from the Fastx Toolkit software (ver. 0.0.14). After removing theprimer sequence (50 bp at the 3 end), chimera sequence, and noise sequence using the dada2
plugin of Qiime2 (ver. 2020.8), effective tags with 97% similarity were classified into one
operational taxonomic unit (OTU). OTUs containing “unidentified” taxonomic annotations
and singleton (only a single sequence across the entire dataset) were removed from data39

analysis. All OTUs with the best BLAST matches to non-bacterial organisms were also
discarded from the analysis.
Before analyzing OTUs, a rarefied OTU table was generated using the QIIME
rarefaction analysis script with the rarefied depth in the minimum sequence number. We
discarded OTUs belonging to chloroplast, mitochondria, cyanobacteria, and archaea from all
samples (Chapter 1). The Shannon diversity index (H) was calculated for the bacterial
community of each sample.
We calculated H to examine the diversity of OTUs in each sample and performed a
non-metric multidimensional scaling (NMDS) analysis to visualize dissimilarities in bacterial
composition among zooplankton’s BP, gut, feces, and ambient water samples using all OTUs.
Heatmap analysis was performed using the dominant bacterial families ( 200 reads for OTUs)
in all samples. Analyses were performed using the R software ver. 4.0.3 (2020) ‘vegan’
package.

3. Results
A total of 472,383 sequences comprising 1,125 bacterial OTUs were generated,
including the gut, body parts other than gut (BP), and feces of the three field-caught
zooplankton, in which 117, 119, and 91 OTUs in D. pulicaria, 49, 39, and 120 OTUs in E.
japonicus, and 63, 19, and 173 OTUs in Cyclopoida spp., respectively. The numbers of OTUs
were not so different between the gut and BP in any taxa, while those in feces were more thandouble than those in the gut and BP for two copepod taxa. In laboratory-rearing D. magna, thenumbers of OTUs were relatively much lower than those in wild animals, 28 and 29 from thegut and BP, respectively. From the ambient waters, the highest bacterial OTUs, 278, were
found. Shannon H diversity indices in feces were higher than those in the gut of the two
copepod taxa while lower than those in the gut of D. pulicaria (Table 2-1). The lowest H was40

found in BP (1.09) of laboratory-rearing D. magna, while the highest one was found (4.16) inambient waters.

Table 2-1. Total numbers of sequences and OTUs, and Shannon’s H indices of bacterial
communities associated with body parts other than gut (BP), gut and feces of Daphnia
pulicaria, Eodiaptomus japonicus, and Cyclopoida spp., and Ambient bacteria (AB) collected
at a pelagic site in the north basin of Lake Biwa on 4 March 2021, and laboratory rearing
Daphnia magna.
Hosttaxa/environment
D. magna
D. magna
D. pulicaria
D. pulicaria
D. pulicaria
E. japonicus
E. japonicus
E. japonicus
Cyclopoida spp.
Cyclopoida spp.
Cyclopoida spp.
Ambient water

Sample type
BP
Gut
BP
Gut
Feces
BP
Gut
Feces
BP
Gut
Feces
AB

Total no. ofsequences
45288
47316
39838
51746
49132
31032
45915
43454
5373
22608
47217
43464

No. of OTU's
29
28
119
117
91
39
49
120
19
63
173
278

Shannon's
H
1.09
1.85
2.47
2.96
2.60
2.21
1.41
2.74
1.94
2.09
2.81
4.16

Bacterial OTU sequences were mostly assigned to 14 identified phyla (99.6%), withthe predominance of Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. These fourphyla accounted for 99% of the total sequences from all host gut, BP, and feces, while 0.44 %of the total sequences were assigned to unknown phyla (termed as unidentified) (Fig. 2-2).
Among the total sequences obtained from BP and the gut of four zooplankton taxa,
Proteobacteria accounted for 68% of all bacterial sequences on average, following
Bacteroidetes, Firmicutes, and Actinobacteria. Among the total sequences obtained from thefeces of the field-collected zooplankton, both Proteobacteria and Bacteroidetes accounted for
21% on average, following Firmicutes and Actinobacteria. In the ambient bacterial assemblage,
Actinobacteria were the most dominant at 27%, while quite low percentages of <6% in gut, BP,
and feces of the field-collected zooplankton. Compositions of Firmicutes were higher in the41

feces (31%–64%) than those in the gut (~2%–19%) of the three field-collected zooplankton,
which was not found in the ambient bacterial assemblage.

Fig. 2-2 Phylum compositions of bacterial OTUs associated with body parts without gut (BP),gut, and feces in Daphnia pulicaria (Dp), Eodiaptomus japonicus (Ej), Cyclopoida spp. (Cy)and ambient bacteria (AB) collected at a pelagic site in the north basin of Lake Biwa on 4
March 2021, and laboratory rearing Daphnia magna (Dm).
The most prevalent bacterial family in all the BP, gut, and feces of D. pulicaria were
Comamonadaceae (mostly including genus Limnohabitans) at 57%, 23%, and 25% in BP, gut,
and feces, respectively (Fig. 2-3). It was mostly followed by Flavobacteriaceae (including
Flavobacterium

alone)

in

the

BP,

by

Flavobacteriaceae,

Moraxellaceae,

andPseudomonadaceae in the gut, and by Flavobacteriaceae and Paenibacillaceae in the feces.
Interestingly, the dominant composition of bacteria associated with laboratory-rearing D.
magna was quite similar to those in D. pulicaria.

42Fig. 2-3 Family compositions of bacterial OTUs ( 200 reads) associated with body parts
without gut (BP), gut, and feces in Daphnia pulicaria (Dp), Eodiaptomus japonicus (Ej),
Cyclopoida spp. (Cy) and ambient bacteria (AB) collected at a pelagic site in the north basinof Lake Biwa on 4 March 2021, and laboratory rearing Daphnia magna (Dm).
In the two copepod taxa, the dominant bacterial compositions were quite different
among BP, gut, and feces. Those in the BP of E. japonicus were Weeksellaceae at 28%,
followed by Flavobacteriaceae at 16% and Oxalobacteraceae at 11%, while those in the gut
were Neisseriaceae at 63%, followed by Weeksellaceae at 8%. Those in the feces were
Paenibacillaceae at 37%, followed by Flavobacteriaceae at 23% and ACK-M1 at 3%. Those in
the BP of Cyclopoida spp. were Oxalobacteraceae at 47%, followed by Burkholderiaceae at
13% and Aeromonadaceae at 10%, while those in the gut were Aeromonadaceae at 13%,
followed by Oxalobacteraceae at 10%. Those in the feces were Paenibacillaceae at 46%,
followed by Aeromonadaceae (7%). On the other hand, the abundant family in the ambient
bacteria was quite different from zooplankton-associated bacterial compositions, being ACKM1 at 17% and C111 at 9% (Fig. 2-3, Table 2-2).
43

44

Dominant bacterial genera identified were Limnohabitans, Flavobacterium,
Brevibacillus, Acinetobacter, and Pseudomonas in both D. pulicaria and D. magna (Table 23). Whereas, those in E. japonicus were Andreprevotia, Chryseobacterium, and Polaromonas,
and those in Cyclopoida spp. were Ralstonia, Burkholderia, Aeromonas, Sphingomonas,
Streptococcus, and Staphylococcus.

Table 2-3. List of dominant genera occurred in the body parts without gut (BP), gut, and fecesof the three zooplankton taxa collected at a pelagic site in the north basin of Lake Biwa, Japan,
on 4 March 2021, including laboratory rearing D. magna. Hyphens: Genus was not detected,
or < 1% of all OUT sequences.

45

Non-metric multidimensional scaling (NMDS) analysis clearly separated the ambient
bacteria from those associated with the gut and BP of the three field-collected zooplankton (Fig.
2-4). Bacterial compositions among the three zooplankton were more similar in feces thanthose in BP and gut. Although those in BP and gut were quite different among the taxa, thosein BP between the two copepod taxa were similar.

Fig. 2-4 Non-metric multidimensional scaling (NMDS) analysis using all OTUs demonstrating
dissimilarities in bacterial composition between ambient bacteria(AB) from ambient water andall zooplankton gut, BP, and feces samples. Ej, Dp, and Cy represent Eodiaptomus japonicus,
Daphnia pulicaria, and Cyclopoida spp., respectively; BP denotes body parts other than thegut of each taxon.
Heatmap analysis showed that the dominant family composition of the bacteria
associated with BP and gut was quite similar between the two daphnid species (Fig. 2-6). Those
associated with feces of D. pulicaria were also similar. Those in the feces of the two copepod
taxa and those in the gut and BP of Cyclopoida spp. were both similar, while those in the gutof E. japonicus were far from the others.

