Practical application of PHB degrading probiotics

Faculty of Bioscience Engineering

Academic Period 2009 – 2010

Practical application of PHB degrading probiotics

Bui Doan Dung

Promotor 1: Prof. Dr ir Peter Bossier

Promotor 2: Prof. Dr ir Willy Verstraete

Thesis submitted in partial fulfillment of the requirements for the academic

degree of Master of Science in Aquaculture

II

COPYRIGHT “The author and promotors give permission to put this thesis to disposal for consultation

and to copy parts of it for personal use. Any other use falls under the limitations of

copyright, in particular the obligation to explicitly mention the source when citing parts

out of this thesis”.

August 2010 Promotor 1…………………………… Prof. Dr ir Peter Bossier Promotor 2…………………………… Prof. Dr ir Willy Verstraete Author………………………………… Bui Doan Dung

III

ACKNOWLEDGEMENT

I had the honor to perform the experiment in both Laboratory of Aquaculture & Artemia

Reference Centre (ARC) and Laboratory of Microbial Ecology and Technology

(LabMET), Ghent University, Belgium.

First of all, I would like to express my deep gratitude to Prof. Dr Patrick Sorgeloos for

offering me a great opportunity to study in such a wonderful country, Belgium. He made

ARC a second home for all international students in general and for me in particular. I am

truly proud of being one of your students.

Secondly, I would like to record my appreciation to Prof. Dr ir Peter Bossier, my thesis

promoter, who has also been our teacher for many courses in the MSc Aquaculture

program. Your advices for my thesis and the knowledge you impart to us during your

class are enormously useful and has played an important part in my MSc study.

I also wish to express my sincerest thank to Prof Willy Verstraete, my co-promotor. I was

very fortunate to have him as my thesis co-promoter. He has a gift in encouraging weak

students like me. I could say, he is the most exciting and awesome professor I have ever

met. I salute him and I am proud to tell people that I had the pleasure of having dinner

with Prof. Willy Verstraete together with his kind, warm hearted and pretty wife!

I would also like to give my deep appreciation to my smart and enthusiastic supervisor

Peter De Schryver. He has been kind and always pushing me forward to my advantage.

His patience and optimism was impressive. I learned a lot at the same time enjoyed

working with him.

My special thanks to my two kind and cool coordinators, Bart Van Delsen and Sebastiaan

Vanopstal. Thanks for your great and enthusiastic support and help during the entire two-

year master programme. You made the time in Ireland one of the most amazing periods

IV

in my life. The delicious food you prepared and the funny drinking moments we had are

unforgettable.

My sincere thanks to ARC staff Anita, Brigite, Tom, Jorg, Geert, and Chris. Without your

help, I would not be able to finish my experiment. Thanks a lot to PHD students in ARC

Toi, Tuan, Thai and Kartik for supporting me always.

I also would like to express thanks to all LabMETERs, particularly Tim and Siska for

your priceless supports and advices during my work there.

I am extremely grateful to all my classmates. I feel lucky and happy to know all of you.

The time with you is unforgettable for me. Hopefully, we could see each other someday!

My deep appreciation to Belgian Technical Cooperation (BTC) for giving me the unique

chance to study in Belgium.

I want to express my great gratitude to my dear family at home - my parents, my younger

brother for supporting and comforting me during the hard times. Thank you and I love

you!

Finally, I would like to thank all the people who helped me and made this thesis possible.

V

TABLE OF CONTENTS LIST OF FIGURES..................................................................................................... VII

LIST OF TABLES .....................................................................................................VIII

LIST OF ABBREVIATIONS ....................................................................................... IX

ABSTRACT...................................................................................................................X

CHAPTER 1. INTRODUCTION.....................................................................................1

CHAPTER 2. LITERATURE REVIEWS........................................................................3

2.1. Disease as the major problem for aquaculture........................................................3

2.2. The use of antibiotics for diseases control .............................................................5

2.3. Most used alternatives for disease management.....................................................6

2.3.1. The use of vaccines ........................................................................................6

2.3.2. The use of immunostimulants.........................................................................7

2.3.3. Bio-control as the best alternative for antibiotic drugs ....................................8

2.4. Artemia biology ..................................................................................................15

