for peer review - pure.knaw.nl · raaijmakers, jos; netherlands institute of ecology, microbial...

For Peer Review

http://mc.manuscriptcentral.com/fems

Exploring fish microbial communities to mitigate emerging

diseases in aquaculture

Journal: FEMS Microbiology Ecology

Manuscript ID FEMSEC-17-05-0231.R1

Manuscript Type: Minireview

Date Submitted by the Author: n/a

Complete List of Authors: de Bruijn, Irene; NIOO-KNAW, Department of Microbial Ecology Liu, Yiying; NIOO-KNAW, Department of Microbial Ecology Wiegertjes, Geert ; Wageningen Universiteit en Researchcentrum, Cell Biology and Immunology group, Department of Animal Sciences Raaijmakers, Jos; Netherlands Institute of Ecology, Microbial Ecology

Keywords: aquaculture, beneficial microbes, microbiomes, emerging diseases, fish

ScholarOne Support 1-434/964-4100

FEMS Microbiology Ecology

For Peer Review

Host immune response Protection against

pathogens

Factors that shape

microbiome

Nutrient acquisition

Host environment

Growth and development

Host genotype

Tolerance to abiotic stress

Functionality of microbiome

Fish microbiome

Page 1 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

1

Exploring fish microbial communities to mitigate 1

emerging diseases in aquaculture 2

3

Irene de Bruijn1^, Yiying Liu1^, Geert F. Wiegertjes2 and Jos M Raaijmakers1,3 4

1 Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands; 5

2 Cell Biology and Immunology group, Department of Animal Sciences, Wageningen University & Research, Wageningen, The Netherlands 6

3 Institute of Biology (IBL), Leiden University, Leiden, The Netherlands 7

8

^Shared first authors 9

Correspondence: I. de Bruijn, Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), 6708PB Wageningen, The 10

Netherlands. E-Mail: [email protected] 11

12

Running title: fish microbiome 13

Keywords: aquaculture, fish, emerging diseases, microbiomes, beneficial microbes 14

15

Page 2 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

2

Abstract 16

Aquaculture is the fastest-growing animal food sector worldwide and expected to further increase to feed the growing human population. 17

However, existing and (re-)emerging diseases are hampering fish and shellfish cultivation and yield. For many diseases, vaccination protocols 18

are not in place and the excessive use of antibiotics and other chemicals is of substantial concern. A more sustainable disease control strategy 19

to protect fish and shellfish from (re-)emerging diseases could be achieved by introduction or augmentation of beneficial microbes. To establish 20

and maintain a ‘healthy’ fish microbiome, a fundamental understanding of the diversity and temporal-spatial dynamics of fish-associated 21

microbial communities and their impact on growth and health of their aquatic hosts is required. This review describes insights in the diversity 22

and functions of the fish bacterial communities elucidated with next-generation sequencing and discusses the potential of the microbes to 23

mitigate (re-)emerging diseases in aquaculture. 24

25

Page 3 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

3

Introduction 26

The United Nations Food and Agricultural Organization (FAO) predicts that the world’s food and feed supply needs to grow by 70% to support 27

the growing human population by 2050. Currently, aquaculture is the fastest-growing animal food sector worldwide (FAO, 2014). However, fish 28

and shellfish production is limited by several emerging diseases caused by viruses, bacteria, fungi, oomycetes, amoebas and other 29

ectoparasites. Bacterial fish diseases are typically addressed by antibiotics (Romero et al., 2012, Cabello et al., 2013), viral diseases by 30

vaccination (Evensen & Leong, 2013) and parasitic diseases by chemical treatment (Burridge et al., 2010). However, the risk of antibiotic 31

resistance development and the transfer of antibiotic resistance genes to other animal pathogens, as well as concerns for environmental 32

impact and consumer safety, have stimulated the need for new sustainable control measures (Brandt et al., 2015). Vaccination is a sustainable 33

control measure, but various viral and bacterial fish diseases, and more specifically fungal and oomycete diseases, cannot yet be controlled by 34

vaccination (Evensen & Leong, 2013, Dhar et al., 2014, Hølvold et al., 2014). In addition, the oral delivery of efficacious and safe vaccines 35

remains a challenge, partly due to antigen breakdown of oral vaccines in the harsh gastric environment, the high tolerogenic gut environment 36

and inadequate vaccine design (reviewed in Embregts & Forlenza, 2016). Additional strategies for sustainable disease control include breeding 37

for disease resistance (Gjedrem, 2012), the use of plant extracts as prebiotics (Akhter et al., 2015, Van Hai, 2015) and augmentation or 38

introduction of beneficial microorganisms. 39

Beneficial microbes introduced into the aquaculture environment, also referred to as probiotics, are considered a highly promising and 40

sustainable strategy to mitigate fish diseases. To date, a range of potential probiotics have been investigated for their application in 41

Page 4 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

4

aquaculture, including bacteria, bacteriophages, fungi and yeasts (Verschuere et al., 2000, Hai, 2015, Newaj-Fyzul & Austin, 2015, Banerjee & 42

Ray, 2017). Despite the large number of promising candidates, however, only a few bacterial probiotics, for example Bactocell® (containing P. 43

acidilactici) (Lallemand-Animal-Nutrition, Gatesoupe, 1999, EFSA-FEEDAP, 2012), Aquastar® (consisting of a mix of Lactobacillus reuteri, 44

Bacillus subtilis, Enterococcus faecium and Pediococcus acidilactici) (Biomin®) and Sanolife (containing Bacillus subtillis, Bacillus licheniformis 45

and Bacillus pumilus) (INVE Aquaculture), have been commercialized (Ibrahem, 2015) or licensed for aquaculture in Europe (EFSA-BIOHAZ, 46

2017). The lack of consistency in performance and persistence under different environmental conditions, their relatively narrow activity 47

spectrum as well as current constraints for commercial application of probiotics, such as strict regulations for registration, shelf life, high 48

production costs, and highly specific storage and transport conditions of the microbial inoculants, are major concerns to the industry (Martínez 49

Cruz et al., 2012, Bentzon‐Tilia et al., 2016, Giatsis et al., 2016). 50

51

Microbiomes 52

In this review the microbiome is defined as described by Joshua Lederberg: “the totality of microbes, their genomes and their interactions in a 53

particular environment”. There is increasing evidence that microbial consortia rather than single microbial species are associated with health 54

and disease (Gilbert et al., 2016). The impact of microbial consortia on early development and health of their eukaryotic hosts is gaining 55

increased attention, with new ‘omics’-based technologies allowing for in-depth characterisations of microbial communities and functions in 56

diverse ecosystems. Indeed, contituents within the human gut microbiomes can significantly drive or suppress disease development (Round et 57

Page 5 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

5

al., 2010, Everard et al., 2013, Gilbert et al., 2016), whereas environmental changes or infections can substantially influence the human gut 58

microbiome by causing blooms of microbes that otherwise are present at low abundance (Stecher et al., 2013). In terrestrial farmed animals, 59

sudden changes like the use of antibiotics or dietary changes can cause substantial shifts in the gut microbial composition leading to disease 60

susceptibility (Gresse et al., Cisek & Binek, 2014, Fecteau et al., 2016, Fouhse et al., 2016). Similar observations have been made for the gut 61

of insects, whose microbiomes can influence insect development by increasing nutrient uptake and stimulating immunity maturation (Weiss & 62

Aksoy, 2011). Also for plants, microbiota play an important role in the protection against biotic and abiotic stress factors (Berendsen et al., 63

2012, Vorholt, 2012, Bulgarelli et al., 2013, Philippot et al., 2013, Turner et al., 2013, Mendes & Raaijmakers, 2015). In line with these findings, 64

microbial communities of fish may harbour substantial potential to modulate health and disease. Due to the complex structure of microbial 65

communities, disentangling interactions and identifying keystone species for specific functions is enormously challenging, especially when 66

environmental influences on population dynamics and activities are taken into account. Microbiome research of humans and other eukaryotes 67

typically describes correlations between microbiome composition and diseases, but it remains a challenge which taxonomic or functional 68

changes in the microbiome are actually causal for disease development or disease protection (Gilbert et al., 2016, Raaijmakers & Mazzola, 69

2016). Hence, a systems-based understanding of the dynamics and functions of microbial communities for their fish and shellfish hosts is 70

needed to enable a more profound selection of beneficial (micro)organisms for augmentation of microbial communities to achieve more 71

sustainable disease control strategies in aquaculture. This review describes the current state-of-the-art of fish microbial community research 72

Page 6 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

6

using next generation sequencing and provides an outlook into the functional potential of fish-associated microbes to control (re-)emerging 73

diseases in aquaculture. 74

75

76

Page 7 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

7

Diversity of fish microbiota 77

Traditionally, microbes associated with aquatic animals and their environment, have been isolated on general and selective agar media. Since 78

only a proportion of the viable microbes in various aquatic environments are culturable (Amann et al., 1995), culture-independent methods 79

have been developed to assess the composition and functional potential of aquatic microbiota. Besides quantitative PCR, denaturing gradient 80

gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), terminal restriction fragment length polymorphism (T-RFLP), 81

clone library sequencing and other molecular techniques (reviewed in Ringø et al., 2016), more advanced ‘omics’ techniques, such as 16S/18S 82

rRNA gene and internal transcribed spacer (ITS) sequencing, metatranscriptomics and metagenomics are now rapidly advancing providing a 83

more in-depth insight in the composition and functions of microbiomes (Llewellyn et al., 2014, Romero et al., 2014, Ghanbari et al., 2015, 84

Merrifield & Rodiles, 2015, Colston & Jackson, 2016, Tarnecki et al., 2017). This review describes the elucidation of the diversity and functions 85

of the fish microbial communities by next generation sequencing methods (Supplementary Table 1), unless mentioned otherwise. 86

87

Microbial diversity of different fish tissues 88

The mucosal tissues, including the skin, olfactory system, gills and also the gut are in direct contact with the environment and thus are the first 89

contact points of the microbes with their host. Mucus covering these tissues can be considered as a primary guardian against external 90

environmental influences and consists of the high molecular weight glycoprotein mucin as well as other proteins, ions and lipids. The mucus 91

contains immune components like lectins, complement proteins, antimicrobial peptides, immunoglobulins, lysozymes and a variety of other 92

Page 8 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

8

enzymes, including proteases (Uribe et al., 2011, Brinchmann, 2016). The mucus provides a carbon source for commensal microbes that can 93

subsequently form a protective shield against invading pathogens (Hansen & Olafsen, 1999, Merrifield & Rodiles, 2015). 94

The mucus of the fish skin and gills generally contains more aerobic than anaerobic microbes (Merrifield & Rodiles, 2015). Although 95

difficult to estimate and compare, the fish skin typically harbours 102-104 bacteria per cm2, whereas the gills harbour 103-106 bacteria per gram 96

of tissue based on cultivation-based methods (Austin, 2006, Merrifield & Rodiles, 2015). The composition of the microbial communities of the 97

gills and skin are different; the protected niches of the gill lamellae contain more microbes that putitatively favor gas-exchange (Hansen & 98

Olafsen, 1999, Wang et al., 2010, Lowrey et al., 2015). For example, the gill microbiota of rainbow trout (Oncorhynchus mykiss) contains 99

mostly Proteobacteria and Bacteroidetes (Flectobacillus and Flavobacterium), while the skin contains more Actinobacteria and Firmicutes 100

(Lowrey et al., 2015). A recent study showed that the gills of common carp (Cyprinus carpio) and zebrafish (Danio rerio) contained ammonia 101

oxidising and denitrifying bacteria, such as Nitrosomas-like bacteria, that are thought to play an important role in detoxifying the excreted 102

ammonia (van Kessel et al., 2016). Another organ of interest in this context is the fish nose: in contrast to mammals, the olfactory system of fish 103

is an external organ not connected to the mouth and mostly consists of a diffuse lymphoid system (Tacchi et al., 2015). The bacterial 104

communities in the olfactory system of rainbow trout as determined by 16S rRNA sequencing, appear highly diverse but relatively comparable 105

to the skin microbiome (containing approximately 100 genera), with Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes as the major 106

phyla (Lowrey et al., 2015). Derived from cultivation-based methods, the fish gut generally houses up to 108 aerobic heterotrophic bacteria 107

represented by approximately 500 species and up to 105 anaerobic bacteria per gram of gut tissue (Austin, 2006, Montalban-Arques et al., 108

Page 9 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

9

2015). For most fish species, the most abundant phyla found in fish guts are typically Proteobacteria, Actinobacteria, Bacteroidetes and 109

Firmicutes (Sullam et al., 2012, Givens et al., 2015, Merrifield & Rodiles, 2015, Tran et al., 2017, Wang et al., 2017a). Lowrey et al (2015) 110

showed that even though the gut of the rainbow trout contains members of the major bacterial phyla, the microbial community gut is much less 111

diverse than that of the skin or gills and harbours mainly Tenericutes (Lowrey et al., 2015). Organs like kidney, brain, liver and muscle are 112

considered to be sterile in healthy individuals, but only few studies have explored this in fish in detail. 113

Furthermore, the diversity and functions of the fungal fish microbiota remain largely understudied. Based on PCR-DGGE, Wang et al. 114