46

Fig. 2-6 Heatmap of major bacterial families ( 200 OUT reads) associated with zooplankton
body parts without gut (BP), gut, and feces in Eodiaptomus japonicus (Ej), Daphnia pulicaria(Dp), Cyclopoida spp. (Cy), Daphnia magna (Dm) and ambient bacteria (AB) were collected
at a pelagic site in the north basin of Lake Biwa on 4 March 2021. Individual lines correspondto bacterial families; each column corresponds to a different sample. The upper dendrogram
represents similarities in bacterial composition among zooplankter gut, BP, and feces; the left
dendrogram represents similarities in patterns of occurrence of a bacterial family in azooplankter sample and SS. ACK-M1 and C111 are candidate Actinobacteria families.
4. Discussion
According to the bacterial composition at the phylum and family level and the NMDS
analysis, the bacterial community associated with BP, gut, and feces in the three field-collected
zooplankton taxa were quite different from those in the ambient waters. Actinobacteria is themost dominant bacterial phyla in the water column in Lake Biwa (Okazaki and Nakano 2016),
while it was less found in the zooplankton bodies and feces in this study. The other abundant47

phylum in the ambient waters, including Chloroflexi, Planctomycetes, Verrucomicrobia,
Gemmatimonadetes, and Chlorobi, were also not found in both BP and gut of the fieldcollected three zooplankton, suggesting that some specific bacteria might be selected from theambient waters by the zooplankton hosts (Macke et al., 2020). On the other hand, a part ofbacterial phyla in the ambient waters, e.g., Verrucomicrobia and Chloroflexi, were also foundin the feces-associated bacteria. As mentioned above, Actinobacteria were found quite lessamount from the host bodies but more abundant from the feces, implying that bacteria in thefeces may include the bacteria from outside of the hosts’ bodies, that is, from the ambient
waters passing through the gut, as described in Chapter 1.
In this study, bacterial compositions in the gut and BP of laboratory-rearing D. magna
were quite similar to those in D. pulicaria collected from Lake Biwa; the dominant families
wereComamonadaceae,

Flavobacteriaceae,

Moraxellaceae,

Paenibacillaceae,

andPseudomonadaceae. These families were also found to be abundant in the whole-body
microbiota of laboratory-rearing D. magna and wild animals of D. galeata and D. obtusa
collected from freshwater lakes and ponds in Italy (Eckert et al. 2021). Freese and Schink(2011) also showed that Comamonadaceae, Moraxellaceae, and Pseudomonadaceae were thedominant bacterial families from the gut of laboratory-rearing D. magna. It has been also
shown that Comamonadaceae, Flavobacteriaceae, and Pseudomonadaceae are the mostabundant families in the host-associated bacteria in laboratory-rearing D. magna, D. pulex, andD. pulicaria originated from different laboratories (Qi et al. 2009). This similarity among
several daphnid species from different regions and laboratories suggest a strict relationshipbetween the daphnid species and the bacterial families, implying that these bacterial families
might construct a stable symbiotic relationship with the daphnids.
Limnohabitans was the most dominant genus in Comamonadaceae from both wild D.
pulicaria and laboratory-reared D. magna in this study and has been proven to be a beneficial

48

symbiont of its daphnid host in several laboratory experiments (Peerakiethajorn 2015, 2016,
Akbar et al. 2020). Limnohabitans spp. possesses various metabolic abilities (Zeng et al. 2012)that may benefit Daphnia nutrition (Cooper and Cressler 2020).
Acinetobacter and Pseudomonas were the dominant genera in Moraxellaceae andPseudomonadaceae, respectively, from the gut of D. pulicaria in this study. Acinetobacter canaccumulate large amounts of phosphorus as phosphate in its cell and release it under anaerobic
conditions (Deinema et al. 1985). A strain of Pseudomonas symbiont aided survival andmaintained the fecundity of its host D. magna under mercury stress conditions in a laboratory
experiment (Fong et al. 2019).
The gut of E. japonicus was constituted by a unique microbiota mostly dominated by
Neisseriaceae, followed by Weeksellaceae, Flavobacteriaceae, Comamonadaceae, andOxalobacteriaceae. Interestingly, all these families were also detected in BP, but the relative
abundance was different. These bacterial families were also found in Chapter 1 on the bacterial
composition of whole-body E. japonicus. Andreprevotia in Neisseriaceae possesses a higher
chitinolytic potential to biodegrade chitin from the exoskeleton of crustaceans (Tran et al.
2018). Chitin-rich crustacean carcasses, exoskeletons from molting, and other chitinous
organisms like fungi may produce an enormous amount of chitinous organic substances
aggregate in the water column (Yu et al. 1991, Grossart and Simon 1998). In addition, basedon the vertical temperature profile of the water column during this sampling time, it was awater mixing period in Lake Biwa, which may facilitate more chitinous particulate organic
matter in the epilimnion from that previously deposited in the hypolimnion during summer
stagnation (Yoshida et al. 2001). E. japonicus are suspension feeders which ingest particulateorganic matter (POM), including chitinous aggregates, and microzooplankton like fungal
zoospores available in the ambient water column, thus leading to a chitin-rich diet which might49

facilitate an increased abundance of Andreprevotia in E. japonicus gut (Tang et al. 2001, Tang
2005, Yeh et al. 2020).
On the contrary, chitin digestion may increase carbon and nitrogen in the intestine, thus
could enhance bacterial proliferation (Pruzzo et al. 2008), consequently, higher prevalence. In
Chapter 1, the reason behind the Flavobacterium association in this host’s gut and feces has
also been described. The genus Chryseobacterium in Weeksellaceae, abundant in BP, has
keratolytic potential and can utilize keratin and biosynthesis of microviridin, a proteaseinhibitor (Kang et al. 2021). Most strains of Chryseobacterium are resistant to a broad rangeof antimicrobial agents (Bernardet et al. 2015) and thus can modulate the host’s interaction
with pathogens and other foulers as an epibiotic bacteria (Wahl et al. 2012). However, there islimited information about the functions of zooplankton’s surface-attached bacteria and theirimpact on the host’s physiology.
Oxalobacteraceae, Sphingomonadaceae, Staphylococcaceae, Neisseriaceae, andBurkholderiaceae dominated the gut and BP microbial composition in host cyclopoids. But theabundance and occurrence of each taxon differed greatly between gut and BP bacterial
compositions. Oxalobacteraceae and Burkholderiaceae were highly abundant in the BP at 47%,
and 13%, respectively, while those in the gut were very low in abundance, at 10% and 1.6 %,respectively. Sphingomonadaceae, Staphylococcaceae, and Neisseriaceae were abundant in the
gut while absent in the BP. This difference can be due to the bacteria in the gut being exposed
to higher amounts of different kinds of substrates ingested by the hosts as food (Flint et al.
2008, Wu et al. 2012) than those exposed by the surface-attached bacteria. On the other hand,
the host’s surface-attached bacteria may use organic matter excreted by the hosts themselves(Tang et al. 2010).
Furthermore, Burkholderia, in the cyclopoids’ body parts without gut, may produce
antibiotics, such as bacteriocin and bioactive secondary metabolites, and can function as a50

biocontrol agent against pathogenic fungi, bacteria, protozoa, and nematodes (Elshafie andCamele 2021). These crustacean hosts do not have adaptive immunity (Vazquez et al. 2009).
Therefore, their association with bacteria that can produce antimicrobial agents may offer
protection against pathogens (Wahl et al. 2012, Elshafie and Camele 2021).

51

CHAPTER 3:“Community structures of symbiotic bacteria associated with the threedominant crustacean zooplankton in different depth habitats of Lake Biwa
during the stagnation period.”52

1. Introduction
The bacteria related to these zooplankters' external and internal body structures arereferred to as bacterial microbiota and are in close physical contact with the host organisms(Macke et al. 2017b). The zooplankton body provides a lucrative microhabitat for its
microbiota as its exterior, while the interior surfaces are rich in organic matter, supporting
bacteria to proliferate rapidly (Bickel and Tang 2014). In turn, abundant microbiota may
benefit its host physiological processes (Tang et al. 2010, Akbar et al. 2020). Such bacterial
microbiota has been found from both gut and body surfaces from marine and freshwaterzooplankton (Sochard et al. 1979, Hansen and Bech 1996, Tang et al. 2010), and also fromdominant crustaceans in Lake Biwa, as already shown in the previous chapters. However, there
is still limited information on the relationship between these bacterial compositions and hosthabitats in different seasons and depths yet.
The availability of foods for host zooplankton and bacterial communities in thesurrounding waters may influence the microbial compositions associated with the hosts(Grossart et al. 2009). The biotic and abiotic environmental factors including temperature, pH,
and food availability greatly vary with season and habitats of the zooplankton hosts (Bickel
and Tang 2014, Velasquez et al. 2022). Such differences can also impact the life history traitsof zooplankton hosts (Liu et al. 2014, Boonmak et al. 2018, Gao et al. 2022) and influence thecommunity structure of both zooplankton-associated and free-living bacteria (Huq et al.1984,
Bickel and Tang 2014). Whereas knowledge of the effects of these factors on zooplanktonassociated microbiota and their consequential influences on physiology and ecology of thehosts still needs to be improved (Samad et al. 2020). Moisander et al. (2015) showed that
temporal changes in food types available for zooplankton were fundamental in controlling hostassociated bacterial composition in a temperate marine coastal habitat. In an aquatic53

environment, temperature and amounts of organic substrates are essential factors for bacterial
proliferation and abundance (Shish and Ducklow 1994, Autio 1998).
On the other hand, zooplankton-associated microbiota may adapt to spatio-temporally
varying environmental conditions (Tang et al. 2010). Gerdts et al. (2013) found no seasonality
in the bacterial communities related to the copepod hosts during two years study period, though
the ambient bacterial community showed clear seasonality. Whereas Qi et al. (2009) showedthat Daphnia spp. from different geographic regions harbored similar bacterial composition.
Symbiotic bacteria are found in every body parts including the gut and should be regarded as
distinct functional units due to their distribution patterns, functions and contributions to their
host's physiology (Tang et al. 2010, Wahl et al. 2012). The bacterial microbiota in a specificsite of the host body can be an unambiguous source for understanding microenvironmental
variation in a host body and the effect of microbiota on the host's biology (Macke et al. 2017b,
Mangus et al. 2015).
In Lake Biwa, Eodiaptomus japonicus, Cyclopoida spp., and Daphnia pulicaria aredominant crustacean zooplankton throughout the year but show different seasonality regarding
their abundance and vertical habitat selection (Kawabata 1989, Yoshida et al. 2001). It is
important to clarify how the host habitat selection influences the microbiota in the different
parts of the host body, especially during the stagnation period, when the environmental factors,
such as temperature, dissolved oxygen, phytoplankton, and microbiota in the surrounding
waters, vary with depth (Tezuka 1982, Okazaki and Nakano 2016, Gurung et al. 2001) andaffect the physiology of the host zooplankton.
Population dynamics, spatio-temporal distributions, and the food availability of these threezooplankton have been well-studied in Lake Biwa (Kawabata, 1987, 1989, Yoshida et al. 2001,
Liu et al. 2020). I’ve already described bacterial community structure associated with thezooplankton hosts in the epi-pelagic zone of the lake in specific seasons in previous chapters.