2.4.1. Artemia taxanomy........................................................................................15

2.4.2. Artemia morphology ....................................................................................15

2.4.3. Artemia reproduction ...................................................................................16

2.4.5. Artemia feeding behavior .............................................................................16

2.4.6. The use of Artemia in aquaculture ................................................................17

CHAPTER 3. MATERIALS AND METHODS.............................................................19

3.1. Places of the experiments ....................................................................................19

3.2. Protocol optimization experiments ......................................................................19

3.2.1. The first protocol optimization experiment ...................................................19

3.2.2 Second protocol optimization experiment ......................................................25

3.3. Main Experiment ................................................................................................27

3.3.1. Overview of the main experiment.................................................................27

3.3.2. Preparation...................................................................................................30

3.3.3. Experiment set-up ........................................................................................34

3.3.4. Sampling......................................................................................................35

3.3.5. Molecular analysis .......................................................................................36

3.4. Data analysis.......................................................................................................37

VI

3.4.1. Survival........................................................................................................37

3.4.2. Molecular fingerprints ..................................................................................37

CHAPTER 4. RESULTS...............................................................................................38

4.1. Main experiment .................................................................................................38

4.1.1. Subtest 1 ......................................................................................................38

4.1.2. Subtest 2 ......................................................................................................41

4.1.3. Subtest 3 ......................................................................................................43

4.2. Protocol optimization experiments ......................................................................46

4.2.1 First protocol optimization experiments.........................................................46

4.2.2. Second protocol optimization experiments ...................................................49

CHAPTER 5. DISCUSSSION.......................................................................................51

5.1. The main experiment ..........................................................................................51

5.1.1. Subtest 1 ......................................................................................................51

5.1.2. Subtest 2 ......................................................................................................53

5.1.3. Subtest 3 ......................................................................................................55

5.2. Sampling protocol and molecular protocol optimization......................................57

5.2.1. Single round PCR versus nested PCR...........................................................57

5.2.2. Method for DNA extraction: CTAB versus adjusted DNeasy .......................57

5.2.3. Minimal amount of Artemia needed for sampling .........................................58

5.2.4. The reproducibility of three batches of Artemia treated in the same way in

non-sterile conditions.............................................................................................58

5.3. The failure of the molecular analysis for the main experiment .............................59

CHAPTER 6. CONCLUSIONS AND FUTURE PERSPECTIVES ...............................60

6.1. Conclusions ........................................................................................................60

6.2. Perspectives ........................................................................................................61

REFERENCES..............................................................................................................62

APPENDICES...............................................................................................................73

VII

LIST OF FIGURES

Figure 2.1. Mechanism of bacteriostatic activity of short-chain fatty acids…..................12 Figure 2.2. Adult Artemia..................................................................................................15

Figure 3.1. Scheme of the main experiment .....................................................................29

Figure 3.2. Sterile aeration of the suspension of cysts.......................................................34

Figure 4.1. Survival of Artemia larvae in different treatments of subtest 1.......................38

Figure 4.2. Survival of Artemia larvae in different treatments of subtest 2.......................42

Figure 4.3. Survival of Artemia larvae in different treatments of subtest 3.......................44 Figure 4.4. DGGE comparison of the PCR amplified DNA extracts from the sterile

Artemia ..............................................................................................................................47

Figure 4.5. DGGE comparison of the PCR amplified DNA extracts from the non-sterile

and non-washed Artemia, the non-sterile and washed Artemia, and the washing water...47

Figure 4.6. DGGE comparison of the CTAB and adjusted DNeasy extraction method...48

Figure 4.7. Comparison of the band pattern intensification of the PCR samples of one,

five, ten, twenty and fifty Artemia.....................................................................................49

Figure 4.8. DNA extracted with adjusted DNeasy method, visualized by means of

DGGE................................................................................................................................50

VIII

LIST OF TABLES

Table 2.1. Examples of socio-economic and other impacts of diseases in shrimp

aquaculture in some Asian and Latin American countries..................................................4

Table 2.2. Mortality caused by luminescent vibrios in different shrimp species...............6

Table 4.1. Growth of bacteria (CFU/mL) on Marine Agar plates during the subtest 1....43

Table 4.2. Growth of bacteria (CFU/mL) on TCBS plates during the subtest 1..............43