(2010) reported the Ascomycota as the most dominant fungal phylum of the gills of gibel carp (Carassius auratus gibelio) and bluntnose black 115

bream (Megalobrama amblycephala Yih). Based on cultivation-based methods, the population density of yeasts ranged up to 107 CFU per 116

gram of gut content and the two most reported fungal phyla were the Ascomycota and Basidiomycota (Gatesoupe, 2007, Romero et al., 2014). 117

To date, only few studies have described Archaea or protozoa in the fish gut. The guts of grass carp (Ctenopharyngodon idellus) mainly 118

contained methanogenic archaea (Wu et al., 2015), while marine flounder (Platichthys flesus) and grey mullet (Mugil cephalus) consisted 119

mainly group II marine archaea based on clone library sequencing (van der Maarel et al., 1998). Ciliates belonging to the order of 120

Clevelandellida and Vestibuliferida were identified in the guts of several marine surgeonfish (Acanthurus achilles, A. gluttatus, Zebrasoma 121

rostsratum, Z. velifenim by microscopy (Grim et al., 2002). Protozoa have shown to be common in the gill when studied by microscopy 122

(Valtonen et al., 2003, Yokoyama et al., 2015), including the pathogenic species like Neoparamoeba perurans causing amoebic gill disease 123

and Ichthyophthirius multifiliis causing the white spot disease. 124

Page 10 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

10

125

Microbial community changes during early development 126

Most fish species lay eggs that are fertilized externally in the aquatic ecosystem. The fish egg surface consists of a thick inner layer (zona 127

radiata) and a thin outer layer (chorion or zona pellucida) that varies between fish species. The permeable eggshell is composed of fibrin-like 128

molecules and glycoproteins, including lectins to which microbes can adhere and colonize the eggs (Hansen & Olafsen, 1999, Olafsen, 2001, 129

Treasurer et al., 2005, Wedekind et al., 2010). In this context, salmonid eggs have been best studied, likely because of the economic 130

importance of Atlantic salmon (Salmo salar) for aquaculture. Population densities of bacteria on fish eggs can range from 103-106 CFU g-1, with 131

Aeromonas, Alteromonas, Arthrobacter, Flavobacterium, Moraxella, Pseudomonas and Streptomyces as the major bacterial genera (Hansen & 132

Olafsen, 1999, Liu et al., 2014). It is very likely that part of the fish egg microbiota was obtained via vertical transmission from the mother, as 133

was shown for the pathogenic bacteria Renibacterium salmoninarum (Bruno & Munro, 1986) and Flavobacterium psychrophilum (Long et al., 134

2014). Not only the surface, but also the internal tissue of eggs of freshwater and marine fish may contain bacteria (Hansen & Olafsen, 1999). 135

The major fungal and oomycete genera reported for Atlantic salmon eggs are Microdochium, Mortierella, Chytriomyces and Saprolegnia (Liu et 136

al., 2014). 137

Only few studies to date have investigated temporal changes in microbiome composition during egg development. For whitefish 138

(Coregenus spp.), the density of bacteria decreased from early (blastoderm visible) to late (late-eyed) stages of egg development with 139

Burkholderiales and Fusobacteria representing the largest groups in the early versus the late stage, respectively (Wilkins et al., 2015a). A large 140

Page 11 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

11

proportion of the 16S rRNA reads obtained with 454 sequencing did not have a match in the database using the stringent quality control (i.e. at 141

least 60% of the reads should have a match with a 16S rRNA gene in the database), suggesting that many unknown microbial species reside 142

on the fish eggs (Wilkins et al., 2015a). The largest group of microbiota on the surface of eggs of lake sturgeon (Acipenser fulvescens) shifted 143

from Betaproteobacteria, to Bacteriodetes to Alphaproteobacteria during early, middle and late development prior to hatching, respectively 144

(Fujimoto et al., 2013). Already before the yolksac is consumed, fish larvae start to ‘drink’ water and the gut becomes colonized by 145

microorganisms from the water. At later developmental stages, larvae are exposed to other microbiota, for example by ingestion of egg debris 146

or by microorganisms present in live and artificial feed (Hansen & Olafsen, 1999, Ringo & Birkbeck, 1999). For example, a shift occurred in 147

microbiota when grass carp larvae started ingesting; in earlier egg and non-feeding egg larval stages the Proteobacteria was the largest group 148

but upon ingestion 80% of the identified bacteria belonged to the Bacteriodetes (Wang et al., 2015). Thus, these initial studies suggest that 149

profound changes can already occur in the microbiome during early development of the fish. 150

Zebrafish is an important model species for research and well suited to study the dynamic changes of the microbiota during 151

development. A recent study showed that the density and diversity of the gut microbial community decreased with age, whereas the relative 152

abundance (eveness) of the different bacterial classes did not change with age (Stephens et al., 2016). Moreover, the microbial community 153

composition of the early life stages was more similar to the ambient water than adult fish and in the adult stages, there was more variation in 154

the gut community between individual fish. The relative abundance of the Fusobacteria (mainly genus Cetobacterium) was increased in the 155

adult gut compared to the juvenile stages (30% vs <1%, respectively) (Stephens et al., 2016). Supporting the observations in salmonids and in 156

Page 12 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

12

zebrafish, also the microbial diversity in grass carp, Chinese perch and Chinese catfish (Ctenopharyngodon idellus, Siniperca chuatsi, Silurus 157

meridionalis, respectively) decreased from larval to adult stages and showed no similarity to the ambient water microbiota (Yan et al., 2016). 158

Also in tilapia larvae, a significant change was observed in gut microbial communities during development (Giatsis et al., 2014). Because 159

larvae have incomplete digestive systems and have more diverse diets, including various planktonic organisms, Yan et al (2016) suggested 160

that they strongly rely on microbes for the digestion of feed. The diet is an important factor in colonization of the guts, as was shown for the 161

guts of rainbow trout larvae where an increase in bacterial abundance and diversity was observed upon first feeding and where Firmicutes 162

were most abundant in plant fed fish and Proteobacteria were most abundant in marine fed fish (Ingerslev et al., 2014b). In gilthead seabream 163

(Sparus aurata), Firmicutes and Bacteroidetes were more abundant in the late, Rotifer and Artemis fed larval stage compared to the early, non-164

feeding larvae (Califano et al., 2017). 165

166

Impact of host genotype on fish microbiota 167

There are over 30,000 fish species worldwide, living in diverse environments. Host genetics is known to be important in shaping the microbial 168

community of fish (reviewed in Romero et al., 2014, Ghanbari et al., 2015). For example, using clone library sequencing, the fish species was 169

the most discriminating factor, besides site and time of sampling, for describing the diversity of the skin microbial communities of six different 170

fish species (Mugil cephalus, Lutjanus campechanus, Cynoscion nebulosus, Cynoscion arenarius, Micropogonias undulatus, and Lagodon 171

rhomboides) that were captured in different seasons in the Gulf of Mexico (Larsen et al., 2013). 172

Page 13 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

13

The importance of the host genetics in shaping the microbiome was demonstrated by Roeselers et al (2011) who identified a core 173

microbiota in the guts of laboratory-reared and wild zebrafish. A core microbiota was also observed on the surface of brown trout eggs reared 174

in different rivers (Wilkins et al., 2015b) and in the guts of rainbow trout kept in either aquarium or at a fish farm (Lyons et al., 2017c). The core 175

microbiota often comprises a small number of operational taxonomic units (OTUs), but which are highly abundant. For example, the core gut 176

microbiota of laboratory-reared and wild zebrafish comprised 21 OTUs of mainly Proteobacteria (Roeselers et al., 2011). Also the core 177

microbiota of three species of laboratory-reared or wild carp (Hypophthalmichthys nobilis, Hypophthalmichthys molitrix, Cyprinus carpio) 178

comprised only 5 OTUs classified to the orders Aeromonadales, Xanthomonadales, and Fusobacteriales but made up 35-40% of the total fecal 179

microbiome (Eichmiller et al., 2016). The core microbiota of the guts of aquaculture-reared and wild fine flounders (Paralichtys adspersus) 180

shared classes like Gammaproteobacteria, Alphaproteobacteria, Bacilli, Clostridia, and Actinobacteria and were present in at least 80% of the 181

samples (Ramírez & Romero, 2017a). The shared bacterial community between aquaculture-reared or laboratory-reared and wild fish suggest 182

that the host genetics plays a role in shaping the fish gut microbial community. In contrast, besides a core community, also a clear difference in 183

composition was observed in the guts of aquaculture-reared and wild fine flounders where the Proteobacteria was the most abundant phylum 184

in the wild species whereas the Firmicutes were the most abundant phylum in the aquaculture-reared species (Ramírez & Romero, 2017a). 185

Significant differences in egg-associated microbial composition and diversity were observed between individual maternal and paternal fish 186

(Wilkins et al., 2016), but the underlying mechanisms of how host genetics influences microbial community structure or whether genetic 187

information is transferred from microbiome to host are not yet understood. 188

Page 14 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

14

189

Effect of environmental conditions on the fish microbiota 190

Despite the importance of host genetics and diet, the composition of the gut microbiota is also determined by the microbiota present in 191

the ambient water and sediment (Sullam et al., 2012, Wu et al., 2012, Xing et al., 2013, Xu et al., 2013, Ghanbari et al., 2015, Kashinskaya et 192

al., 2015). The gut microbiota of Atlantic salmon reared in a recirculated aquarium facility and an open freshwater loch cage system in the West 193

of Scotland shared 19 out of 1850 OTUs, indicating that the environment significantly contributes to assembly of the gut microbiome (Dehler et 194

al., 2017). Due to environmental fluctuations, the ambient water conditions like temperature and nutrient levels change and affect microbiome 195

composition (Zarkasi et al., 2014, Bentzon‐Tilia et al., 2016, Zarkasi et al., 2016). The microbial community composition and densities of natural 196

waters are influenced by the seasons, as was shown for the bacterioplankton in the Western English Channel where annual day length was the 197

strongest determining factor (Gilbert et al., 2012). Also other studies showed seasonal effects on aquatic microbiota. For example, a higher 198

microbial diversity was observed in a Portuguese marine aquaculture system in the warmer season (Pereira et al., 2011) and variations in 199

Vibrio populations were correlated to the season and plankton composition (Turner et al., 2009). Potential mechanisms underlying seasonal 200

effects on fish microbiota are diverse. One of the seasonal factors involved is temperature. For example, the difference between the bacterial 201

community of brown trout eggs collected at different spawning locations in rivers in Switzerland were correlated with increasing temperature 202

and not with river location or with small genetic variations within the brown trout populations (Wilkins et al., 2015b). When investigating the 203

Page 15 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

15

Pseudomonas and Vibrio isolates from the Atlantic salmon gut, specific lineages were found only at colder temperatures during the seasons 204

(Hatje et al., 2014). 205

A second environmental factor determining the fish microbiome composition is water chemistry. To simulate the effect of acid rain and 206

subsequent acid stress, the Amazonian tambaqui (Colossoma macropomum) was exposed to acidic water (pH 4) for two weeks and 207

substantial shifts were observed in the gut and skin bacterial community, where the gut bacterial community appears more resilient (Sylvain et 208

al., 2016). Also salinity is an important factor; freshwater and seawater contain different microbes which have a major impact on the community 209

composition of the fish. This is best studied in salmon where the adults live in the sea and migrate to freshwater to spawn. During development, 210

the juvenile fish undergo a physiological process called smoltification and adapt from freshwater to their adult life in seawater. Upon transfer 211

from freshwater to seawater under controlled laboratory conditions, the skin microbiota of the mucus of salmon showed substantial differences 212

in relative frequencies of the bacterial taxa (decrease in bacterial eveness) and a higher diversity in seawater (Lokesh & Kiron, 2016). 213

Proteobacteria were abundant on the skin of fish living in both freshwater and seawater, but the Bacteroidetes, Actinobacteria, Firmicutes, 214

Cyanobacteria and Verrucomicrobia phyla became less abundant when salmon was transferred to seawater (Lokesh & Kiron, 2016). 215

Interestingly, after 4 weeks in seawater, only 23 out of 952 OTUs were in common with the freshwater community (Lokesh & Kiron, 2016), 216

indicating that the transition from freshwater to seawater has major and rapid effects on the skin microbiota. Also the total amount of taxa in the 217

intestinal microbiota from an adult marine salmon was significantly affected, with less Bacteroidetes, Firmicutes and Actinobacteria compared 218

to freshwater adults or other developmental stages (parr and smolt) (Llewellyn et al., 2016). Even though the Proteobacteria remained 219

Page 16 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

16

abundant, shifts in composition occurred with increasing salinity. In entire Black Molly (Poecilia sphenops) fish, the Gammaproteobacteria (in 220

particular Vibrio and Aeromonas) were detected at higher relative abundance at higher salinity levels (Schmidt et al., 2015). Vibrionales were 221

also more abundant in the guts of marine fish, while Aeromonodales and Enterobacteriales were more abundant in freshwater fish (Sullam et 222

al., 2012). Collectively, these studies indicate that water salinity has major effects on the skin and gut micriobiota. 223