54

In this chapter, the zooplankton-associated bacterial compositions were compared between two
different depth layers, i.e., epilimnion (0–20 m) and hypolimnion (20–50 m), in June and
September 2021, to clarify the depth-mediated effects on the composition of the zooplanktonassociated bacteria during the vertically stagnated water column in Lake Biwa.
2. Materials and methods
2.1 Sample collection and preparation

Zooplankton were collected from the two different depths of the water column, from
20 m to the surface and from just above the bottom to 20 m, with a vertical net haul using a
NORPAC net (mesh size, 200 µm; mouth diameter, 45 cm), and with oblique tow of a closing
net (mesh size, 100 µm; mouth diameter, 30 cm), respectively, at a pelagic site (3518.793ʺN,
1368.854ʺE, 70 m deep) in the north basin of Lake Biwa, Japan on 17 June and 21 September
2021. Another zooplankton samples for enumerating individual numbers were simultaneously
collected from the same two depth layers with vertical discrete haul using the closing net. The
zooplankton were immediately killed with Lugol’s solution and preserved with sugar-formalin
at a final concentration of 4%. Samples were collected in the morning between 9:30 and 11:30.
Vertical profiles of water temperature, dissolved oxygen (DO), and chlorophyll a concentration
were obtained with a conductivity-temperature-depth profiler (AAQ-Rinko, JFE advantec).
Lake waters for determining ambient bacteria were collected from 0, 10, 20, 30, 40, and 50 mwith a Van-Dorn bottle (volume, 6 L), and the lake waters from 0, 10, and 20 m and those from30, 40 and 50 m were combined with the same volume of each depth water for following
metagenomic analysis on the ambient bacteria. The live zooplankton and lake waters collected
were transferred in an insulated container to the laboratory within 1 h.

55

Approximately 100 adult individuals of each zooplankton taxon, i.e., Daphnia
pulicaria (>2 mm), Eodiaptomus japonicus, and Cyclopoida spp., were sorted from the live
zooplankton sample under a dissecting microscope (Olympus, SZX12, Japan), washed twicewith aged tap water filtered through a glass-fiber filter (Whatman, GF/F), autoclaved at 120 °C
for 20 min (ATW), and incubated separately into 1-L glass jars filled with ATW for evacuating
the gut contents. The zooplankton sorted from each depth of 0–20 m and 20–50 m wereincubated separately in a growth chamber (Sanyo, MLR-350, Japan) at ambient watertemperatures, i.e., 17 °C in June and 22 °C in September above 20 m, and 10 °C below 20 m
in both months, for 24 h, picked up from the jar and washed twice with Milli-Q water, and thenused for following DNA extraction.
For enumerating zooplankton abundance, each of the three dominant zooplankton taxa,
E. japonicus adult males and females, adult cyclopoids, and large D. pulicaria (>2 mm) was
counted under a dissecting microscope (Olympus, SZH10, Japan) at a magnification of 40x.
For the following DNA extraction, 30 adult E. japonicus, 35 adult cyclopoids, and 15
adult D. pulicaria (>2 mm) were sorted from the zooplankton specimens evacuated the gut
contents, and removed the gut from the body with a sterile tungsten needle under a dissecting
microscope (Olympus, SZX9) at a magnification of 40–50x,. Each of the gut removed and allother body parts excluding the gut was transferred into a sterile 1.5-mL micro-tube (Eppendorf,
Germany) filled with sodium phosphate (978 µL) and MT buffer (122 µL) in a Fast DNA®
Spin Kit for Soil (MP Biomedicals, LLC, USA), and then frozen at -80˚C for the following
analysis.
To analyze bacterial compositions in the ambient waters, each 1 L of the combined
waters from the two different depth strata (0–20 m and 30–50 m) was filtered with a 20-µmmesh sieve. A 300 ml of the pre-sieved water was filtered with a 0.2-µm pore polycarbonate
membrane filter (Advantec, K020A047A, Japan). The filter was transferred into a 5-mL tube

56

(Labcon, U.S.A.) filled with sodium phosphate (978 µL) and MT buffer (122 µL) in a Fast
DNA® Spin Kit for Soil (MP Biomedicals, LLC, USA) for DNA extraction. All samples werefrozen at −80 °C until the following analysis.

2.2 Metabarcoding analysis using Illumina MiSeq
Samples were thawed and homogenized using a sterile disposable plastic pellet pestle.
DNA was directly extracted from each sample using a Fast DNA ® Spin Kit for Soil.
Metabarcoding targeted the V4 region of the 16S rRNA gene with primers 515F (5ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGTGCCAGCMGCCGCGGT
AA-3)

and

806R

(5-

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNGGACTACHVGGGTWTCTAAT-3) (Glenn et al. 2019). All PCR reactions were performed in each sample with a 10
L mixture of an Ex TaqHS-mix (TaKaRa Bio Inc., Japan), 10 M of each forward and reverse
primer, and about 5 ng of extracted DNA. Initial denaturation proceeded at 94C for 2 min,followed by 30 cycles of denaturation at 94C for 30 s, annealing at 50C for 30 s, elongation
at 72C for 30 s, followed by a final extension at 72C for 5 min. Before the second PCR was
performed, the first PCR product was purified by Beckman Coulter AMPure XP (Beckman
Coulter, Inc., USA). To attach the index sequence to the products of the first PCR, products ofthesecondPCR

wereprocessed

with

the

primerset

F:

AATGATACGGCGACCACCGAGATCTACAC-Index-2(8bp)ACACTCTTTCCCTACACGACGC

and

R:

CAAGCAGAAGACGGCATACGAGAT-

Index1(8bp)-GTGACTGGAGTTCAGACGTGTG (Tanaka et al. 2020). Initial denaturation
was at 94 C for 2 min, followed by 12 cycles of denaturation at 94 C for 30 s, annealing at
60 C for 30 s, elongation at 72 C for 30 s, and a final extension at 72 C for 5 min. After
purification using the Beckman Coulter AMPure XP, library quality was assessed using an

57

Agilent fragment analyzer system and dsDNA 915 reagent kit (Advanced Analytical
Technologies). Libraries were sequenced on an Illumina MiSeq platform, generating 2  300
bp paired-end reads. A total of 12,22,427 raw reads were obtained.

2.3 Data analysis
Raw data collected from the Illumina MiSeq sequencing platform were considered raw
tags. Basic tags were prepared after removing the barcode and PCR primer sequences using
the fastq_barcode_splitter from the Fastx Toolkit software (ver. 0.0.14). After removing theprimer sequence (50 bp at the 3 end), chimera sequence, and noise sequence using the dada2
plugin of Qiime2 (ver. 2021.11), effective tags with 97% similarity were classified into one
amplicon sequence variant (ASV), hereinafter operational taxonomic unit (OTU) (FrankelBricker et al. 2020). OTUs containing “unidentified” taxonomic annotations and singleton(only a single sequence across the entire dataset) were removed from data analysis. All OTUs
with the best Blast matches to non-bacterial organisms were also discarded from the analysis.
Before analyzing OTUs, a rarefied OTU table was generated using the QIIME
rarefaction analysis script with the rarefied depth in the minimum sequence number. We
discarded the OTUs belonging to cyanobacteria, chloroplast, mitochondria, and archaea fromall samples (Chapters 1, 2).
The DNA samples of this study were included in three separate sequencing runs, i.e.,
E. japonicus, Cyclopoida spp., and D. pulicaria and ambient water samples (AB). The Shannon
diversity index (H) was calculated for the bacterial community of each sample. Non-metricmultidimensional scaling (NMDS) analysis was performed to compare bacterial composition
between sample types for each zooplankton taxa and water samples using all OTUs. To
examine statistical differences in bacterial communities between sampling months andbetween habitats, I performed analysis of similarities (ANOSIM). Heatmap analysis was also

58

performed in family level using  200 reads of OTUs. All statistical analyses were performed
using the R software package ‘vegan’ ver. 4.0.3 (2020).

3. Results
3.1 Vertical structure of the water column
In both June and September 2021, the temperature and dissolved oxygen (DO) profiles in
the water column showed that lake water was stratified, with a shallower mixing layer in Junethan that in September (Fig 3-1). Thermoclines were developed at 4–15 m in June, while at
13–15 m in September. The lake waters below 15 m were considered as hypolimnion in bothmonths. I separated the water column to two layers, i.e., above 20 m and below 20 m, andtherefore former layer included epilimnion and metalimnion while later one just hypolimnion.
Average water temperatures above 20 m were 17.1 C on 17 June while 22.0 C on 21
September, being 5 C higher in September than that in June. The average water temperature
below 20 m was around 10 C in both months. Chlorophyll a concentration above 20 m werehigher in June (2.86 g/L) than that in September (0.98 g/L). Although percentages of DO
saturation were higher throughout the water column in June, ca. 26% higher above thethermocline than in September. More than 100% above 10 m in June might probably be
attributed to photosynthesis of phytoplankton. The three zooplankton taxa tested were mainly
distributed above 20 m during the sampling period (Table 3-1). E. japonicus was mostly
colonized in epilimnion, >10 fold than that in the hypolimnion, while not so large difference
between the depth layers in Cyclopoida spp., >3 fold. D. pulicaria was relatively low in number
in both layers compared to those two copepods.