224

Effect of feeding strategy on the fish microbiota 225

Diet can have a major impact on the fish gut microbiota. In a recent study with 15 different fish species, a more varied diet, i.e. omnivorous 226

versus piscivorous, resulted in a higher bacterial diversity in the guts (Givens et al., 2015). The gut microbiota of different wild fish species 227

caught from the same river showed different community between omnivorous, herbivorous, carnivorous and filter-feeding fish. Fusobacteria 228

were more abundant in carnivorous fish species, while Cyanobacteria were more abundant in filter-feeders (Liu et al., 2016). In another study, 229

Fusobacteria were more abundant in the guts of omnivorous fish compared to carnivorous and herbivorous fish, Bacteroidetes were more 230

abundant in marine herbivorous fish and Firmicutes were more abundant in carnivorous fish compared to the other feeding strategies (Sullam 231

et al., 2012). The microbial gut communities of salmon or rainbow trout fed with either fish meal or fish meal free diets resulted in shifts in 232

specific bacterial genera such as a higher abundance of Lactobacillus and Staphylococcus in a grain-based diet (Wong et al., 2013, Schmidt et 233

al., 2016). The microbial gene composition in the guts of rye-grass fed grass carp showed a higher abundance of genes involved in the 234

biosynthesis and metabolism of carbohydrates, lipids and amino acids compared to fish fed on diets containing fish meal (Ni et al., 2014). 235

Page 17 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

17

These results support the findings that also the type of feed, including lipid and fatty acid content (reviewed in Ringø et al., 2016), is an 236

important factor in shaping the gut microbial community. 237

238

Effects of the microbiota on fish health 239

Pathogenic microorganisms are an integral component of fish microbiomes, but despite their presence they often do not cause disease. 240

Similar to mammals and plants, pathogens can become more prevalent and cause infection and disease, a process referred to as dysbiosis, 241

when the fish commensal microbial community balance is disturbed (Sagvik et al., 2008, Stecher et al., 2013, Turner et al., 2013, Romero et 242

al., 2014, Montalban-Arques et al., 2015, Moya & Ferrer, 2016). For example, in the guts of healthy largemouth bronze gudgeon (Coreius 243

guichenoti) pathogenic Aeromonas species were present, but in higher abundance in furunculosis diseased fish (Li et al., 2016), indicating that 244

in healthy individuals the virulence and the prevalence of pathogens are suppressed. A similar phenomen was observed in the guts of 245

seahorse (Hippocampus spp.) by PCR-DGGE where pathogenic Vibrio spp were suppressed in healthy individuals (Li et al., 2015a). In 246

general, the microbial population is less diverse in diseased fish compared to healthy fish. This was also observed in the guts of “red-247

operculum” diseased crucian carp, and in addition, Cetobacterium, Cyanobacterium, and Clostridiaceae OTUs were more abundant in healthy 248

fish whereas Aeromonas, Vibrio and Shewanella OTUs were more abundant in diseased individuals (Li et al, 2017). 249

The imbalances in the protective commensal microbial community can be induced by changes in the environment, including water 250

conditions, temperature, seasonal changes, climate change, antibiotic usage or changes in rearing conditions. Another disturbance could be 251

Page 18 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

18

infection by a primary pathogen that causes a microbial imbalance, and which allows a secondary pathogen to invade. This was shown in a 252

large study where bacterial community shifts occurred in the skin mucus during salmon lice infection in Atlantic salmon post-smolts and a 253

higher abundance of genera containing potential secondary bacterial pathogens (Pseudomonas, Flavobacterium) was observed in fish with 254

high lice infestation (Llewellyn et al., 2017). A similar phenomenon was observed in Atlantic salmon eggs in which the Saprolegnia-infected 255

eggs showed higher abundance of potential bacterial fish pathogens (Vibrio, Aeromonas) (Liu et al., 2014). 256

Next to host factors involved in protection against pathogens, commensal microbes play important functions that contribute to host 257

health and protection against pathogens (Merrifield & Rodiles, 2015, Parra et al., 2015, Salinas, 2015, Xu et al., 2016). These functions include 258

direct protective effects against pathogens via antibiosis, competition for resources or niche exclusion, but also indirect effects by stimulating 259

the host immune response and nutrient uptake thereby increasing fish health (Figure 1). 260

261

Direct effects of microbiota on pathogens 262

Commensal microbes may prevent pathogen infection by several mechanisms, including 1) niche exclusion, as was reported for several 263

probiotic bacteria which colonized mucosal tissues and occupied infection sites; 2) competition for essential resources, including nutrients and 264

iron; and 3) antibiosis (Verschuere et al., 2000, Ibrahem, 2015, Banerjee & Ray, 2017). Iron is essential for growth and microorganisms acquire 265

iron by producing iron-chelating compounds (siderophores). Commensal microbes can compete with pathogens via siderophores with a higher 266

affinity for iron (Ahmed & Holmström, 2014) or by cross-feeding on the siderophores produced by the pathogen (Raaijmakers et al., 1995). 267

Page 19 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

19

Next to niche exclusion and competition, commensal microorganisms can antagonize pathogens via the production of antimicrobial 268

compounds, also referred to as antibiosis. Various studies have shown that several members of the fish gut microbiome produce compounds 269

that inhibit the growth of pathogens (Sugita et al., 1997, Ringø et al., 2000, Liu et al., 2010, Caruffo et al., 2015). For example, the cell-free 270

supernatant of more than 70% of the lactic acid isolates obtained from the gut of Atlantic salmon had the ability to inhibit growth of the fish 271

pathogen Aeromonas salmonicida (Ringø & Holzapfel, 2000). In addition, metagenomic analyses of the seabass gut microbiome showed an 272

increased proportion of genes encoding antimicrobial compounds, like beta-lactamases, upon stress caused by starvation (Xia et al., 2014). 273

Several skin bacterial isolates of rainbow trout, for example Arthrobacter, Psychrobacter and Pseudococcus isolates, inhibited the growth of 274

Saprolegnia australis and Mucor hiemalis in in vitro dual culture assays, or when testing cell-free supernatants (Lowrey et al., 2015, Salinas, 275

2015). Also, Frondihabitans and Pseudomonas species protected Atlantic salmon eggs from Saprolegnia diclina infection, most likely by 276

competitive niche exclusion and the production of lipopeptide surfactants with zoosporidical and growth-inhibitory activities (Liu et al., 2014, Liu 277

et al., 2015). Collectively, these studies indicate that direct effects of commensal members of the microbiome contribute to the protection of fish 278

against pathogens. 279

280

Indirect effects of microbiota on pathogens: modulation of host immune responses 281

Fish possess an immune system similar to other vertebrates combining innate (natural) and acquired (adaptive) immunity. However, the 282

immune system of fish also has several differences compared to the mammalian immune system, and even has differences between fish 283

Page 20 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

species. For example, in fish, bone marrow and lymph nodes are not present and the major immune organs are the thymus, the head kidney 284

(bone marrow equivalent) and spleen (Secombes & Wang, 2012). In general, innate immunity does not require prior exposure to be effective 285

and cells of the innate immune system (neutrophils, macrophages, natural killer cells amongst others) can thus respond immediately. Fish are 286

considered to rely relatively strongly on innate immunity, especially at lower temperatures (Magnadottir, 2006). Adaptive immunity, in contrast, 287

is mediated largely by lymphocytes and is characterised by memory and therefore is slower in its response. This is not any different for the fish 288

immune system (Zhu et al., 2013). Innate immune cells of fish, in particular phagocytic cell types such as neutrophils and macrophages, which 289

are similar to innate immune cells of other vertebrates, can express several different receptors for recognition of a variety of patterns expressed 290

by bacterial, viral and fungal pathogens (microbial-associated molecular patterns (MAMPs)). These patterns can include, among others, 291

microbe-derived surface molecules such as lipoproteins, lipopolysaccharides, peptidoglycan, and bacterial flagellin, but can also include 292

microbe-derived double-stranded RNA or unmethylated CpG motifs in bacterial DNA (Pietretti & Wiegertjes, 2014). In general, phagocytic 293

receptors will mediate ingestion, whereas sensing receptors mediate recognition; the complete process is targeted to destroy phagocytosed 294

microbes (Neumann et al., 2001). Subsequently, antigen-presenting cells, including macrophages, present fragments of the ingested 295

pathogens to T lymphocytes which subsequently secrete cytokines to direct protective immunity, which is similar to warm-blooded vertebrates. 296

Both T and B lymphocytes typically employ receptors based on rearranged genes, building unique and highly specific T- and B-cell receptors 297

on lymphocytes (Magadan et al., 2015). With the appropriate cytokine help, B lymphocytes differentiate into plasma cells, secreting large 298

Page 21 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

quantities of specific immunoglobulins (Ig, or antibodies). Both T and B lymphocytes can differentiate into memory cells, leading to highly 299

specific secondary immune responses. 300

Commensal microbes play an important role in shaping the immune response against pathogens. Several yeasts have been shown to 301

stimulate the immune response, growth, development and metabolism of the fish hosts due to cell wall components like β-glucans and chitin 302

(Gatesoupe, 2007, Petit & Wiegertjes, 2016). As in humans (Nutsch & Hsieh, 2012), the immune system is able to distinguish between 303

commensals and pathogens in fish, by activating specific pathways and cytokines upon pathogen attack (Gomez et al., 2013, Kelly & Salinas, 304

2017). It has been proposed that Igs are important for the immune system to differentiate between commensal microbiota and pathogens, 305

since the majority of the microbes in mucosal tissues of fish have been found coated with host Igs (Zhang et al., 2010, Xu et al., 2013). The 306

microbiota are coated with different Ig isotypes, depending on the exact mucosal tissue (Gomez et al., 2013, Tacchi et al., 2014), suggesting 307

that mucosal tissues in fish may have their own specific adaptive immunity (Salinas, 2015). Upon colonization of zebrafish larvae, commensal 308

bacteria were shown to elicit a mild inflammatory reaction, induce genes involved in priming of the innate immunity (Galindo-Villegas et al., 309

2012, Chu & Mazmanian, 2013), and activate the innate immune response (Nayak, 2010, Maynard et al., 2012, Montalban-Arques et al., 310

2015). The zebrafish gut microbiota, although different from the mammalian microbiota in terms of dominant phyla, induces a conserved host 311

response during colonization and development and the composition of the gut microbiota clearly affects the inflammatory response (reviewed 312

in Brugman et al., 2009). With regard to the very early stages of fish development, fish eggs lack adaptive immunity and have to rely on 313

maternally transferred Ig and components of the complement system or rely on innate immune factors of their own, such as lectins and 314

Page 22 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

22

lysozyme (Hansen & Olafsen, 1999, Huttenhuis et al., 2006, Løvoll et al., 2006). Therefore, it is expected that the microbiome of fish eggs have 315

a more direct effect in the defence against pathogens, which was shown for the pathogen Saprolegnia on salmon eggs (Liu et al., 2014). 316

317

Indirect effects of microbiota on fish health: modulation of nutrient uptake 318

Commensal gut microbes also aid the fish in nutrient acquisition (Nayak, 2010, Montalban-Arques et al., 2015, Borrelli et al., 2016). Germ-free, 319

gnotobiotic zebrafish have a reduced number of goblet and enteroendocrine cells in the gut, resulting in larvae defective in the uptake of 320

protein macromolecules (Bates et al., 2006). As a consequence, germ-free zebrafish had a lower body weight, lived shorter and exhibited 321

anxiety-like behaviour (Montalban-Arques et al., 2015). The gut microbes can produce exogenous enzymes to facilitate the digestion of food 322

and degradation of large and complex molecules, such as chitin, protein and starch (Austin, 2006, Nayak, 2010, Ray et al., 2012, Merrifield & 323

Rodiles, 2015, Montalban-Arques et al., 2015). The presence of short chain fatty acids (SFCA), which is implicated to be important for gut 324

functioning and immune stimulation in both mammalian and fish gut, indicate fermentation of sugars by the gut microbes, mainly lactic acid 325

bacteria (Montalban-Arques et al., 2015). In a study combining 16S rRNA gene sequencing and metabolomics, the gut microbial and 326

metabolome profiles of five species of carp significantly correlated and showed for example that lactic and phosphoric acid could be correlated 327

with a higher Clostridium content (Li et al., 2017b). The gut microbes can also produce vitamins and eicosapentaenoic acid (EPA, an omega-3 328

fatty acid that is essential for metabolism) to enhance the health of the host (Austin, 2006). Protozoa have been detected microscopically in the 329

fish gut of surgeonfish (Naso tonganus) that contain food vacuoles ingesting bacteria and flagellates (Grim, 2006) and are postulated as 330

Page 23 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

23

potential contributors to the fish gut composition and nutrient uptake, but more research is needed to determine their functions for the hosts 331

(Romero et al., 2014). The gut community of rainbow trout contained more Firmicutes in fish with a plant-fed diet compared to a high 332

abundance of Bacteriodetes, Proteobacteria and Actinobacteria in the guts of fish with a marine-fed diet. When challenged with pathogenic 333

Yersinia ruckeri, the survival was not affected by diet type, however, a higher abundance was observed of the genus Yersinia in the guts of the 334

marine-fed fish, as well as higher IL1β and Mbl2 gene transcription (Ingerslev et al., 2014a). This suggests that the type of diet influences the 335

microbial population and could potentially promote the microbial subpopulation providing protection against pathogens. 336

337

Outlook 338

Fish tissues are home to diverse microbial communities whose composition is influenced by ambient water quality (i.e. pH, oxygen levels, 339

salinity), environmental factors (i.e. season, temperature), host age, host genotype, feeding strategy and rearing conditions (feed, stress, 340

antibiotics usage) (Figure 1). Similar to the human microbiome, a high percentage of variation in microbiome composition is observed between 341

individual fish belonging to the same species (Bolnick et al., 2014b, Givens et al., 2015, Schmidt et al., 2015). For example, the relative 342

abundance of the Proteobacteria ranged from 1% in one individual up to 98% of another individual in stickleback (Bolnick et al., 2014b). Even 343

under controlled laboratory conditions, it can be difficult to control the composition of microbial populations between replicate tanks (Giatsis et 344

al., 2014, Giatsis et al., 2016, Schmidt et al., 2016). This high variation between and within studies complicates the identification of keystone 345

microbial species involved in fish disease protection. 346

Page 24 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

24

Comparing the microbiota of healthy and diseased fish belonging to the same species that are in the same developmental stage and 347

are kept under the same conditions is one of the approaches to identify microbiota and mechanisms involved in disease protection. Only few 348

microbiome studies comparing healthy and diseased fish have been described (Table 1) and therefore it is difficult to get a comprehensive 349

picture which microbes or microbial consortia are most promising for disease protection. Moreover, classic microbiological techniques, 350

including isolations and functional characterisation of single microbial isolates or consortia, will be required to validate the potential functions of 351

the microbiota in fish health and to test their efficacy in aquaculture. 352

Microbes are diverse and several can perform the same function. Hence, it might not be that important who is there, but more what they 353

are doing (Louca et al., 2016, Moya & Ferrer, 2016). Therefore, the challenge is to study ‘function over phylogeny’ (Gibbons, 2017) by moving 354

away from only a taxonomic description of the microbiota. Instead, studying functional characteristics that are most important in the direct or 355

indirect protection of fish against pathogens should be investigated. This can be achieved by studying the frequency and diversity of 356

biosynthetic gene clusters, their transcription, and the in situ production of the corresponding proteins and metabolites (Franzosa et al., 2015). 357

Combining these technologies will minimize the over- or underrepresentation of certain bacterial families caused by DNA extraction methods 358

(Kashinskaya et al., 2017) and PCR-bias when only investigating rRNA amplicon sequencing (Chakravorty et al., 2007, Sinclair et al., 2015, 359

Stoddard et al., 2015). These approaches will provide a better insight in the disease ecology and microbial functional potential of the fish 360

microbiota and enable to search for keystone microbes, microbial consortia and traits involved in disease protection that can be either 361

Page 25 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

25

augmented in the indigenous microbial population or introduced as probiotics for sustainable protection against (re-)emerging fish diseases 362

(Bentzon‐Tilia et al., 2016, Stentiford et al., 2017). 363

The selection of probiotics is tedious and need to fulfil many requirements, including pH tolerance, colonization ability and efficacy 364

(Banerjee & Ray, 2017). Prebiotics are indigestible carbohydrates that stimulate specific beneficial microbes during hydrolysis in the gut, 365

stimulating fish health (Song et al., 2014, Akhter et al., 2015). Synbiotics (Gibson & Roberfroid, 1995) entail a combination of pre- and 366

probiotics where the prebiotic is included to stimulate the colonization and/or immune-stimulatory activity of the probiotic, and is currently 367

receiving increased attention (Ringø & Song, 2016, Huynh et al., 2017). The underlying mechanisms of the pre-, pro- or synbiotics will still need 368

clarification, but these approaches, in combination with the production shifts to recirculation aquaculture systems (RAS) to stabilise the 369

environmental conditions like temperature and salinity, will have a high potential towards more sustainable land-based forms of aquaculture. 370

Another approach is host-mediated microbiome engineering (Mueller & Sachs, 2015), which describes the steering of the microbiome towards 371

an increased host performance by artificial selection of the microbiomes over multiple generations. Nowadays, the use of probiotics focuses on 372

the application in fish feed, however, also external applications should be considered to boost the skin immune response against skin diseases 373

caused by fungi and other parasites. 374

Overall, it is evident that the fish microbiome has enormous potential for fish health, helping the host in its defence against pathogen 375

colonization and infection. A mechanistic understanding of how specific microbes or microbial consortia, including their metabolites, act against 376

Page 26 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

26

pathogens and trigger the fish immune response may provide practical means to engineer the indigenous fish microbiome for enhancing 377

disease control and fish health. 378

379

Acknowledgements 380

This work was financially supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 381

634429 ParaFishControl (Advanced Tools and Research Strategies for Parasite Control in European farmed fish) and the NWO Applied and 382

Engineering sciences (project 14224). This output reflects only the authors’ view and the European Union cannot be held responsible for any 383

use that may be made of the information contained therein. This manuscript is publication number xxxx of Netherlands Institute of Ecology 384

(NIOO-KNAW). All authors declare no conflict of interest. 385

386

References 387

Adeoye AA, Yomla R, Jaramillo-Torres A, Rodiles A, Merrifield DL & Davies SJ (2016) Combined effects of exogenous enzymes and probiotic 388

on Nile tilapia (Oreochromis niloticus) growth, intestinal morphology and microbiome. Aquaculture 463: 61-70. 389

Ahmed E & Holmström SJM (2014) Siderophores in environmental research: roles and applications. Microbial Biotechnology 7: 196-208. 390

Akhter N, Wu B, Memon AM & Mohsin M (2015) Probiotics and prebiotics associated with aquaculture: a review. Fish & shellfish immunology 391

45: 733-741. 392

Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. 393

Microbiological Reviews 59: 143-169. 394

Austin B (2006) The bacterial microflora of fish, revised. Scientific World Journal 6: 931-945. 395

Bakke I, Coward E, Andersen T & Vadstein O (2015) Selection in the host structures the microbiota associated with developing cod larvae 396

(Gadus morhua). Environmental Microbioogyl 17: 3914-3924. 397

Page 27 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

27

Baldo L, Riera JL, Tooming-Klunderud A, Albà MM & Salzburger W (2015) Gut microbiota dynamics during dietary shift in eastern African 398

cichlid fishes. PLOS ONE 10: e0127462. 399

Banerjee G & Ray AK (2017) The advancement of probiotics research and its application in fish farming industries. Research in veterinary 400

science 115: 66-77. 401

Bates JM, Mittge E, Kuhlman J, Baden KN, Cheesman SE & Guillemin K (2006) Distinct signals from the microbiota promote different aspects 402

of zebrafish gut differentiation. Developmental Biology 297: 374-386. 403

Bentzon‐Tilia M, Sonnenschein EC & Gram L (2016) Monitoring and managing microbes in aquaculture – Towards a sustainable industry. 404

Microbial Biotechnology 9: 576-584. 405

Berendsen RL, Pieterse CMJ & Bakker PAHM (2012) The rhizosphere microbiome and plant health. Trends in Plant Science 17: 478-486. 406

Biomin® http://www.biomin.net/en/products/aquastar/. p.^pp. 407

Bolnick DI, Snowberg LK, Caporaso JG, Lauber C, Knight R & Stutz WE (2014a) Major Histocompatibility Complex class IIb polymorphism 408

influences gut microbiota composition and diversity. Molecular Ecology 23: 4831-4845. 409

Bolnick DI, Snowberg LK, Hirsch PE, Lauber CL, Org E, Parks B, Lusis AJ, Knight R, Caporaso JG & Svanbäck R (2014b) Individual diet has 410

sex-dependent effects on vertebrate gut microbiota. Nature Communications 5: 4500. 411

Borrelli L, Aceto S, Agnisola C, De Paolo S, Dipineto L, Stilling RM, Dinan TG, Cryan JF, Menna LF & Fioretti A (2016) Probiotic modulation of 412

the microbiota-gut-brain axis and behaviour in zebrafish. Scientific reports 6: 30046. 413

Brandt KK, Amezquita A, Backhaus T, et al. (2015) Ecotoxicological assessment of antibiotics: A call for improved consideration of 414

microorganisms. Environment International 85: 189-205. 415

Brinchmann MF (2016) Immune relevant molecules identified in the skin mucus of fish using -omics technologies. Molecular bioSystems 12: 416

2056-2063. 417

Brugman S, Liu KY, Lindenbergh-Kortleve D, Samsom JN, Furuta GT, Renshaw SA, Willemsen R & Nieuwenhuis EE (2009) Oxazolone-418

induced enterocolitis in zebrafish depends on the composition of the intestinal microbiota. Gastroenterology 137: 1757-1767.e1751. 419

Bruno DW & Munro ALS (1986) Observations on Renibacterium salmoninarum and the salmonid egg. Diseases of Aquatic Organisms 1: 83-420

87. 421

Bulgarelli D, Schlaeppi K, Spaepen S, Ver Loren van Themaat E & Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota 422

of plants. Annual Review of Plant Biology 64: 807-838. 423

Burridge L, Weis JS, Cabello F, Pizarro J & Bostick K (2010) Chemical use in salmon aquaculture: A review of current practices and possible 424

environmental effects. Aquaculture 306: 7-23. 425

Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dolz H, Millanao A & Buschmann AH (2013) Antimicrobial use in aquaculture re-examined: its 426

relevance to antimicrobial resistance and to animal and human health. Environmental Microbiology 15: 1917-1942. 427

Califano G, Castanho S, Soares F, Ribeiro L, Cox CJ, Mata L & Costa R (2017) Molecular taxonomic profiling of bacterial communities in a 428

gilthead seabream (Sparus aurata) hatchery. Frontiers in Microbiology 8. 429

Page 28 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

28

Carda-Dieguez M, Mira A & Fouz B (2014) Pyrosequencing survey of intestinal microbiota diversity in cultured sea bass (Dicentrarchus labrax) 430

fed functional diets. FEMS Microbiolology Ecology 87: 451-459. 431

Carlson JM, Hyde ER, Petrosino JF, Manage ABW & Primm TP (2015) The host effects of Gambusia affinis with an antibiotic-disrupted 432

microbiome. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 178: 163-168. 433

Caruffo M, Navarrete N, Salgado O, Díaz A, López P, García K, Feijóo CG & Navarrete P (2015) Potential probiotic yeasts isolated from the 434

fish gut protect zebrafish (Danio rerio) from a Vibrio anguillarum challenge. Frontiers in Microbiology 6: 1093. 435

Chakravorty S, Helb D, Burday M, Connell N & Alland D (2007) A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of 436

pathogenic bacteria. Journal of microbiological methods 69: 330-339. 437

Chu H & Mazmanian SK (2013) Innate immune recognition of the microbiota promotes host-microbial symbiosis. Nature immunology 14: 668-438

675. 439

Cisek AA & Binek M (2014) Chicken intestinal microbiota function with a special emphasis on the role of probiotic bacteria. Polish journal of 440

veterinary sciences 17: 385-394. 441

Colston TJ & Jackson CR (2016) Microbiome evolution along divergent branches of the vertebrate tree of life: what is known and unknown. 442

Molecular Ecology 25: 3776-3800. 443

Davis DJ, Bryda EC, Gillespie CH & Ericsson AC (2016) Microbial modulation of behavior and stress responses in zebrafish larvae. 444

Behavioural brain research 311: 219-227. 445

Dehler CE, Secombes CJ & Martin SA (2017) Environmental and physiological factors shape the gut microbiota of Atlantic salmon parr (Salmo 446

salar L.). Aquaculture 467: 149-157. 447

Desai AR, Links MG, Collins SA, Mansfield GS, Drew MD, Van Kessel AG & Hill JE (2012) Effects of plant-based diets on the distal gut 448

microbiome of rainbow trout (Oncorhynchus mykiss). Aquaculture 350: 134-142. 449

Dhar AK, Manna SK & Thomas Allnutt FC (2014) Viral vaccines for farmed finfish. Virus Disease 25: 1-17. 450

Di Maiuta N, Schwarzentruber P, Schenker M & Schoelkopf J (2013) Microbial population dynamics in the faeces of wood-eating loricariid 451

catfishes. Letters in applied microbiology 56: 401-407. 452

EFSA-BIOHAZ (2017) Scientific Opinion on the update of the list of QPS-recommended biological agents intentionally added to food or feed as 453

notified to EFSA. EFSA Journal 15: 4664. 454

EFSA-FEEDAP (2012) Scientific Opinion on the efficacy of Bactocell (Pediococcus acidilactici) when used as a feed additive for fish. EFSA 455

Journal 10: 2286. 456

Eichmiller JJ, Hamilton MJ, Staley C, Sadowsky MJ & Sorensen PW (2016) Environment shapes the fecal microbiome of invasive carp 457

species. Microbiome 4: 44. 458

Embregts CW & Forlenza M (2016) Oral vaccination of fish: Successes, challenges and future perspectives. Developmental and Comparative 459

Immunology 64: 118-137. 460

Page 29 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

29

Estruch G, Collado MC, Peñaranda DS, Tomás Vidal A, Jover Cerdá M, Pérez Martínez G & Martinez-Llorens S (2015) Impact of fishmeal 461

replacement in diets for gilthead sea bream (Sparus aurata) on the gastrointestinal microbiota determined by pyrosequencing the 16S 462

rRNA gene. PLOS ONE 10: e0136389. 463

Etyemez M & Balcazar JL (2015) Bacterial community structure in the intestinal ecosystem of rainbow trout (Oncorhynchus mykiss) as 464

revealed by pyrosequencing-based analysis of 16S rRNA genes. Research in veterinary science 100: 8-11. 465