59

Figure 3-1. Vertical profiles of temperature, chlorophyll a concentration, and dissolved oxygen
content throughout the water column at a pelagic site in the north basin of Lake Biwa
(3518.793ʺN, 1368.854ʺE) on 17 June and 21 September 2021.
Table 3-1. Average water temperature, chlorophyll a concentration (Chl-a), % of dissolved
oxygen, and individual (adults) densities of three zooplankton hosts at a pelagic site(3518.793ʺN, 1368.854ʺE) of the north basin of Lake Biwa on 17 June and 21 September
2021.
Environmental parameters and densities ofzooplankton hosts
Water temperature ( C)
Chl-a (g/L)% saturation of dissolved oxygen
Eodiaptomus japonicus (indiv. / m3)
Cyclopoida spp. (indiv. / m3)
Daphnia pulicaria (indiv. / m3)June
0–20 m
17.09
2.86
109.3
3950
1500
450

20–50 m
10.34
0.46
90.59
450
350
100

September
0–20 m20–50 m
22.02
10.49
0.98
0.22
85.18
71.81
8650
850
1800
500
100
50

60

3.2 Bacterial compositions in zooplankton and ambient waters
A total of 1,091,297 sequences, comprising 2,850 bacterial OTUs, were generated from 24
samples of the gut (Gut) and body parts other than gut (BP) of the three zooplankton species
and ambient bacteria (AB) from two different depth layers in June and September (Table 3-2).
A total of 876,673 sequences and 1,396 OTUs were found from Gut and BP, while 214,624
sequences and 1,454 OTUs from AB. The bacterial communities in AB showed extremely
higher diversity than those associated with any zooplankton taxa and higher in the hypolimnion
than those in the epilimnion regarding both numbers of OTU and Shannon’s H (Table 3-2).
The diversities of the bacteria associated with zooplankton were higher above 20 m than thosebelow 20 m except for the two copepod taxa in June, where Shannon’s Hs were almost similar
between the depths though the number of OTUs was relatively higher in the upper layer.
Shannon’s Hs of the BP-associated bacteria were mostly higher than those of the Gutassociated bacteria except for D. pulicaria collected below 20 m in June when the diversity
was higher in the Gut-associated bacteria.

61

Table 3-2. Total numbers of sequences and identified OTUs, and Shannon’s H index ofbacteria associated with body parts without gut (BP), and gut of Daphnia pulicaria,
Eodiaptomus japonicus, and Cyclopoida spp., and ambient bacteria (AB), collected from Lake
Biwa on 17 June, and 21 September 2021.
Host taxa/
environment
D. pulicaria

E. japonicus

Cyclopoida spp.

Ambient waters

Month

Depth

Site

Jun
Jun
Jun
Jun
Sep
Sep
Sep
Sep
Jun
Jun
Jun
Jun
Sep
Sep
Sep
Sep
Jun
Jun
Jun
Jun
Sep
Sep
Sep
Sep
Jun
Jun
Sep
Sep

0–20m
0–20m
20–50m20–50m0–20m
0–20m
20–50m20–50m0–20m
0–20m
20–50m20–50m0–20m
0–20m
20–50m20–50m0–20m
0–20m
20–50m20–50m0–20m
0–20m
20–50m20–50m0–20m30–50m0–20m30–50m

BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
BP
Gut
-----

Total no. ofsequences
45,78849,801
41,26642,53939,21638,82338,30929,47736,89942,42017,092
28,02831,81020,903
26,96330,80136,35648,19637,99739,28331,344
41,58738,96242,81352,96857,774
48,981
54,901

No. of
OTU's
102
77
55
55
75
78
42
63
39
68
51
49
59
66
45
18
45
84
34
43
76
83
43
46
317
380
357
400

Shannon's H
3.20
2.79
2.00
2.5
2.50
2.15
1.64
1.50
2.41
2.04
2.79
2.50
2.61
2.52
1.97
1.27
2.22
1.73
2.25
1.87
2.80
2.46
2.30
1.77
3.98
4.46
3.77
4.10

More than 99.6% of bacterial OTU sequences generated were assigned to 16 identified
phyla. A few sequences with an unknown taxonomic assignment at the phylum level (0.36%
of the total sequences) were designated as unidentified. The dominant bacterial phyla in BP
and Gut of all three hosts were Proteobacteria and Bacteroidetes (82% and 14% of the total
sequences, respectively, on average), while those in AB were Proteobacteria, Actinobacteria
and Bacteroidetes (35%, 22%, and 14% of the total sequences, respectively) (Fig. 3-2).

62Chloroflexi and Planctomycetes additionally dominated in AB from hypolimnion in both Juneand September. No Firmicutes and Tenericutes were found from AB, while no Chloroflexi,
Planctomycetes, Nitrospirae, Chlorobi, Armatimonadetes, and Gemmatimonadetes were foundfrom zooplankton hosts.

Fig. 3-2 Phylum composition of bacterial OTUs associated with body parts without gut (BP)and gut (Gut) in Daphnia pulicaria, Eodiaptomus japonicus, Cyclopoida spp., and ambient
bacteria (AB) in ambient water, collected from a pelagic site in the north basin of Lake Biwa
on 17 June and 21 September 2021.
The most abundant bacterial family associated with D. pulicaria from epilimnion inJune were Aeromonadaceae (41%) and Flavobacteriaceae (15%) in the Gut, andFlavobacteriaceae (24%) and Comamonadaceae (22%) in the BP. Whereas, in hypolimnion,
those of the Gut was dominated by Neisseriaceae (30%) and Flavobacteriaceae (22%), andthose in BP were Neisseriaceae (41%) and Comamonadaceae (9%), being quite different fromthose of both Gut and BP in epilimnion. In September, dominant bacterial families in
epilimnion were Aeromonadaceae (51%) and Oxalobacteraceae (25%) in the Gut,
Oxalobacteraceae (40%) and Neisseriaceae (12%) in the BP, being different from those in June.
63

In hypolimnion, Oxalobacteraceae and Aeromonadaceae were dominated in both the Gut (71%
and 13%, respectively) and BP (60% and 13%, respectively), being also quite different fromthose in June. The Major abundant bacterial components in the Gut and BP from both epi- andhypolimnion in September were similar, but different between the depths. Flavobacteriaceae
was abundant from both Gut and BP in June at 17% on average, while that in September was<1% (Fig. 3-3). Oxalobacteraceae, Aeromonadaceae, Comamonadaceae, Flavobacteriaceae
and Neisseriaceae were also found in Gut and BP at 3-28% and 7-28%, respectively, on
average from epi- and hypolimnion in both June and September. Moraxellaceae was commonly
found from the BP in both months.

Fig. 3-3 Family composition of bacterial OTUs ( 200 reads) associated with body parts
without gut (BP) and gut (Gut) in Daphnia pulicaria, Eodiaptomus japonicus, Cyclopoida spp.
and ambient bacteria (AB) in ambient water, collected from a pelagic site in the north basin of
Lake Biwa on 17 June and 21 September 2021. ACK-M1 and C111 are candidate
Actinobacteria families.
Dominant bacterial families in E. japonicus from epilimnion in June werePseudomonadaceae (56%) and Comamonadaceae (14%) in the Gut, Comamonadaceae (25%)

64and Weeksellaceae (24%) in the BP, while those from hypolimnion were Flavobacteriaceae
and Neisseriaceae in the Gut (30% and 20%, respectively) and the BP (27% and 20%,
respectively), being quite different from those from epilimnion. On the contrary, in September,
Oxalobacteraceae was the most dominant family in the Gut and BP from both epi- andhypolimnion; 51% and 40%, respectively, in epilimnion, while 46% and 53%, respectively, in
hypolimnion. Next dominant bacterial family in the Gut were Moraxellaceae (13%) andfacultative anaerobic Streptococcaceae (7%), in epilimnion, while Aeromonadaceae (45%)
from hypolimnion, greatly contrasting with those in June. Flavobacteriaceae was found fromboth the Gut and BP in June but absent in September. Oxalobacteraceae, Comamonadaceae,
Neisseriaceae, Weeksellaceae and Aeromonadaceae were found from the Gut and BP in bothJune and September.
In Cyclopoida spp., the most dominant bacterial family in epilimnion of June were
Aeromonadaceae and Comamonadaceae from the Gut (66% and 20%, respectively) and BP(21% and 28%, respectively). Whereas, those in hypolimnion were Comamonadaceae (32%),
Oxalobacteraceae (27%) and Chromatiaceae (25%) from the Gut, Neisseriaceae (42%),
Comamonadaceae (27%) and Oxalobacteraceae (13%) from the BP. On the contrary, those inepilimnion of September was dominated by Pseudoalteromonadaceae in both of Gut and BP at
33% and 28%, respectively, followed by Aeromonadaceae at 33% in the Gut andComamonadaceae at 17% in BP. Those in hypolimnion were Aeromonadaceae (41%) andOxalobacteraceae (14%) from the Gut and Comamonadaceae (28%) and Aeromonadaceae(20%) from the BP.
The dominant bacterial families in AB were very different from those associated withthe three hosts, but almost similar between the months and depths, being Pelagibacteraceae,
ACK-M1,

C111,

Comamonadaceae,

Methylophilaceae,

Chthoniobacteraceae

andChitinophagaceae.