Evensen O & Leong J-AC (2013) DNA vaccines against viral diseases of farmed fish. Fish & shellfish immunology 35: 1751-1758. 466

Everard A, Belzer C, Geurts L, et al. (2013) Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced 467

obesity. Proceedings of the National Academy of Sciences of the United States of America 110: 9066-9071. 468

Fan L, Chen J, Meng S, Song C, Qiu L, Hu G & Xu P (2017) Characterization of microbial communities in intensive GIFT tilapia (Oreochromis 469

niloticus) pond systems during the peak period of breeding. Aquaculture Research 48: 459-472. 470

FAO (2014) The state of world fisheries and aquaculture 2014. Rome, Italy. 471

Fecteau M-E, Pitta DW, Vecchiarelli B, Indugu N, Kumar S, Gallagher SC, Fyock TL & Sweeney RW (2016) Dysbiosis of the fecal microbiota in 472

cattle infected with Mycobacterium avium subsp. paratuberculosis. PLOS ONE 11: e0160353. 473

Fouhse JM, Zijlstra RT & Willing BP (2016) The role of gut microbiota in the health and disease of pigs. Animal Frontiers 6: 30-36. 474

Franchini P, Fruciano C, Frickey T, Jones JC & Meyer A (2014) The gut microbial community of midas cichlid fish in repeatedly evolved 475

limnetic-benthic species pairs. PLOS ONE 9: e95027. 476

Franzosa EA, Hsu T, Sirota-Madi A, Shafquat A, Abu-Ali G, Morgan XC & Huttenhower C (2015) Sequencing and beyond: integrating 477

molecular 'omics' for microbial community profiling. Nature Reviews Microbiology 13: 360-372. 478

Fujimoto M, Crossman JA, Scribner KT & Marsh TL (2013) Microbial community assembly and succession on lake sturgeon egg surfaces as a 479

function of simulated spawning stream flow rate. Microbial Ecology 66: 500-511. 480

Gajardo K, Rodiles A, Kortner TM, Krogdahl Å, Bakke AM, Merrifield DL & Sørum H (2016) A high-resolution map of the gut microbiota in 481

Atlantic salmon (Salmo salar): A basis for comparative gut microbial research. Scientific reports 6: 30893. 482

Galindo-Villegas J, Garcia-Moreno D, de Oliveira S, Meseguer J & Mulero V (2012) Regulation of immunity and disease resistance by 483

commensal microbes and chromatin modifications during zebrafish development. Proceedings of the National Academy of Sciences of 484

the United States of America 109: E2605-E2614. 485

Gatesoupe FJ (1999) The use of probiotics in aquaculture. Aquaculture 180: 147-165. 486

Gatesoupe FJ (2007) Live yeasts in the gut: Natural occurrence, dietary introduction, and their effects on fish health and development. 487

Aquaculture 267: 20-30. 488

Gaulke CA, Barton CL, Proffitt S, Tanguay RL & Sharpton TJ (2016) Triclosan exposure is associated with rapid restructuring of the 489

microbiome in adult zebrafish. PLOS ONE 11: e0154632. 490

Page 30 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

30

Geraylou Z, Souffreau C, Rurangwa E, De Meester L, Courtin CM, Delcour JA, Buyse J & Ollevier F (2013a) Effects of dietary arabinoxylan-491

oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut microbiota of juvenile 492

Siberian sturgeon (Acipenser baerii). Fish & shellfish immunology 35: 766-775. 493

Geraylou Z, Souffreau C, Rurangwa E, Maes GE, Spanier KI, Courtin CM, Delcour JA, Buyse J & Ollevier F (2013b) Prebiotic effects of 494

arabinoxylan oligosaccharides on juvenile Siberian sturgeon (Acipenser baerii) with emphasis on the modulation of the gut microbiota 495

using 454 pyrosequencing. FEMS Microbiology Ecology 86: 357-371. 496

Ghanbari M, Kneifel W & Domig KJ (2015) A new view of the fish gut microbiome: Advances from next-generation sequencing. Aquaculture 497

448: 464-475. 498

Giatsis C, Sipkema D, Smidt H, Verreth J & Verdegem M (2014) The colonization dynamics of the gut microbiota in tilapia larvae. PLOS ONE 499

9: e103641. 500

Giatsis C, Sipkema D, Smidt H, Heilig H, Benvenuti G, Verreth J & Verdegem M (2015) The impact of rearing environment on the development 501

of gut microbiota in tilapia larvae. Scientific reports 5: 18206. 502

Giatsis C, Sipkema D, Ramiro-Garcia J, Bacanu GM, Abernathy J, Verreth J, Smidt H & Verdegem M (2016) Probiotic legacy effects on gut 503

microbial assembly in tilapia larvae. Scientific reports 6: 33965. 504

Gibbons SM (2017) Microbial community ecology: Function over phylogeny. Nature Ecology & Evolution, News and Views, http://rdcu.be/w3ky. 505

Gibson GR & Roberfroid MB (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. The Journal of 506

nutrition 125: 1401-1412. 507

Gilbert JA, Quinn RA, Debelius J, Xu ZZ, Morton J, Garg N, Jansson JK, Dorrestein PC & Knight R (2016) Microbiome-wide association studies 508

link dynamic microbial consortia to disease. Nature 535: 94-103. 509

Gilbert JA, Steele JA, Caporaso JG, et al. (2012) Defining seasonal marine microbial community dynamics. The ISME journal 6: 298-308. 510

Givens CE, Ransom B, Bano N & Hollibaugh JT (2015) Comparison of the gut microbiomes of 12 bony fish and 3 shark species. Marine 511

Ecology Progress Series 518: 209-223. 512

Gjedrem T (2012) Genetic improvement for the development of efficient global aquaculture: A personal opinion review. Aquaculture 344–349: 513

12-22. 514

Gomez D, Sunyer JO & Salinas I (2013) The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens. 515

Fish & shellfish immunology 35: 1729-1739. 516

Gresse R, Chaucheyras-Durand F, Fleury MA, Van de Wiele T, Forano E & Blanquet-Diot S Gut microbiota dysbiosis in postweaning piglets: 517

understanding the keys to health. Trends in Microbiology 25: 851-873. 518

Grim JN (2006) Food vacuole contents in the ciliate, Balantidium jocularum (Balantididae), a symbiont in the intestine of the surgeonfish, Naso 519

tonganus (Acanthuridae). The Journal of eukaryotic microbiology 53: 269-274. 520

Grim JN, Clements KD & Byfield T (2002) New species of Balantidium and Pamcichttdotherus (Ciliophora) inhabiting the intestines of four 521

surgeonfish species from the Tuvalu Islands, Pacific Ocean. Journal of Eukaryotic Microbiology 49: 146-153. 522

Page 31 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

31

Hai NV (2015) The use of probiotics in aquaculture. Journal of Applied Microbiology 119: 917-935. 523

Hansen GH & Olafsen JA (1999) Bacterial interactions in early life stages of marine cold water fish. Microbial Ecology 38: 1-26. 524

Hatje E, Neuman C, Stevenson H, Bowman JP & Katouli M (2014) Population dynamics of Vibrio and Pseudomonas species isolated from 525

farmed tasmanian Atlantic salmon (Salmo salar L.): a seasonal study. Microbial Ecology 68: 679-687. 526

Hennersdorf P, Mrotzek G, Abdul-Aziz MA & Saluz HP (2016a) Metagenomic analysis between free-living and cultured Epinephelus 527

fuscoguttatus under different environmental conditions in Indonesian waters. Marine pollution bulletin 110: 726-734. 528

Hennersdorf P, Kleinertz S, Theisen S, Abdul-Aziz MA, Mrotzek G, Palm HW & Saluz HP (2016b) Microbial diversity and parasitic load in 529

tropical fish of different environmental conditions. PLoS One 11: e0151594. 530

Hølvold LB, Myhr AI & Dalmo RA (2014) Strategies and hurdles using DNA vaccines to fish. Veterinary Research 45: 21-21. 531

Huttenhuis HBT, Grou CPO, Taverne-Thiele AJ, Taverne N & Rombout JHWM (2006) Carp (Cyprinus carpio L.) innate immune factors are 532

present before hatching. Fish & shellfish immunology 20: 586-596. 533

Huynh TG, Shiu YL, Nguyen TP, Truong QP, Chen JC & Liu CH (2017) Current applications, selection, and possible mechanisms of actions of 534

synbiotics in improving the growth and health status in aquaculture: a review. Fish & shellfish immunology 64: 367-382. 535

Ibrahem MD (2015) Evolution of probiotics in aquatic world: potential effects, the current status in Egypt and recent prospectives. Journal of 536

Advanced Research 6: 765-791. 537

Ingerslev HC, Strube ML, Jorgensen L, Dalsgaard I, Boye M & Madsen L (2014a) Diet type dictates the gut microbiota and the immune 538

response against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). Fish & shellfish immunology 40: 624-633. 539

Ingerslev HC, von Gersdorff Jørgensen L, Lenz Strube M, Larsen N, Dalsgaard I, Boye M & Madsen L (2014b) The development of the gut 540

microbiota in rainbow trout (Oncorhynchus mykiss) is affected by first feeding and diet type. Aquaculture 424–425: 24-34. 541

INVE Aquaculture http://www.inveaquaculture.com/our-solutions/health/. 542

Kashinskaya EN, Andree KB, Simonov EP & Solovyev MM (2017) DNA extraction protocols may influence biodiversity detected in the intestinal 543

microbiome: a case study from wild Prussian carp, Carassius gibelio. FEMS Microbiology Ecology 93: fiw240. 544

Kashinskaya EN, Belkova NL, Izvekova GI, Simonov EP, Andree KB, Glupov VV, Baturina OA, Kabilov MR & Solovyev MM (2015) A 545

comparative study on microbiota from the intestine of Prussian carp (Carassius gibelio) and their aquatic environmental compartments, 546

using different molecular methods. Journal of Applied Microbiology 119: 948-961. 547

Kelly C & Salinas I (2017) Under pressure: interactions between commensal microbiota and the teleost immune system. Frontiers in 548

Immunology 8: 559. 549

Kohl KD, Amaya J, Passement CA, Dearing MD & McCue MD (2014) Unique and shared responses of the gut microbiota to prolonged fasting: 550

a comparative study across five classes of vertebrate hosts. FEMS Microbiology Ecology 90: 883-894. 551

Kormas KA, Meziti A, Mente E & Frentzos A (2014) Dietary differences are reflected on the gut prokaryotic community structure of wild and 552

commercially reared sea bream (Sparus aurata). Microbiology Open 3: 718-728. 553

Lallemand-Animal-Nutrition http://lallemandanimalnutrition.com/products/bactocell-aquaculture/. 554

Page 32 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

32

Larsen A, Tao Z, Bullard SA & Arias CR (2013) Diversity of the skin microbiota of fishes: evidence for host species specificity. FEMS 555

Microbiology Ecology 85: 483-494. 556

Larsen AM, Mohammed HH & Arias CR (2014) Characterization of the gut microbiota of three commercially valuable warmwater fish species. 557

Journal of Applied Microbiology 116: 1396-1404. 558

Li F, Wang K, Luo W, Huang L & Lin Q (2015a) Comparison of the intestinal bacterial flora in healthy and intestinal-diseased seahorses 559

Hippocampus trimaculatus, Hippocampus erectus, and Hippocampus spinosissimus. Journal of the World Aquaculture Society 46: 263-560

272. 561

Li T, Long M, Gatesoupe FJ, Zhang Q, Li A & Gong X (2015b) Comparative analysis of the intestinal bacterial communities in different species 562

of carp by pyrosequencing. Microbial Ecology 69: 25-36. 563

Li T, Li H, Gatesoupe F-J, She R, Lin Q, Yan X, Li J & Li X (2017a) Bacterial signatures of “Red-Operculum” disease in the gut of crucian carp 564

(Carassius auratus). Microbial Ecology 74: 510-521. 565

Li T, Long M, Li H, Gatesoupe F-J, Zhang X, Zhang Q, Feng D & Li A (2017b) Multi-Omics analysis reveals a correlation between the host 566

phylogeny, gut microbiota and metabolite profiles in cyprinid fishes. Frontiers in Microbiology 8: 454. 567

Li T, Long M, Ji C, et al. (2016) Alterations of the gut microbiome of largemouth bronze gudgeon (Coreius guichenoti) suffering from 568

furunculosis. Scientific reports 6: 30606. 569

Li X, Yan Q, Xie S, Hu W, Yu Y & Hu Z (2013) Gut microbiota contributes to the growth of fast-growing transgenic common carp (Cyprinus 570

carpio L.). PLoS One 8: e64577. 571

Li XM, Zhu YJ, Yan QY, Ringo E & Yang DG (2014) Do the intestinal microbiotas differ between paddlefish (Polyodon spathala) and bighead 572

carp (Aristichthys nobilis) reared in the same pond? Journal of Applied Microbiology 117: 1245-1252. 573

Liu H, Guo X, Gooneratne R, Lai R, Zeng C, Zhan F & Wang W (2016) The gut microbiome and degradation enzyme activity of wild freshwater 574

fishes influenced by their trophic levels. Scientific reports 6: 24340. 575

Liu Y, De Schryver P, Van Delsen B, Maignien L, Boon N, Sorgeloos P, Verstraete W, Bossier P & Defoirdt T (2010) PHB-degrading bacteria 576

isolated from the gastrointestinal tract of aquatic animals as protective actors against luminescent vibriosis. FEMS Microbiology Ecology 577

74: 196-204. 578

Liu Y, Rzeszutek E, van der Voort M, Wu C-H, Thoen E, Skaar I, Bulone V, Dorrestein PC, Raaijmakers JM & de Bruijn I (2015) Diversity of 579

aquatic Pseudomonas species and their activity against the fish pathogenic oomycete Saprolegnia. PLOS ONE 10: e0136241. 580

Liu Y, de Bruijn I, Jack ALH, et al. (2014) Deciphering microbial landscapes of fish eggs to mitigate emerging diseases. The ISME journal 8: 581

2002-2014. 582

Llewellyn MS, Boutin S, Hoseinifar SH & Derome N (2014) Teleost microbiomes: the state of the art in their characterization, manipulation and 583

importance in aquaculture and fisheries. Frontiers in Microbiology 5: 207. 584

Llewellyn MS, McGinnity P, Dionne M, Letourneau J, Thonier F, Carvalho GR, Creer S & Derome N (2016) The biogeography of the atlantic 585

salmon (Salmo salar) gut microbiome. The ISME journal 10: 1280-1284. 586

Page 33 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

33

Llewellyn MS, Leadbeater S, Garcia C, et al. (2017) Parasitism perturbs the mucosal microbiome of Atlantic salmon. Scientific reports 7: 587

43465. 588

Lokesh J & Kiron V (2016) Transition from freshwater to seawater reshapes the skin-associated microbiota of Atlantic salmon. Scientific reports 589

6: 19707. 590

Long A, Call DR & Cain KD (2014) Investigation of the link between broodstock infection, vertical transmission, and prevalence of 591

Flavobacterium psychrophilum in eggs and progeny of Rainbow trout and Coho salmon. Journal of Aquatic Animals and Health 26: 66-592

77. 593

Louca S, Jacques SMS, Pires APF, Leal JS, Srivastava DS, Parfrey LW, Farjalla VF & Doebeli M (2016) High taxonomic variability despite 594

stable functional structure across microbial communities. Nature Ecology & Evolution 1: 0015. 595

Løvoll M, Kilvik T, Boshra H, Bøgwald J, Sunyer JO & Dalmo RA (2006) Maternal transfer of complement components C3-1, C3-3, C3-4, C4, 596

C5, C7, Bf, and Df to offspring in rainbow trout (Oncorhynchus mykiss). Immunogenetics 58: 168-179. 597

Lowrey L, Woodhams DC, Tacchi L & Salinas I (2015) Topographical mapping of the rainbow trout (Oncorhynchus mykiss) microbiome reveals 598

a diverse bacterial community with antifungal properties in the skin. Applied and Environmental Microbiology 81: 6915-6925. 599

Lyons PP, Turnbull JF, Dawson KA & Crumlish M (2017a) Effects of low-level dietary microalgae supplementation on the distal intestinal 600

microbiome of farmed rainbow trout Oncorhynchus mykiss (Walbaum). Aquaculture Research 48: 2438-2452. 601

Lyons PP, Turnbull JF, Dawson KA & Crumlish M (2017b) Exploring the microbial diversity of the distal intestinal lumen and mucosa of farmed 602

rainbow trout Oncorhynchus mykiss (Walbaum) using next generation sequencing (NGS). Aquaculture Research 48: 77-91. 603

Lyons PP, Turnbull JF, Dawson KA & Crumlish M (2017c) Phylogenetic and functional characterization of the distal intestinal microbiome of 604

rainbow trout Oncorhynchus mykiss from both farm and aquarium settings. Journal of Applied Microbiology 122: 347-363. 605

Magadan S, Sunyer OJ & Boudinot P (2015) Unique features of fish immune repertoires: particularities of adaptive immunity within the largest 606

group of vertebrates. Results and problems in cell differentiation 57: 235-264. 607

Magnadottir B (2006) Innate immunity of fish (overview). Fish & shellfish immunology 20: 137-151. 608

Martínez Cruz P, Ibáñez AL, Monroy Hermosillo OA & Ramírez Saad HC (2012) Use of probiotics in aquaculture. ISRN Microbiology 2012: 609

916845. 610

Maynard CL, Elson CO, Hatton RD & Weaver CT (2012) Reciprocal interactions of the intestinal microbiota and immune system. Nature 489: 611

231-241. 612

Mendes R & Raaijmakers JM (2015) Cross-kingdom similarities in microbiome functions. The ISME journal 9: 1905-1907. 613

Merrifield DL & Rodiles A (2015) The fish microbiome and its interactions with mucosal tissues. Mucosal Health in Aquaculture,(Peatman BHB, 614

ed.) Academic Press, San Diego. pp. 273-295. 615

Michl SC, Ratten J-M, Beyer M, Hasler M, LaRoche J & Schulz C (2017) The malleable gut microbiome of juvenile rainbow trout 616

(Oncorhynchus mykiss): Diet-dependent shifts of bacterial community structures. PLOS ONE 12: e0177735. 617

Miyake S, Ngugi DK & Stingl U (2015) Diet strongly influences the gut microbiota of surgeonfishes. Molecular Ecology 24: 656-672. 618

Page 34 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

34

Montalban-Arques A, De Schryver P, Bossier P, Gorkiewicz G, Mulero V, Gatlin III DM & Galindo-Villegas J (2015) Selective manipulation of 619

the gut microbiota improves immune status in vertebrates. Frontiers in Immunology 6. 620

Moya A & Ferrer M (2016) Functional redundancy-induced stability of gut microbiota subjected to disturbance. Trends in Microbiology 24: 402-621

413. 622

Mueller UG & Sachs JL (2015) Engineering microbiomes to improve plant and animal health. Trends in Microbiology 23: 606-617. 623

Narrowe AB, Albuthi-Lantz M, Smith EP, Bower KJ, Roane TM, Vajda AM & Miller CS (2015) Perturbation and restoration of the fathead 624

minnow gut microbiome after low-level triclosan exposure. Microbiome 3: 6. 625

Nayak SK (2010) Role of gastrointestinal microbiota in fish. Aquaculture Research 41: 1553-1573. 626

Neumann NF, Stafford JL, Barreda D, Ainsworth AJ & Belosevic M (2001) Antimicrobial mechanisms of fish phagocytes and their role in host 627

defense. Developmental and Comparitive Immunology 25: 807-825. 628

Newaj-Fyzul A & Austin B (2015) Probiotics, immunostimulants, plant products and oral vaccines, and their role as feed supplements in the 629

control of bacterial fish diseases. Journal of fish diseases 38: 937-955. 630

Ni J, Yan Q, Yu Y & Zhang T (2014) Factors influencing the grass carp gut microbiome and its effect on metabolism. FEMS Microbiology 631

Ecoogyl 87: 704-714. 632

Nutsch KM & Hsieh C-S (2012) T cell tolerance and immunity to commensal bacteria. Current opinion in immunology 24: 385-391. 633

Olafsen JA (2001) Interactions between fish larvae and bacteria in marine aquaculture. Aquaculture 200: 223-247. 634

Parra D, Reyes-Lopez FE & Tort L (2015) Mucosal immunity and B cells in teleosts: Effect of vaccination and stress. Frontiers in Immunology 635

6: 354. 636

Pereira C, Salvador S, Arrojado C, Silva Y, Santos AL, Cunha A, Gomes NC & Almeida A (2011) Evaluating seasonal dynamics of bacterial 637

communities in marine fish aquaculture: a preliminary study before applying phage therapy. Journal of Environmental Monitoring 13: 638

1053-1058. 639

Petit J & Wiegertjes GF (2016) Long-lived effects of administering beta-glucans: Indications for trained immunity in fish. Developmental and 640

Comparative Immunology 64: 93-102. 641

Philippot L, Raaijmakers JM, Lemanceau P & van der Putten WH (2013) Going back to the roots: the microbial ecology of the rhizosphere. 642

Nature Reviews Microbiology 11: 789-799. 643

Pietretti D & Wiegertjes GF (2014) Ligand specificities of Toll-like receptors in fish: indications from infection studies. Developmental and 644

Comparative Immunology 43: 205-222. 645

Raaijmakers JM & Mazzola M (2016) ECOLOGY. Soil immune responses. Science 352: 1392-1393. 646

Raaijmakers JM, van der Sluis I, Koster M, Bakker PAHM, Weisbeek PJ & Schippers B (1995) Utilization of heterologous siderophores and 647

rhizosphere competence of fluorescent Pseudomonas spp. Canadian Journal of Microbiology 41: 126-135. 648

Ramírez C & Romero J (2017a) Fine flounder (Paralichthys adspersus) microbiome showed important differences between wild and reared 649

specimens. Frontiers in Microbiology 8. 650

Page 35 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

35

Ramírez C & Romero J (2017b) The microbiome of Seriola lalandi of wild and aquaculture origin reveals differences in composition and 651

potential function. Frontiers in Microbiology 8: 1844. 652

Ran C, Huang L, Liu Z, Xu L, Yang Y, Tacon P, Auclair E & Zhou Z (2015) A comparison of the beneficial effects of live and heat-inactivated 653

baker's yeast on Nile tilapia: suggestions on the role and function of the secretory metabolites released from the yeast. PLOS One 10: 654

e0145448. 655

Ran C, Hu J, Liu W, Liu Z, He S, Dan BC, Diem NN, Ooi EL & Zhou Z (2016) Thymol and carvacrol affect hybrid tilapia through the 656

combination of direct stimulation and an intestinal microbiota-mediated effect: insights from a germ-free zebrafish model. The Journal of 657

nutrition 146: 1132-1140. 658

Ray AK, Ghosh K & Ringø E (2012) Enzyme-producing bacteria isolated from fish gut: a review. Aquaculture Nutrition 18: 465-492. 659

Ringo E & Birkbeck TH (1999) Intestinal microflora of fish larvae and fry. Aquaculture Research 30: 73-93. 660

Ringø E & Holzapfel W (2000) Identification and characterization of Carnobacteria associated with the gills of Atlantic salmon (Salmo salar L.). 661

Systematic and Applied Microbiology 23: 523-527. 662

Ringø E & Song SK (2016) Application of dietary supplements (synbiotics and probiotics in combination with plant products and β-glucans) in 663

aquaculture. Aquaculture Nutrition 22: 4-24. 664

Ringø E, Bendiksen HR, Wesmajervi MS, Olsen RE, Jansen PA & Mikkelsen H (2000) Lactic acid bacteria associated with the digestive tract of 665

Atlantic salmon (Salmo salar L.). Journal of Applied Microbiology 89: 317-322. 666

Ringø E, Zhou Z, Vecino JLG, et al. (2016) Effect of dietary components on the gut microbiota of aquatic animals. A never-ending story? 667

Aquaculture Nutrition 22: 219-282. 668

Roeselers G, Mittge EK, Stephens WZ, Parichy DM, Cavanaugh CM, Guillemin K & Rawls JF (2011) Evidence for a core gut microbiota in the 669

zebrafish. The ISME journal 5: 1595-1608. 670

Romero J, Feijoó CG & Navarrete P (2012) Antibiotics in aquaculture – use, abuse and alternatives. Health and environment in aquaculture. 671

(Carvalho E, ed.) InTech, pp. 159-198. 672

Romero J, Ringø E & Merrifield DL (2014) The gut microbiota of fish. Aquaculture Nutrition, pp. 75-100. John Wiley & Sons, Ltd. 673

Round JL, O'Connell RM & Mazmanian SK (2010) Coordination of tolerogenic immune responses by the commensal microbiota. J Autoimmun 674

34: J220-225. 675

Rurangwa E, Sipkema D, Kals J, Ter Veld M, Forlenza M, Bacanu GM, Smidt H & Palstra AP (2015) Impact of a novel protein meal on the 676

gastrointestinal microbiota and the host transcriptome of larval zebrafish Danio rerio. Frontiers in physiology 6: 133. 677

Sagvik J, Uller T & Olsson M (2008) A genetic component of resistance to fungal infection in frog embryos. Proc Biol Sci 275: 1393-1396. 678

Salinas I (2015) The mucosal immune system of teleost fish. Biology 4: 525. 679

Schmidt V, Amaral-Zettler L, Davidson J, Summerfelt S & Good C (2016) Influence of fishmeal-free diets on microbial communities in Atlantic 680

salmon (Salmo salar) recirculation aquaculture systems. Applied and Environmental Microbiology 82: 4470-4481. 681

Page 36 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

36

Schmidt VT, Smith KF, Melvin DW & Amaral-Zettler LA (2015) Community assembly of a euryhaline fish microbiome during salinity acclimation. 682

Molecular Ecology 24: 2537-2550. 683

Secombes CJ & Wang T (2012) The innate and adaptive immune system of fish. Infectious disease in aquaculture, (Austin B, ed.), pp. 3-48. 684