65Non-metric multidimensional scaling (NMDS) analysis using all bacterial OTUs foreach zooplankton taxa in June and September showed different dissimilarity patterns of thebacterial compositions associated with the host among the taxa (Fig. 3-4). In D. pulicaria,
bacterial compositions were similar between the Gut and BP but different between the twomonths and depths. In E. japonicus, those in June were also similar between the Gut and BP
but different between the depths, while those from the Gut were quite different from others in
September. In Cyclopoida spp., bacterial compositions were different between two months,
two depths, and body parts, i.e., Gut and BP. Those in AB also differed between months anddepths.
Table 3-3. ANOSIM results comparing dissimilarities between June and September andbetween epi- (0–20m) and hypolimnion (20–50m) for zooplankton-associated bacterial
community compositions, collected from Lake Biwa on 17 June and 21 September 2021. Rvalue (1, -1), R-value close to 1 indicates significant differences between groups, -1 indicateswithin the group in more different than between groups.
Zooplankton taxa

Treatments

Replicatesin eachgroup

Permutations

ANOSIMR
statistic

P-valueGroup 1

Group 2

D. pulicaria

June

September

4

999

0.74

0.03

D. pulicaria

0–20m

20–50m

4

999

0.36

0.05

E. japonicus

June

September

4

999

0.74

0.03

E. japonicus

0–20m

20–50m

4

999

-0.04

0.57

Cyclopoida spp.

June

September

4

999

0.44

0.03

Cyclopoida spp.

0–20m

20–50m

4

999

0.14

0.29

ANOSIM showed that dissimilarities of bacterial composition associated with D.
pulicaria (Gut and BP) were significantly different between the two months and two depths,
while those with two copepod taxa were also significantly different between the two months
but not different between the two depths (Table 3-3).

66

Fig. 3-4 Non-metric multidimensional scaling (NMDS) analysis using all OTUs demonstrating
dissimilarities among samples from June and September for each zooplankton taxa; D.
pulicaria (a), E. japonicus (b), Cyclopoida spp. (c), and ambient bacteria (AB) (d) fromambient water (d). Dp, Ej, and Cy represent Daphnia pulicaria, Eodiaptomus japonicus, andCyclopoida spp., respectively, BP denotes body parts other than the gut of each taxon.
Heatmap analysis showed that the dominant family composition of bacteria associatedwith zooplankton hosts were similar between the two depths in each month, and therefore, fivelarge clusters were constructed (Fig. 3-6). In each cluster, those in the same month were mostly
constituted. Cluster 1 constitutes dominant family compositions with E. japonicus and D.
pulicaria in both depths of September; those associated with the Gut and BP of E. japonicus
in the epilimnion and with the Gut in the hypolimnion, those with the Gut and BP of D.
pulicaria in the hypolimnion and with the BP in epilimnion were relatively similar. The
dominant families were Oxalobacteraceae, Aeromonadaceae, and Comamonadaceae. Cluster67

2 included those with E. japonicus and D. pulicaria in both epi- and hypolimnion of June; those
associated with the Gut and BP of E. japonicus and D. pulicaria and BP of Cyclopoida spp.
from hypolimnion were relatively similar. The dominant families were Neisseriaceae,
Flavobacteriaceae, Comamonadaceae, and Oxalobacteraceae. In cluster 3, those with E.
japonicus and Cyclopoida spp. in both depths of both months were constituted with dominantfamilies of Comamonadaceae and Oxalobacteraceae. In cluster 4, those with the Gut of E.
japonicus and Cyclopoida spp. in the hypolimnion and the Gut of D. pulicaria and Cyclopoidaspp. in the epilimnion of September, dominating Aeromonadaceae and Oxalobacteraceae. In
cluster 5, those with the Gut of D. pulicaria and Cyclopoida spp. in the epilimnion of Junewere constituted with the dominance of Aeromonadaceae, Comamonadaceae, andNeisseriaceae.

68Fig. 3-6 Heatmap of abundant bacterial families ( 200 OTU reads) associated withzooplankter BP and gut of Daphnia pulicaria (Dp), Eodiaptomus japonicus (Ej), Cyclopoidaspp. (Cy); BP denotes body parts other than the gut. Individual lines correspond to bacterialfamilies; each column corresponds to a different sample. The upper dendrogram represents
similarities in bacterial composition among zooplankter BP and gut; the left dendrogram
represents similarities in patterns of occurrence of a bacterial family in zooplankter samples.
The number at the top indicates each cluster.
4. Discussion
In this chapter, I examined how the bacterial compositions associated with the Gut and
BP of the three zooplankton hosts differed between the two months and the depth habitats at apelagic site in the north basin of Lake Biwa. The lake water was tightly stratified during thestudy period, and June was start of the stagnation period while September was the end of theperiod. Therefore, abiotic and biotic conditions of the environment, including temperature,
dissolved oxygen, phytoplankton, and zooplankton community, and environmental bacterial69

compositions differed between the months and the depths (Tezuka 1984, Kawabata 1989,
Hsieh et al. 2010, Okazaki and Nakano 2016). Ambient bacterial compositions observed wereremarkably different between two different water layers, 0−20 m and 30−50 m. Dominat
phylum of ambient bacteria were Chloroflexi and Planctomycetes in hypolimnion in both Juneand September, and it is similar to those previously reported by Okazaki et al. (2013) andOkazaki and Nakano (2016). The Gut and BP of D. pulicaria, E. japonicus, and Cyclopoidaspp. in epilimnion contained higher microbial diversity (H) than those in hypolimnion. It might
be associated with consistently higher bacterial abundance in epilimnion with higher
temperature and DO than those in hypolimnion during the summer stagnation period in Lake
Biwa (Gurung et al. 2001). Moreover, during the stagnation period, dissolved organic carbon(Maki et al. 2010) and phytoplankton (Tezuka 1984) become more abundant in the epilimnion
compared to the hypolimnion. These conditions might be considered to facilitate a favorable
condition for diverse bacterial attachment and proliferation in the gut and body surface of thethree zooplankton hosts in epilimnion (Huq et al. 1984, Bickel and Tang 2014). Whereas
symbiotic bacteria with the zooplankton may utilize organic matter imported and produced by
the hosts themselves through ingestion and egestion (M øller 2005, Møller et al. 2007), being
independent of the availability of organic substrates in their surrounding environment (Bickel
and Tang 2014).
Chl-a concentrations were mainly distributed at 020 m depth in Lake Biwa andabundant in June while low in September (Fig. 3-1). NMDS and ANOSIM showed that thebacterial communities in the BP and Gut of the three zooplankton hosts differed significantly
between June and September. Flavobacteriaceae, which is a type of bacteria specialized indigesting phytoplankton-derived substrates (Qi et al. 2009), in the Gut and BP of D. pulicaria
and E. japonicus, had a lower contribution in September than in June. This may be associatedwith the phytoplankton diet available for D. pulicaria and E. japonicus in June but not in70September. Commamonadaceae, which has been shown to be a widely occurring bacterialfamily in Daphnia as also shown in Chapter 2, and ubiquitous during phytoplankton bloom invarious freshwater bodies (Eckert and Pernthaler 2014), was lower in September than in June.
In September, Oxalobacteraceae was detected in the Gut, and BP of E. japonicus andD. pulicaria in the hypolimnion, where water temperatures were lower than 10˚C in bothmonths, may be attributed to the availability of nutrients in the host microhabitat (Tang et al.
2010) that fosters the growth of this mesophilic bacterial group disregarding temperature
limitation (Pomeroy and Wiebe 2001). Bickel and Tang (2014) reported that temperature had
a limited impact on Acartia tonsa and Balanus sp.-associated bacterial abundance in
Chesapeake Bay. During the stratification period, the availability of organic substrate forheterotrophic microbial processes in the hypolimnion may be rather more important thantemperature (Bickel and Tang 2014). The members of Oxalobacteraceae are heterotrophic andcan be aerobic or microaerobic to anaerobic (Baldani et al. 2014). Thus, abundantOxalobacteraceae detected in the Gut and BP of E. japonicus and D. pulicaria inhabiting in
hypolimnion may be attributed to the availability of nutrients in the host microhabitat (Tang etal. 2010, Bickel and Tang 2014) that may foster the growth of this bacterial group disregarding
temperature and oxygen limitation (Pomeroy and Wiebe 2001).
In the Gut and BP of Cyclopoida spp., Pseudoalteromonadaceae including Vibrio
cholerae was dominant just from the epilimnion in September. Water temperature might be
one of the key parameters affecting the spatio-temporal distribution of Vibrio cholerae
associated with Cyclopoida spp. in Lake Biwa. Vibrio spp. are predominant symbiotic
bacterium in the gut and body surface of marine copepods (Sochard et al. 1979, Huq et al. 1983,
Tang et al. 2010) and are prevalent in warm water conditions (Huq et al. 1984). The presenceof Vibrio sp. in the gut of cyclopoids from the epilimnion may be attributed to theirconsumption of chitin-rich diets. Vibrio spp. are specialized to digest chitin (Cottrell et al.

71

2000). The adult Mesocyclops is a carnivore and preys on nauplii and juvenile E. japonicus(Kawabata 1991), having a chitinous body mainly distributed in epilimnion during thestagnation period (Kawabata 1987). Consequently, Cyclopoida spp. inhabiting in hypolimnion
might face a lack of such food and may feed on other microzooplankton available (Kawabata
1987).
The microbial difference in Cyclopoida spp. between two habitats also could correlatewith a species-specific difference and their habitat selection. In Lake Biwa, Cyclopoida spp.
mainly include two species, Mesocyclops dissimilis and Cyclops vicinus (Kawabata 1989). The
adult M. dissimilis is abundant during the stagnation period and restricted to epilimnion,
whereas C. vicinus is mainly distributed to the deeper layer (Kawabata 1989). Therefore,
interspecies variability and their different habitat selection in the study period can be importantreasons for harboring diverse microbiota among individuals from different habitats (Datta etal. 2018, Sadaiappon et al. 2021).

72

CHAPTER 4:“Taxonomic and ecological aspects on epibiotic ciliates and flagellates
attached to cyclopoid copepods in Lake Biwa.”73

1. Introduction
Epibiosis is a type of symbiotic relationship where one organism spends its sessile life
stage attached to the other organism's surface. The organism that attaches to another livingorganism, which is referred to as the basibiont or host, is called epibiont (Wahl, 1989). Suchepisymbiotic relationships can have both positive and negative effects for both epibiont andhost (Wahl 1989, Wahl et al.1997, Cabral et al. 2017), or it could be impartial, depending on
the ecological aspect (Wahl 2008). Occasionally, this relationship could be detrimental to hosts(Herman and Mihursky 1964, Turner et al. 1979, Visse 2007), decreasing their life span
(Gilbert and Schroder 2003) and fecundity (Henebry and Ridgeway 1979, Threlkeld andWilley 1993), increasing burden and interfering host’s motility (Willey et al. 1990, Jones et al.
2016); altering their physiology and behavior (Barea-Arco et al. 2001, Willey and Threlkeld
1993); competing for the food, if size range of ingestible food particles overlaps (Kankaala andEloranta 1987). On the contrary, epibionts may protect the host (basibiont) against predators(Wahl 1989) and sometimes provide a food source for the host when usual planktonic feed is
at low levels (Barea-Arco et al. 2001). By attaching to the host bodies, epibionts may evade
zooplankton predators (Henebry and Ridgeway 1979), travel to areas with available foods(Kankaala and Eloranta 1987, Wahl 1989), and have optimal conditions for growth (RegaliSeleghim and Godinho 2004).
Epibiosis involving a relationship between epizoic protists and zooplankton is a
widespread phenomenon in aquatic environments (Chiavelli 2003, Jones et al. 2016). The
flagellated green alga Colacium attaches and colonizes on the body surface of crustacean
zooplankton, including Daphnia spp., cyclopoid and calanoid copepods (Willey et al. 1990,
Threlkeld et al. 1993, Zalocar et al. 2011) from both fresh- (Huber-Pestalozzi 1955) andbrackish waters (Møhlenberg and Kaas 1990). Previous studies showed that Colacium
vesiculosum was mutualistic to its calanoid hosts (Møhlenberg and Kaas 1990, Zalocar et al.

74

2011). Epizoic Colacium spp. was prevalent when their zooplankton hosts were abundant(Møhlenberg and Kaas 1990, Chiavelli et al. 1993), and the infestation rate was positively
correlated with ambient temperature (Møhlenberg and Kaas 1990). However, the ecologicalcontext and significance of the relationship between these zooplankton hosts and Colacium spp.
is poorly understood. The epizoic flagellate Colacium may prefer zooplankton taxa as a single
host for colonization over other taxa (Chiavelli et al. 1993, Møhlenberg and Kaas 1993).
Many species of peritrich ciliates have been shown to attach as epibionts on the bodyof various crustacean zooplankton species (Khal 1935; Sprague and Couch 1971; FernandezLeborans and Tato-Porto 2000; Bickel et al. 2012). There are many reports on colonial peritrich
ciliate Epistylis spp. attached to the exoskeletons of cyclopoid and calanoid copepods andcladocerans (Foissner et al. 1999, Bickel et al. 2012, Clamp et al. 2016, De Souza Santos et al.
2020). However, knowledge on the effect of Epistylis spp. on its crustacean hosts is very
limited, though high infestation by Epistylis sp. has been shown to reduce the survival of its
host Pseudodiaptomus stuhlmanni in a laboratory experiment (Jones et al. 2016). De Souza
Santos et al. (2020) showed that the prevalence of epibiont Epistylis sp. on calanoid andcyclopoid hosts was related to its host size and abundance but not to any ambient abiotic
variables. On the contrary, Utz and Coats (2005) didn’t find any relationship between hostabundance and the prevalence of Epistylis infestation. Cabral et al. (2017) reported that both
physico-chemically and biologically (food availability and host abundance) environmental
parameters regulated the populations of epibiotic ciliate Epistylis spp. in the floodplain.
Epistylis sp. also shows host-specificity (Utz and Coats 2005, Clamp et al. 2016, Lu et al.
2020) as shown in Colacium, and tends to attach to just a couple of host species even whenother zooplankton species coexist (Xie et al. 2001, Cabral et al. 2017).
In this chapter, I focused on the following steps, 1) species of epibionts occurred wereidentified based on their morphological features, 2) seasonal changes and vertical distribution75

of the epibiont and infestation rate were determined with environmental variables, and 3)
factors affecting these spatio-temporal variation in infestation of the epibionts were discussed.

2. Materials and Methods
Zooplankton samples were collected monthly with a vertical net haul using a conicalplankton net (mouth diameter, 30 cm: mesh size, 0.1 mm) with a flowmeter from just above
the bottom to the surface at two fixed pelagic sites, K4 (3518′56.6′′N, 13611′27.0′′E; depth~50 m) from 15 January 2018 to 19 December 2019, and a station near Japan water agency(JWA) observatory buoy (3518.672′ N, 13608.593′ E; depth ~70 m) from 17 March 2020 to
16 December 2021 and from 20 June to 21 December 2022 in north basin of Lake Biwa. To
determine vertical distribution of zooplankton and epibionts, zooplankton samples were alsocollected with discrete vertical net hauls from 8 depth layers, 0–5, 5–10, 10–15, 15–20, 20–25,
25–30, 30–50 m and 50 m – just above the bottom, using a closing plankton net (mouthdiameter, 30 cm; mesh size, 0.1 mm) in 24 August, 14 September, 17 October, and 15
November at the same time with the monthly sampling in 2022. The zooplankton collected
were immediately killed with Lugol's Iodine solution and preserved with 5% sugar formalin.
Vertical profiles of water temperature, dissolved oxygen (DO), and chlorophyll a concentration
throughout the water column were determined with a CTD profiler (AAQ-Rinko, JFE
advantec) on each occasion.
Each crustacean zooplankton species and individuals attached by epibionts wereidentified and enumerated under a dissecting microscope (OLYMPUS SZH10) at themagnification of 30−70x. Infestation prevalence as total infestation rate (total number of
infested individuals), co-infestation rate of ciliates and flagellates, and infection rate of each
epibiont was calculated as the number of zooplankton infested by epibionts, i.e., ciliates andflagellates, divided by the total number of the host zooplankton (Bush et al. 1997). The

76

epibionts attached to just cyclopoid copepods (see Results in detail), and then the epibionts
attached to the body parts of the host cyclopoids, i.e., antennule, cephalothorax, thorax
segments, swimming legs, caudal rami, and setae were also examined.
Another zooplankton sample was collected with a vertical net haul from 30 m to thesurface using a Norpac net (mouth diameter, 45 cm; mesh size, 0.2 mm) to examine themorphological features of the epibionts. Live zooplankton collected were immediatelytransferred to the laboratory with an insulated container. In the laboratory, the body size andshape of the epibionts and types of colonies were examined with a phase contrast microscope(OLYMPUS IX70, Japan) at a magnification of 100−400x, equipped with a digital camera(WRAYCAM-NF1000) connected to a computer. Measurements were performed using thesoftware WraySpect ver. 2.1. and identified the species of the flagellate and ciliate epibionts.
Silver-nitrate staining was performed to identify some specific characteristics of the zooid,
including pellicular striation, telotroch band, infundibular ciliature, and shape of macronucleusof the ciliate epibionts (Waggoner et al. 2016). Identification of epibionts was made based on
mainly Foissner et al. (1999) and Lu et al. (2020) for ciliates, and Huber-Pestalozzi (1955),
Rosowski and Kugrens (1973) and Zalocar et al. (2011) for flagellates.

3. Results
Two types of protistan epibionts, i.e., stalked colonial peritrich ciliate Epistylis sp. andepizoic flagellate Colacium sp., were exclusively found on adults and copepodites of
Cyclopoida spp. There were three types of infestation observed; cyclopoids were infested by
the ciliate alone, the flagellate alone, or both (hereinafter co-infestation). Colacium sp. wascommonly attached to the cephalosome, metasome, urosome, antennule, caudal rami and setae,
and swimming legs of the cyclopoid hosts (Fig. 4-1), while Epistylis sp. was mainly attached
to metasome and urosome of the adult and late copepodite (Fig. 4-2).

77

Fig. 4-1 Photographs of cyclopoid copepods infested by flagellate epibiont, Colacium
vesiculosum, in Lake Biwa; different sites of the body, cephalosome and antennae (a), urosome
and caudal rami (b) and right antennae of adult male (c), and a co-infestation (both flagellates
and ciliates infected a cyclopoid) (d).

78

Fig. 4-2 Photographs of colonies and zooids of ciliate epibionts, Epistylis anastatica, attached
to Cyclopoida spp. in Lake Biwa; a–g, and i (live specimen), h (silver nitrate staining specimen).
A contractile dichotomously branched stalk with a smooth surface (a), shape of colonies
attached to the metasome and urosome of cyclopoid copepods (b–d), zooids with characteristicfeatures, i.e., peristomial lip, peristomial collar, shape of peristomial disc, contractile vacuole,and its position (e–g), transversely striated pellicle (h), and c-shaped macronucleus (i).