Woodhead Publishing Limited, Cambridge. 685

Semova I, Carten JD, Stombaugh J, Mackey LC, Knight R, Farber SA & Rawls JF (2012) Microbiota regulate intestinal absorption and 686

metabolism of fatty acids in the zebrafish. Cell Host Microbe 12: 277-288. 687

Sinclair L, Osman OA, Bertilsson S & Eiler A (2015) Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating 688

the Illumina platform. PLOS ONE 10: e0116955. 689

Smith CC, Snowberg LK, Gregory Caporaso J, Knight R & Bolnick DI (2015) Dietary input of microbes and host genetic variation shape 690

among-population differences in stickleback gut microbiota. The ISME journal 9: 2515-2526. 691

Song SK, Beck BR, Kim D, Park J, Kim J, Kim HD & Ringo E (2014) Prebiotics as immunostimulants in aquaculture: a review. Fish & shellfish 692

immunology 40: 40-48. 693

Standen BT, Rodiles A, Peggs DL, Davies SJ, Santos GA & Merrifield DL (2015) Modulation of the intestinal microbiota and morphology of 694

tilapia, Oreochromis niloticus, following the application of a multi-species probiotic. Applied Microbiology and Biotechnology 99: 8403-695

8417. 696

Star B, Haverkamp TH, Jentoft S & Jakobsen KS (2013) Next generation sequencing shows high variation of the intestinal microbial species 697

composition in Atlantic cod caught at a single location. BMC Microbiology 13: 248. 698

Stecher B, Maier L & Hardt W-D (2013) 'Blooming' in the gut: how dysbiosis might contribute to pathogen evolution. Nature Reviews 699

Microbiology 11: 277-284. 700

Stentiford GD, Sritunyalucksana K, Flegel TW, Williams BA, Withyachumnarnkul B, Itsathitphaisarn O & Bass D (2017) New paradigms to help 701

solve the global aquaculture disease crisis. PLOS pathogens 13: e1006160. 702

Stephens WZ, Burns AR, Stagaman K, Wong S, Rawls JF, Guillemin K & Bohannan BJM (2016) The composition of the zebrafish intestinal 703

microbial community varies across development. The ISME journal 10: 644-654. 704

Stoddard SF, Smith BJ, Hein R, Roller BRK & Schmidt TM (2015) rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and 705

archaea and a new foundation for future development. Nucleic Acids Research 43: D593-D598. 706

Sugita H, Matsuo N, Hirose Y, Iwato M & Deguchi Y (1997) Vibrio sp. strain NM 10, isolated from the intestine of a Japanese coastal fish, has 707

an inhibitory effect against Pasteurella piscicida. Applied and Environmental Microbiology 63: 4986-4989. 708

Sullam KE, Essinger SD, Lozupone CA, O'Connor MP, Rosen GL, Knight R, Kilham SS & Russell JA (2012) Environmental and ecological 709

factors that shape the gut bacterial communities of fish: a meta-analysis. Molecular Ecology 21: 3363-3378. 710

Sylvain F-É, Cheaib B, Llewellyn M, Gabriel Correia T, Barros Fagundes D, Luis Val A & Derome N (2016) pH drop impacts differentially skin 711

and gut microbiota of the Amazonian fish tambaqui (Colossoma macropomum). Scientific reports 6: 32032. 712

Page 37 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

37

Tacchi L, Larragoite Erin T, Muñoz P, Amemiya Chris T & Salinas I (2015) African lungfish reveal the evolutionary origins of organized mucosal 713

lymphoid tissue in vertebrates. Current Biology 25: 2417-2424. 714

Tacchi L, Musharrafieh R, Larragoite ET, Crossey K, Erhardt EB, Martin SAM, LaPatra SE & Salinas I (2014) Nasal immunity is an ancient arm 715

of the mucosal immune system of vertebrates. Nature communications 5: 5205-5205. 716

Tarnecki AM, Burgos FA, Ray CL & Arias CR (2017) Fish intestinal microbiome: diversity and symbiosis unravelled by metagenomics. Journal 717

of Applied Microbiology 123: 2-17. 718

Tran NT, Wang G-T & Wu S-G (2017) A review of intestinal microbes in grass carp Ctenopharyngodon idellus (Valenciennes). Aquaculture 719

Research 48: 3287-3297. 720

Treasurer JW, Cochrane E & Grant A (2005) Surface disinfection of cod Gadus morhua and haddock Melanogrammus aeglefinus eggs with 721

bronopol. Aquaculture 250: 27-35. 722

Turner JW, Good B, Cole D & Lipp EK (2009) Plankton composition and environmental factors contribute to Vibrio seasonality. The ISME 723

journal 3: 1082-1092. 724

Turner TR, James EK & Poole PS (2013) The plant microbiome. Genome Biology 14: 209. 725

Uribe C, Folch H, Enriquez R & Moran G (2011) Innate and adaptive immunity in teleost fish: a review. Veterinarni Medicina 56: 486-503. 726

Valtonen ET, Holmes JC, Aronen J & Rautalahti I (2003) Parasite communities as indicators of recovery from pollution: parasites of roach 727

(Rutilus rutilus) and perch (Perca fuviatilis) in central Finland. Parasitology 126 Suppl: S43-52. 728

van der Maarel MJEC, Artz RRE, Haanstra R & Forney LJ (1998) Association of marine archaea with the digestive tracts of two marine fish 729

species. Applied and Environmental Microbiology 64: 2894-2898. 730

Van Hai N (2015) The use of medicinal plants as immunostimulants in aquaculture: A review. Aquaculture 446: 88-96. 731

van Kessel MA, Dutilh BE, Neveling K, Kwint MP, Veltman JA, Flik G, Jetten MS, Klaren PH & Op den Camp HJ (2011) Pyrosequencing of 16S 732

rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.). AMB Express 1: 41. 733

van Kessel MAHJ, Mesman RJ, Arshad A, et al. (2016) Branchial nitrogen cycle symbionts can remove ammonia in fish gills. Environmental 734

Microbiology Reports 8: 590-594. 735

Verschuere L, Rombaut G, Sorgeloos P & Verstraete W (2000) Probiotic bacteria as biological control agents in aquaculture. Microbiology and 736

Molecular Biology Reviews 64: 655-671. 737

Vorholt JA (2012) Microbial life in the phyllosphere. Nature Reviews Microbiology 10: 828-840. 738

Wang AR, Ran C, Ringø E & Zhou ZG (2017a) Progress in fish gastrointestinal microbiota research. Reviews in Aquaculture. 739

doi:10.1111/raq.12191 740

Wang J, Tao Q, Wang Z, Mai K, Xu W, Zhang Y & Ai Q (2017b) Effects of fish meal replacement by soybean meal with supplementation of 741

functional compound additives on intestinal morphology and microbiome of Japanese seabass (Lateolabrax japonicus). Aquaculture 742

Research 48: 2186-2197. 743

Page 38 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

38

Wang W, Wu S, Zheng Y, Cheng Y, Li W, Zou H & Wang G (2015) Characterization of the bacterial community associated with early-744

developmental stages of grass carp (Ctenopharyngodon idella). Aquaculture Research 46: 2728-2735. 745

Wang W, Zhou Z, He S, Liu Y, Cao Y, Shi P, Yao B & Ringø E (2010) Identification of the adherent microbiota on the gills and skin of poly-746

cultured gibel carp (Carassius auratus gibelio) and bluntnose black bream (Megalobrama amblycephala Yih). Aquaculture Research 41: 747

e72-e83. 748

Wedekind C, Gessner MO, Vazquez F, Maerki M & Steiner D (2010) Elevated resource availability sufficient to turn opportunistic into virulent 749

fish pathogens. Ecology 91: 1251-1256. 750

Weiss B & Aksoy S (2011) Microbiome influences on insect host vector competence. Trends in Parasitology 27: 514-522. 751

Wilkins LGE, Fumagalli L & Wedekind C (2016) Effects of host genetics and environment on egg-associated microbiotas in brown trout (Salmo 752

trutta). Molecular Ecoogyl 25: 4930-4945. 753

Wilkins LGE, Rogivue A, Fumagalli L & Wedekind C (2015a) Declining diversity of egg-associated bacteria during development of naturally 754

spawned whitefish embryos (Coregonus spp.). Aquatic Sciences 77: 481-497. 755

Wilkins LGE, Rogivue A, Schutz F, Fumagalli L & Wedekind C (2015b) Increased diversity of egg-associated bacteria on brown trout (Salmo 756

trutta) at elevated temperatures. Scientific reports 5: 17084. 757

Wong S, Stephens WZ, Burns AR, Stagaman K, David LA, Bohannan BJM, Guillemin K & Rawls JF (2015) Ontogenetic differences in dietary 758

fat influence microbiota assembly in the zebrafish gut. mBio 6: e00687-00615. 759

Wong S, Waldrop T, Summerfelt S, et al. (2013) Aquacultured rainbow trout (Oncorhynchus mykiss) possess a large core intestinal microbiota 760

that is resistant to variation in diet and rearing density. Applied and Environmental Microbiology 79: 4974-4984. 761

Wu S, Wang G, Angert ER, Wang W, Li W & Zou H (2012) Composition, diversity, and origin of the bacterial community in grass carp intestine. 762

PLOS One 7: e30440. 763

Wu S, Ren Y, Peng C, Hao Y, Xiong F, Wang G, Li W, Zou H & Angert ER (2015) Metatranscriptomic discovery of plant biomass-degrading 764

capacity from grass carp intestinal microbiomes. FEMS Microbiology Ecology 91. 765

Wu S-G, Tian J-Y, Gatesoupe F-J, Li W-X, Zou H, Yang B-J & Wang G-T (2013) Intestinal microbiota of gibel carp (Carassius auratus gibelio) 766

and its origin as revealed by 454 pyrosequencing. World Journal of Microbiology and Biotechnology 29: 1585-1595. 767

Xia JH, Lin G, Fu GH, Wan ZY, Lee M, Wang L, Liu XJ & Yue GH (2014) The intestinal microbiome of fish under starvation. BMC Genomics 768

15. 769

Xing M, Hou Z, Yuan J, Liu Y, Qu Y & Liu B (2013) Taxonomic and functional metagenomic profiling of gastrointestinal tract microbiome of the 770

farmed adult turbot (Scophthalmus maximus). FEMS Microbiology Ecology 86: 432-443. 771

Xu Z, Takizawa F, Parra D, Gómez D, von Gersdorff Jørgensen L, LaPatra SE & Sunyer JO (2016) Mucosal immunoglobulins at respiratory 772

surfaces mark an ancient association that predates the emergence of tetrapods. Nature Communications 7: 10728. 773

Page 39 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

39

Xu Z, Parra D, Gomez D, Salinas I, Zhang Y-A, Jorgensen LvG, Heinecke RD, Buchmann K, LaPatra S & Sunyer JO (2013) Teleost skin, an 774

ancient mucosal surface that elicits gut-like immune responses. Proceedings of the National Academy of Sciences of the United States 775

of America 110: 13097-13102. 776

Yan Q, Li J, Yu Y, et al. (2016) Environmental filtering decreases with fish development for the assembly of gut microbiota. Environmental 777

Microbiology. 778

Ye L, Amberg J, Chapman D, Gaikowski M & Liu WT (2014) Fish gut microbiota analysis differentiates physiology and behavior of invasive 779

Asian carp and indigenous American fish. The ISME journal 8: 541-551. 780

Yokoyama H, Itoh N & Ogawa K (2015) Fish and shellfish diseases caused by marine protists. Springer, Tokyo. 781

Zarkasi KZ, Taylor RS, Abell GC, Tamplin ML, Glencross BD & Bowman JP (2016) Atlantic salmon (Salmo salar L.) gastrointestinal microbial 782

community dynamics in relation to digesta properties and diet. Microbial Ecology 71: 589-603. 783

Zarkasi KZ, Abell GCJ, Taylor RS, Neuman C, Hatje E, Tamplin ML, Katouli M & Bowman JP (2014) Pyrosequencing-based characterization of 784

gastrointestinal bacteria of Atlantic salmon (Salmo salar L.) within a commercial mariculture system. Journal of Applied Microbiology 785

117: 18-27. 786

Zhang M, Sun Y, Liu Y, Qiao F, Chen L, Liu W-T, Du Z & Li E (2016) Response of gut microbiota to salinity change in two euryhaline aquatic 787

animals with reverse salinity preference. Aquaculture 454: 72-80. 788

Zhang Y-A, Salinas I, Li J, Parra D, Bjork S, Xu Z, LaPatra SE, Bartholomew J & Sunyer JO (2010) IgT, a primitive immunoglobulin class 789

specialized in mucosal immunity. Nature Immunology 11: 827-835. 790

Zhu LY, Nie L, Zhu G, Xiang LX & Shao JZ (2013) Advances in research of fish immune-relevant genes: a comparative overview of innate and 791

adaptive immunity in teleosts. Developmental and Comparative Immunology 39: 39-62. 792

793

Page 40 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Table 1. Overview of studies comparing the fish microbiomes of healthy and diseased individuals.

Host Tissue Disease Technique Reference

Atlantic salmon (Salmo salar) eggs Saprolegniosis 16S rRNA - Phylochip Liu et al., 2014

skin Salmon lice 16S rRNA - Illumina MiSeq

Llewellyn et al., 2017

Largemouth bronze gudgeon (Coreius guichenoti)

gut Furunculosis 16S rRNA - Illumina MiSeq

Li et al., 2016

Seahorse (Hippocampus spp.) gut Intestinal disease 16S rRNA - DGGE Li et al., 2015 Crucian carp (Carassius auratus)

gut Red-Operculum 16S rRNA - Illumina MiSeq

Li et al., 2017

Rainbow trout (Oncorhynchus mykiss)

gut Yersinia 16S rRNA - Illumina HiSeq

Ingerslev et al., 2014a

Page 41 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure legends Figure 1. Schematic overview of different life-support functions of the fish microbiome and the impact of the host and ambient environment on the microbiome diversity, assembly and functions.

Page 42 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Host immune response Protection against

pathogens

Factors that shape

microbiome

Nutrient acquisition

Host environment

Growth and development

Host genotype

Tolerance to abiotic stress

Functionality of microbiome

Fish microbiome

Page 43 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Supplementary table 1. Overview of studies describing fish microbiome analyses by next generation sequencing methods.

Host Tissue Aim of study Technique Reference

Atlantic cod (Gadus morhua) gut host development, origin of microbiota

16S rRNA - 454 Bakke et al., 2015

variation between individuals 16S rRNA - 454 Star et al., 2013

Atlantic salmon (Salmo salar) gut description community 16S rRNA - Ion Torrent Gajardo et al., 2016

diet 16S rRNA - 454 Zarkasi et al., 2016

diet 16S rRNA - Illumina HiSeq Schmidt et al., 2016

geographical origin 16S rRNA - Illumina MiSeq Llewellyn et al., 2016

rearing environment 16S rRNA - Illumina MiSeq Dehler et al., 2017

temperature environment 16S rRNA - 454 Zarkasi et al., 2014

skin rearing environment 16S rRNA - Ion Torrent Lokesh & Kiron, 2016

Salmon lice-diseased vs healthy 16S rRNA - Illumina MiSeq Llewellyn et al., 2017

Atlantic sharpnose (Rhizoprionodon terraenovae)

gut host comparison 16S rRNA - 454 Givens et al., 2015

Bighead carp (Hypophthalmichthys nobilis)

gut feeding strategy 16S rRNA - Illumina MiSeq Liu et al., 2016

host comparison 16S rRNA - 454 Li et al., 2014


host genotype 16S rRNA - 454 Li et al., 2017a

rearing environment, diet, host 16S rRNA - Illumina HiSeq Eichmiller et al., 2016

Black molly (Poecilia sphenops)

entire fish

salinity 16S rRNA - Illumina HiSeq Schmidt et al., 2015

Black sea bass (Centropristis striata)


Bluegill (Lepomis macrochirus) gut host comparison 16S rRNA - 454 Larsen et al., 2014

Blunt snout bream (Megalobrama amblycephala)


host genotype 16S rRNA - 454 Li et al., 2017b

Brown trout (Salmo trutta) eggs host genotype, environment 16S rRNA - Illumina MiSeq Wilkins et al., 2016

geographical origin 16S rRNA - 454 Wilkins et al., 2015

Butterflyfish (Chaetodonlunulatus lunulatus, C. ornatissimus, C. reticulatus and C. vagabundus)

gill host comparison 16S rRNA - 454 Reverter et al., 2017

Page 44 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Catfish (Panaque) gut diet 16S rRNA - 454 Di Maiuta et al., 2013

Channel catfish (Ictalurus punctatus)

gut host comparison 16S rRNA - 454 Larsen et al., 2014

Chinese perch (Siniperca chuatsi)


host comparison, host development 16S rRNA - Illumina MiSeq Yan et al., 2016

Cichlid (Amphilophus astorquii, A. zaliosus, A. amarillo, A. sagittae)

gut geographic origin, limnetic vs benthic

16S rRNA - Illumina MiSeq Franchini et al., 2014

Cichlid (Haplotaxodon microlepis, H. trifasciatus, Plecodus straeleni, Perissodus microlepis, P. eccentricus)

gut host comparison, diet 16S rRNA - 454 Baldo et al., 2015

Clownfish (Amphiprion percula)

heads effect suspended sediment 16S rRNA - 454 Hess et al., 2015

Common carp (Cyprinus carpio)

gut transgenic gcGH 16S rRNA - 454 Li et al., 2013

description community 16S rRNA - 454 van Kessel et al., 2011

feeding strategy 16S rRNA - Illumina MiSeq Liu et al., 2016


Crevalle jack (Caranx hippos) gut host comparison 16S rRNA - 454 Givens et al., 2015

Crucian carp (Carassius cuvieri)

gut host genotype 16S rRNA - 454 Li et al., 2017b

Red-Operculum-diseased vs healthy

16S rRNA - Illumina MiSeq Li et al., 2017b


Eurasian perch (Perca fluviatilis)

gut diet (littoral carbon), sex 16S rRNA - 454 Bolnick et al., 2014b

Fathead minnows (Pimephales promelas)

gut antimicrobial treatment 16S rRNA - Illumina MiSeq Narrowe et al., 2015

Fine flounder (Paralichthys adspersus)

gut rearing environment 16S rRNA - Ion Torrent Ramírez & Romero, 2017a

Freshwater drum (Aplodinotus grunniens)

gut rearing environment, diet, host comparison

16S rRNA - Illumina HiSeq Eichmiller et al., 2016

Gibel carp (Carassius auratus gibelio)

gut environmental origin of microbiota 16S rRNA - 454 Wu et al., 2013

Gilthead seabream (Sparus gut diet 16S rRNA - 454 Estruch et al., 2015

Page 45 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

aurata) diet 16S rRNA - 454 Kormas et al., 2014

host development 16S rRNA - 454 Califano et al., 2017

Gizzard shad (Dorosoma cepedianum)

gut geographical origin 16S rRNA - 454 Ye et al., 2014

Goldfish (Carassius auratus) gut feeding strategy 16S rRNA - Illumina MiSeq Liu et al., 2016


Grass carp (Ctenopharyngodon idellus)

gut diet 16S rRNA - Illumina HiSeq Ni et al., 2014

environmental origin of microbiota 16S rRNA - 454 Wu et al., 2012




host comparison, host development 16S rRNA - Illumina MiSeq Yan et al., 2016

Great barracuda (Sphyraena barracuda)


Hogchoker (Trinectes maculatus)


Japanese seabass (Lateolabrax japonicus)

gut diet 16S rRNA - 454 Wang et al., 2017

King mackerel (Scomberomorus cavalla)


Largemouth bass (Micropterus salmoides)

gut host comparison 16S rRNA - 454 Larsen et al., 2014

Largemouth bronze gudgeon (Coreius guichenoti)

gut Furunculosis-diseased vs healthy 16S rRNA - Illumina MiSeq Li et al., 2016

Mahi-mahi (Coryphaena hippurus)


Mummichog (Fundulus heteroclitus)


Nile tilapia (Oreochromis niloticus; genetically improved farmed)

gut origin of microbes 16S rRNA - 454 Fan et al., 2017

Nile tilapia (Oreochromis niloticus)

gut diet 16S rRNA - 454 Ran et al., 2016

enzyme and probiotic supplementation

16S rRNA - Ion Torrent Adeoye et al., 2016

Page 46 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

host development and environment 16S rRNA - 454 Giatsis et al., 2014

host development, environment and probiotic

16S rRNA - 454 Giatsis et al., 2016

probiotic 16S rRNA - 454 Ran et al., 2015

probiotic 16S rRNA - Ion Torrent Standen et al., 2015

rearing environment 16S rRNA - 454 Giatsis et al., 2015

salinity 16S rRNA - Illumina HiSeq Zhang et al., 2016

vertebrate comparison 16S rRNA - Illumina HiSeq Kohl et al., 2014

Paddlefish (Polyodon spathala)

gut host comparison 16S rRNA - 454 Li et al., 2014

Parrotfish (Chlorurus sordidus, Scarus niger)

gut host comparison, feeding strategy 16S rRNA - 454 Miyake et al., 2015

Pinfish (Lagodon rhomboides) gut host comparison 16S rRNA - 454 Givens et al., 2015

Prussian carp (Carassius gibelio)

gut

environmental origin of microbiota 16S rRNA - Illumina MiSeq Kashinskaya et al., 2015

validation DNA extraction methods 16S rRNA - 454 Kashinskaya et al., 2017

Rabbitfish (Siganus stellatus) gut host, feeding strategy 16S rRNA - 454 Miyake et al., 2015

Rainbow trout (Oncorhynchus mykiss)

gut description community 16S rRNA - 454 Etyemez & Balcazar, 2015

description community 16S rRNA - Illumina MiSeq Lyons et al., 2017a

diet 16S rRNA - Illumina HiSeq Ingerslev et al., 2014b

diet 16S rRNA - Illumina MiSeq Lyons et al., 2017b

diet 16S rRNA - Illumina MiSeq Michl et al., 2017

diet 16S rRNA - 454 Desai et al., 2012

diet, Yersinia-infection 16S rRNA - Illumina HiSeq Ingerslev et al., 2014a

diet, rearing density 16S rRNA - 454 Wong et al., 2013

rearing environment 16S rRNA - Illumina MiSeq Lyons et al., 2017a

skin, gill, gut, olfactory organ

tissue specificity 16S rRNA - 454 Lowrey et al., 2015

Red drum (Sciaeops ocellatus) gut host comparison 16S rRNA - 454 Givens et al., 2015

Red Snapper (Lutjanus campechanus)

gill, gut, blood

description community 16S rRNA - 454 Tarnecki et al., 2016

Sandbar shark (Carcharhinus gut host comparison 16S rRNA - 454 Givens et al., 2015

Page 47 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

plumbeus)

Seabass (Dicentrarchus labrax)

gut diet 16S rRNA - 454 Carda-Dieguez et al., 2014

skin mucus

geographical origin 16S rRNA - 454 Pimentel et al., 2017

Siberian sturgeon (Acipenser baerii)

gut

prebiotic, probiotic 16S rRNA - 454 Geraylou et al., 2013a

prebiotic 16S rRNA - 454 Geraylou et al., 2013b

Silver carp (Hypophthalmichthys molitrix)

gut




geographical origin 16S rRNA - 454 Ye et al., 2014

Silver perch (Bairdiella chrysoura)


Southern catfish (Silurus meridionalis)

gut host comparison, host development 16S rRNA - Illumina MiSeq Yan et al., 2016

Southern flounder (Paralichthys lethostigma)


Spanish mackerel (Scomberomorus maculatus)


Spinner shark (Carcharhinus brevipinna)


Spotted coral grouper (Epinephelus sexfasciatus)

gut host comparison, polluted environment

16S rRNA - Illumina MiSeq Hennersdorf et al., 2016b

Striped burrfish (Chilomycterus schoepfi)


Surgeonfish (Acanthurus gahhm, A. nigrofuscus, A. sohal, Ctenochaetus striatus, Naso elegans, N. hexacanthus, N. unicornis, Zebrasoma desjardinii, Z. xanthurum)

gut host comparison, feeding strategy 16S rRNA - 454 Miyake et al., 2015

Tambaqui (Colossoma macropomum)

gut, skin pH effect 16S rRNA - Illumina MiSeq Sylvain et al., 2016

Page 48 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Threespine stickleback (Gasterosteus aculeatus)

gut diet (littoral carbon), sex 16S rRNA - 454 Bolnick et al., 2014b

MHC genotype 16S rRNA - Illumina HiSeq Bolnick et al., 2014a

probiotics and host genotype 16S rRNA - Illumina MiSeq Smith et al., 2015

Tiger grouper (Epinephelus fuscoguttatus)

gut

environment 16S rRNA - Illumina MiSeq Hennersdorf et al., 2016a

host comparison, polluted environment

16S rRNA - Illumina MiSeq Hennersdorf et al., 2016b

Topmouth culter (Culter alburnus)


Turbot (Scophthalmus maximus)

gut taxonomic and functional diversity Total DNA - Illumina HiSeq Xing et al., 2013

Western mosquitofish (Gambusia affinis)

gut antibiotic treatment 16S rRNA - Illumina HiSeq Carlson et al., 2015

Yellowtail amberjack (Seriola lalandi)

gut rearing environment 16S rRNA - Ion Torrent Ramírez & Romero, 2017b

Yellowtail scad (Atule mate) gut host, polluted environment 16S rRNA - Illumina MiSeq Hennersdorf et al., 2016b

Zebrafish (Danio rerio) gut behavior, stress, probiotic 16S rRNA - Illumina MiSeq Davis et al., 2016

diet 16S rRNA - 454 Rurangwa et al., 2015

diet 16S rRNA - Illumina HiSeq Wong et al., 2015

diet 16S rRNA - Illumina MiSeq Gaulke et al., 2016

diet 16S rRNA - 454 Semova et al., 2012

host development 16S rRNA - Illumina HiSeq Stephens et al., 2016

probiotic treatment 16S rRNA - Ion Torrent Borrelli et al., 2016

rearing environment 16S rRNA - 454 Roeselers et al., 2011

Page 49 of 49



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

for peer review - pure.knaw.nl · raaijmakers, jos; netherlands institute of ecology, microbial...

Documents