3.1 Morphological features of the epibionts
3.1.1 Flagellates
Solitary or colonial non-motile green vegetative cells without flagellum attached to thesurface of the outer shell of the cyclopoid body by a mucilaginous dichotomously branchedstalk. Cells often grouped together, forming amorphous colonies. Vegetative cells were ovoid
or spindle to somewhat pyriform, having paramylon bodies, and 13.86−23.28 m long and79

6.00−9.98 m width on average. Cell apices obtuse-shaped. It was difficult to observechloroplast and pyrenoid in the cell body due to excess food reserved (Rosowski and Kugrens1973) (Table 4-1, Fig. 4-1). According to the morphological features of this flagellate withdescriptions by Huber-Pestalozzi (1955), Rosowski and Kugrens (1973), and Zalocar et al.
(2011), this epizoic flagellate should be identified Colacium vesiculosum (Ehrenberg 1833).
Table 4-1. Morphological features of flagellate epibiont infested Cyclopoida spp. in Lake
Biwa.
Identifying
features

Morphological features of Colacium sp. in thisstudy

Colacium vesciculosum
described by Ehrenberg (1833)

Vegetative cell

Solitary or colonial

Solitary or colonial

Shape of cell
body

Ovoid to spindle, cell apices obtuse

Ovoid to spindle

Length of cell13.86–23.28 µm long (mean = 18.612.50, n=34)
 6–9.98 µm wide (mean = 8.071.01, n=34)

Sessile cell 15-21 and 11-14
wide

Stalk

Stalk formed and dichotomously branched

Gelatinous stalk forming
dichotomously branching colony

Paramylon
bodiesPresent

Present

Chloroplastand pyrenoid

Not observed

Chloroplast saucer-shaped with apyrenoid, 5 to 10 in number

FlagellaAbsentSessile cell without flagella3.1.2 Ciliates
Acontractile stalk was dichotomously branched with a smooth surface, and thebranching pattern of the colony is umbellate. The height of the colony ranged from 241.6−482.0
m, and the stalk were 41.1−113.1 m long and 6.5−13.0 m wide. Extended zooids wereelongated and bell-shaped, 45.8−79.2 m long and 18.8−39.2 m wide. Pellicle was striatedtransversely with a well-defined peristomial lip and peristomial collar as wide as the zooid’s
body Peristomial disc was slightly convex, and a single contractile vacuole was located at theventral wall of the infundibulum underneath the peristomial collar. C-shaped macronucleus
was longitudinally oriented. Infundibular polykinety, including oral ciliature, was not studied

80

well in this study (Table 4-2, Fig. 4-2). According to the morphological features of this ciliate
with descriptions by Foissner et al. (1999) and Lu et al. 2020, this peritrich ciliate might beidentified as Epistylis anastatica (Linnaeus 1767, Ehrenberg 1830).
Table 4-2. Morphological features of ciliate epibiont infested Cyclopoida spp. in Lake Biwa.
Characteristics

Morphological featuresof ciliate in this study

Epistilis anastatica
Ehrenburg (1830)

References other thanEhrenburg (1830)

Size of zooids (invivo)

45.79–79.19 µm long(mean = 56.937.40, n=40)
18.75–39.3 µm wide
(mean = 24.204.99, n=40)

60–100 µm  20–35µm

Foissner et al. 1999,
Lu et al. 2020

Shape of zooids

Conical or elongate bellshaped, pellicletransversely striatedSlightly conical,
elongate bell-shaped,
pellicle with transverse
striaeFoissner et al. 1999, Lu
et al. 2020

Colonyshape/height

Umbellate, 241.62–482µm (mean = 347.9165.43,
n=25)

Umbellate, usually<500 µm

Foissner et al. 1999, Lu
et al. 2020

Stalk

Dichotomously branched,acontractile, and smooth

Dichotomously
branched, acontractile,and smooth

Foissner et al. 1999

Macronucleus

C-shaped andlongitudinally oriented

longitudinally oriented,
slightly curved to Cshaped, upper partlying under peristome
and lower part curved

Foissner et al. 1999

Contractile vacuole

Slightly underneath theperistomal collar

Slightly underneath theperistomal collar, at theventral wall of thevestibulum

Foissner et al. 1999

Peristomal collar

Narrow and about as wide
as body

Narrow and about aswide as body

Foissner et al. 1999

Peristomal disc

Slightly convex

Slightly convex

Foissner et al. 1999

3.2 Seasonal changes in temperature, dissolved oxygen, and chlorophyll a concentration
Lake water temperature increased from late April at the surface, and the thermocline
developed at 10–20 m from June to October every year during the study period (Fig. 4-3). DO
saturations were >100% due to photosynthesis above 10 m throughout the study period but

81decreased to <80% from June to September, probably because of decomposition of sinking
organic matters originated by phytoplankton bloom in June. Chl-a concentration increased
above 20 m from June to July and October to November in 2018 to 2020. Another increase
throughout the water column in March and a weak increase in October in 2021. No such large
seasonality was found in 2022.

Fig. 4-3 Vertical profiles of water temperatures (a) dissolved oxygen (DO) saturation (b), andChlorophyll a concentration (c) from January 2018 to December 2019 at site K4 and fromMarch 2020 to December 2022 at JWA bouy in the North basin of Lake Biwa.
Average water temperature above 30 m increased from May onwards, peaked in Augustand remained almost the same until September, and then gradually decreased toward the next
March (Fig. 4-4). On the contrary, average DO saturation above 30 m gradually decreased82during the stratification period to September and then increased up to 100% by the next May
every year. Average chlorophyll-a concentration above 30 m increased in June and October in
2018–2021, while no such increase was found in 2022.

Fig. 4-4 Seasonal changes in total infestation rate (infestation prevalence) of epibionts andaverage water temperature (Temp), dissolved oxygen (DO) saturation, and chlorophyll a (Chla) concentrations above 30 m at a pelagic site in the north basin of Lake Biwa from 15 January
2018 to 21 December 2022.
3.3 Seasonal changes of host abundance and the prevalence of the epibionts
The epibionts occurred just in cyclopoid copepods from July to November in 2018 and2019, May to November in 2021, and July to December in 2020 and 2022. The infestation was
mostly found in the adult and copepodite stages but not in the naupliar stage, except for July
2022, when the nauplii infected by flagellates was found. Total infestation rates gradually
increased from June or July every year (Fig. 4-5). The maximum rates were usually found in
October or November and increased year to year from 18% in 2018 to 71% in 2022, thendeclined toward December. The total number of cyclopoid copepods varied from
October/November to December with a mean value of 3.7 to 1.8 ind. L-1 with clear seasonality

83

(Fig. 4-5). The timing of the increase was different among the year; mostly from June except
for 2020 and 2022, when it was September. Infested cyclopoids increased from July almost
every year except for 2020 when they increased from September. During the study period, nosuch infection was detected in other crustacean zooplankton taxa, including the most dominantcalanoid copepod E. japonicus and Daphnia spp.

Fig. 4-5 Seasonal distributions in abundance of total Cyclopoida spp. (adult and copepodites)
and Cyclopoida spp. infested by both ciliates and flagellates at a pelagic site in the north basinof Lake Biwa from 15 January 2018 to 21 December 2022.

Infestation rates of the flagellate C. vesiculosum alone increased year to year, from
20% in 2018 to 53% in 2022, while those of ciliate E. anastatica alone showed no such yearto-year variation. Seasonally, the highest infestation rate of the flagellates was found in July at
61% on average among the five years and the lowest at 2.5% in December, while the highest
infestation rate of the ciliates alone was found in November at 46%, and the lowest in June.
Co-infestation, i.e., infested by both flagellates and ciliates, was found throughout the study
period with no seasonality and varied from 5.19 to 10.34 on the annual mean (Fig. 4-6).

84

Fig. 4-6 Compositions in co-infestation (both flagellates and ciliates), flagellates alone, andciliates alone infestation for Cyclopoid spp. at a pelagic site in the north basin of Lake Biwa
from 15 January 2018 to 21 December 2022.

In 2022, vertical distributions of the dominant zooplankton species and epibiontsinfested cyclopoids were investigated. All zooplankton species including infested cyclopoids
were mostly distributed above 20 m in August and September, expanded to 25 m in Octoberand 30 m in November, depending on the thermocline (Fig. 4-7).
Total infestation rate was positively correlated with cyclopoid abundance and averagewater temperature above 30 m while negatively correlated with average DO saturation (Fig. 48). There was no such relationship between the infestation rate and average chlorophyll aconcentration above 30 m.

85

Fig. 4-7 Vertical distributions of dominant zooplankton, Eodiaptomus japonicus, Daphnia spp.,
Cyclopoida spp., and infected cyclopoids at a station (3518.672′ N, 13608.593′ E) near the
JWA buoy in the north basin of Lake Biwa on 24 August, 14 September, 17 October and 15
November 2022.

86Fig. 4-8 Scatter plots showing the relationship between infestation prevalence (%) and hostabundance (a), temperature (b), dissolved oxygen (%) (c), and chlorophyll-a (d). For
temperature, dissolved oxygen, and chlorophyll-a, an average value (from 0 to 30 m depth)
was used.
4. Discussion
This is the first report on epibiosis for crustacean zooplankton in Lake Biwa.
Identification of the epibionts based on morphological features might play a crucial role instudying the interaction of the organisms and their ecological aspects. Since the oral ciliatureof the identified species of E. anastatica and the number of chloroplasts with or without
pyrenoid in the C. vesiculosum cell have not been appropriately studied, an integrated approachby combining these morphological features with DNA sequencing is recommended.

87

The epibiont flagellate C. vesiculosum and ciliate E. anastatica strictly attached tocyclopoid copepods even when other zooplankton species were co-existent throughout thewater column. Several previous studies reported that species of Epistylis and Colacium showed
specificity for some species of zooplankton hosts for colonization (De Souza Santos et al. 2020,
Corre et al. 2020, Gilbert and Schroder 2003, Utz and Coats 2005, Xie et al. 2001). According
to Clamp et al. (2016), only Mesocyclops isabellae was infested with Epistylis anastatica
despite the coexistences of calanoid Neodiaptomus lindbergi and Heliodiaptomus viduus in an
Indian pond. Lu et al. (2020) also reported that E. anastatica exclusively infested cyclopoid
copepods in two freshwater ponds in Qingdao, China. Jones et al. (2016) observed that Epistylis
sp. attached to Pseudodiaptomus stuhlmanni but not to Acartiella natalensis and Oithonabrevicornis in St. Lucia Estuary, South Africa. Cabral et al. (2017) found that each of the twodifferent species of Epistylis was specific to Notodiaptomus henseni and Thermocyclops
minutus in a river floodplain in Brazil.
It can be assumed that the hydrodynamic disturbance created by continuous swimmingor feeding currents of suspension feeder E. japonicus and Daphnia spp. may trigger an escaping
response in E. anastatica and C. vesiculosum epibionts roaming in the ambient water(Threlkeld et al. 1993). Vavra (1963) mentioned that characteristics of swimming movements
by hosts are related to the survival of Epistylis spp. The dominant zooplankton taxa in thepresent study, E. japonicus, D. pulicaria, and Cyclopoida spp., have distinct swimming
characteristics compared to each other. D. pulicaria moves in a well-known characteristic“hop-and-sink” style ( Uttieri et al. 2014, CO'Keefe et al. 1998). Hwang and Turner (1995)
found that Cyclopoida spp. moved in a jerking pattern with frequent rest, while calanoid
Temora turbinata swam continuously without rest. Additionally, Drenner and McComas(1980) demonstrated that diaptomid copepod moves in a zigzag or erratic motion. It is crucial
to examine the distinct movement characteristics of these three zooplankters in Lake Biwa to88

understand why cyclopoids are the only hosts preferred by both epibionts for adherence andcolonization.
There are many previous reports on photosynthetic C. vesiculosum colonizing bodysurface of both copepods and daphnids (Møhlenberg and Kaas 1990, Chiavelli et al. 1993,
Zalocar et al. 2011, Cabral et al. 2014). In this study, C. vesiculosum is also attached tocyclopoids. For the photosynthetic flagellate like C. vesiculosum, attachment to otherorganisms may benefit from its mobile hosts, which may allow the epibionts to accessdissolved nutrients and the photic environment (Threlkeld et al. 1993). On the other hand,motile cells released from both sessile epibionts can be good food sources even for the host
cyclopoids (Barea-Arco et al. 2001).
C. vesiculosum attached to cyclopoids dominated in the epilimnetic waters of Lake
Biwa, implying that warm water with higher solar irradiation during summer to late autumn
may provide a favorable environment for the attached alga (Salmaso and Tolotti 2009). A highabundance of cyclopoid hosts during the growing seasons for the alga might increase adhesion
sites available for new colonization (Genc and Bozkurt 2009), consequently increasing
infestation prevalence. In this study, though the cyclopoid hosts were distributed even below
the euphotic zone, C. vesiculosum was also attached there. It can be related to the trophic stateof the genus Colacium, which are facultative heterotroph and can grow in dark hypolimnion
using dissolved organic carbon (DOC) (Salmaso and Tolotti 2009).
The ciliate epibiont Epistylis mainly feed on bacteria, pico- and nano-sized plankton, andparticulate organic matter (Kankaala and Eloranta 1987, Bickel et al. 2012), while flagellates
may take dissolved nutrient and DOC (Salmaso and Tolotti 2009). In this study, the low DO
concentrations in the mid-depth layer during the stagnation period might be associated withincreased microbial activities related to deposited organic materials from the phytoplankton

89

bloom. The stratified lake water in the early summer of every year may induce favorable
conditions in metalimnion for this epizoic ciliate and flagellate.

90Summary
Chapter 1
I report bacterial communities from bodies and feces of the three most abundant
zooplankton taxa (Eodiaptomus japonicus, Daphnia pulicaria, Cyclopoida spp.) from Lake
Biwa using meta-barcoding analysis targeting the prokaryote-specific V4 region of 16S rDNA.
A total of 293,018 sequences comprising 669 bacterial OTUs were generated, including threezooplankton bodies, of which 54 (D. pulicaria), 88 (E. japonicus), and 84 (Cyclopoida spp.)
OTUs were identified, and in their feces, 132, 156, and 155 OTUs, respectively. Among theidentified OTUs, 7 (D. pulicaria), 48 (E. japonicus), and 51 (Cyclopoida spp.) OTUs wereunique in the host bodies, and 13, 36, and 31 OTUs were in their feces, respectively. Bacteria
associated with the body and feces of E. japonicus were most divergent. The bacterial
compositions of the bodies and feces in all three host taxa differed significantly. Proteobacteria,
Bacteroidetes, Firmicutes, and Actinobacteria dominated the bacterial phyla in the zooplankton
bodies and feces. At lower taxonomic levels, the bacteria of bodies (particularly) and feces
differed among zooplankton taxa. Prevalent bacteria of D. pulicaria and Cyclopoida spp.
bodies were Aeromonadales (Gammaproteobacteria), and those of E. japonicus were
dominated by Burkholderiales (Betaproteobacteria). Aeromonadaceae was the most dominantbacterial family in zooplankton bodies, while Pelagibacteraceae was dominant in feces. The
bacterial communities present in the feces were similar among the taxa but differed from thosefound in the bodies of the animals themselves, implying that the feces contained transient
bacteria passing through the digestive system. The bacterial composition associated with thezooplankton bodies may be related to the host’s feeding habits, habitat selection, and behavior.

91

Chapter 2
In this chapter, the resident bacteria in the gut and body parts without gut (BP) of thethree dominant zooplankton with different feeding habits were identified, characterized,
and compared with bacterial compositions in their feces and ambient waters. To determine
dominant and stable symbiotic bacteria in the gut and other body parts, the gut wasdissected from the animals’ bodies. zooplankton and water samples were collected fromthe epilimnion (0–20 m) at a pelagic site in the north basin of Lake Biwa on 4 March 2021.
Symbiotic bacterial communities in the gut, body other than the gut, and feces differedfrom those in the ambient waters. The gut, body other than the gut, and feces of the threezooplankton contained different bacterial communities, which also differed among thethree species. The most dominant family in the gut and BP of D. pulicaria was
Comamonadaceae for each site, at 23% and 57%, in E. japonicus Neisseriaceae at 63%,
and Weeksellaceae at 28%, and in Cyclopoida spp. Aeromonadaceae at 13% andOxalobacteraceae at 47%, respectively. These results suggested that the differences in thesymbiotic bacterial communities in the gut and body may depend on feeding habits and thehost’s specific preferences and immune responses in the three taxa living in the same habitat.

Chapter 3

To analyze bacterial composition in zooplankton gut and body excluding gut, andambient waters, samples were collected from the two different depths of the water column,
from 20 m to the surface and from the 50 m to 20 m at the same pelagic site in the northbasin of Lake Biwa, on 17 June, and 21 September 2021. Vertical profiles of watertemperature, dissolved oxygen (DO), and chlorophyll a concentrations were recorded at
the same time. The lake water was vertically stagnated in June and September. Water
temperature, dissolved oxygen, and chlorophyll a concentration were very different

92between the two depths and months. The gut and body parts without gut (BP) of D.
pulicaria, E. japonicus, and Cyclopoida spp. inhabited 0−20 m contained higher microbial
diversity than their 20−50 m inhabiting counterpart. The symbiotic bacterial community inthe gut and body parts of each zooplankton taxa were significantly different between Juneand September, while different between habitats in D. pulicaria and not significantly
different in the two copepods. These results suggested that changes in the abiotic and biotic
factors of the ambient environment affect bacterial communities in the gut and body partsof the three zooplankton.

Chapter 4This report presents findings on the presence of epibiotic infestation on cyclopoid
copepods in Lake Biwa. From January 2018 to December 2022, zooplankton samples werecollected monthly at two pelagic sites in the north basin of Lake Biwa using a vertical net haul.
At the same time, temperature, dissolved oxygen (DO), and Chlorophyll-a were measured
throughout the water column. The epibionts identified through its gross morphological features
were the epizoic flagellate Colacium vesiculosum and Peritrich ciliate Epistylis anastatica,
which infested both adult and copepodites of Cyclopoida spp. To confirm the morphological
identification, a DNA base phylogenetic analysis is recommended. These two epibionts were
specific only to cyclopoid copepods, despite the presence of E. japonicus and Daphnia spp. in
the water layers. Three types of epibiotic infestation were observed: cyclopoids infested with
only flagellate, ciliate, or both. The epibiotic infestation was generally found from May to
December, with no infestation found from January to April. The infestation prevalence washighest from August to November of each year. A positive relationship was found between
infestation prevalence and temperature and host abundance, while dissolved oxygen showed anegative association, indicating a stable interaction between abiotic and biotic ecologicalprocesses that regulate this episymbiotic relationship in Lake Biwa.
93

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