IX

LIST OF ABBREVIATIONS

/: Per

ANOVA: Analysis of variance

CFU: Colony forming unit

DGGE: Denaturating gradient gel electrophoresis

FAO: Food and Agricultural Organization of the United Nations

FASW: Filtered and autoclaved seawater

FOS: Fructooligosaccharides

MA: Marine agar

MOS: Mannanoligosaccharides

OD: Optical density

p: Statistical p-value

PCR: Polymerase chain reaction

PHB: Poly-β-hydroxybutyrate

SCFAs: Short chain fatty acids

TAE: Tris-Acetyl-Ethyleendiaminetetraatcetaat

TCBS: Thiosulfate Citrate Bile Salts Sucrose

UV: Ultraviolet

µl: Micro litre

Practical application of PHB degrading probiotics Bui Doan Dung

X

ABSTRACT

The biological control of diseases by environmental and friendly methods such

as probiotics and prebiotics has become an alternative for the use of antibiotics in

aquaculture production. Poly-β-hydroxybutyrate (PHB) has been known as a prebiotic as

it could increase the pathogen resistance and beneficially influence the growth of aquatic

animals. Moreover, the addition of PHB-degraders together with PHB particles has been

shown to significantly increase the survival of brine shrimp larvae infected with the

virulent pathogen strain Vibrio campbellii under gnotobiotic conditions.

In this study, we tried to apply that method in non-sterile conditions. Two pure strains of

PHB-degrading bacteria isolated from European sea bass and giant river prawn were

chosen. PHB particles were added to the Artemia cultures at the concentration of 100 mg

L-1. Challenge tests, performed with the pathogenic Vibrio campbellii LMG 21365,

showed the increase of survival for infected Artemia franciscana nauplii to be 2-3 times

higher than that of the control due to the PHB-degrading activities. Additionally, the

strain M13 isolated from giant river prawn was shown to be a probiotic in non-axenic

cultures of Artemia by providing a defensive effect against pathogenic Vibrio campbellii.

Moreover, in non-sterile cultures, at the concentration of 100 mg L-1, PHB particles could

also double the survival of challenged brine shrimp compared to the control. All

treatments in this experiment decreased Vibrio campbellii LMG 21365 virulence.

However they were not able to inhibit Vibrio campbellii growth in the culture water.

The dose of the PHB particles (100 mg L-1) is economically reasonable and the involved

isolates are easily developed; both make this research highly applicable in the real

production.

Keywords: PHB, PHB-degrader, Artemia, non-sterile


1

CHAPTER 1. INTRODUCTION

Aquaculture is the fastest growing food-producing sector in the world, with an average

annual growth rate of 8.9% since 1970, compared to only 1.2% for capture fisheries and

2.8% for terrestrial farmed meat production systems over the same period (FAO, 2004).

In 2002, the total world aquaculture production was reported to be 51.4 million tonnes by

volume and US$ 60.0 billion by value. This represents an annual increase of 6.1% in

volume and 2.9% in value, respectively, over the reported figures for 2000. The majority

of aquaculture production of fish, crustaceans and mollusks continues to come from the

freshwater environment (57.7% by volume and 48.4% by value). Mariculture constitutes

36.5% of the production and 35.7% of the total value. Although brackish water

production represented only 5.8% of the production volume in 2002, it contributed 15.9%

of the total value, reflecting the prominence of high-value crustaceans and finfish (FAO,

2004).

The current trend in aquaculture expansion is focused on intensification of aquatic

production. This has led to Aquaculture’s major problem, disease, which is holding back

the aquaculture development, impeding both economic and social development in many

countries.

Antibiotics have been used in attempts to control bacterial disease in aquaculture. In

Thailand, for shrimp production, 500 - 600 tonnes antibiotic were used annually

(Moriarty, 1999). The effectiveness of antibiotics is undeniable, but apart from that, the

excessive use of antibiotics in aquaculture also causes a threat to human health and to the

environment (Martinez, 2009). The residual antibiotics in aquaculture products can lead

to an alteration of the normal human gut microbiota and can generate problems

of allergy and toxicity (Cabello, 2006). A lot of global efforts have been made to

decrease the use of antibiotics in aquaculture and thus, to make this industry more

sustainable.


2

There is an increasing demand for alternatives to antibiotics. Various solutions have been

applied such as vaccination, immunostimulation, the use of probiotics, prebiotics,

bacteriophages, microalgae or green–water. It has been widely published that short chain

fatty acids and poly–β–hydroxybutyrate (PHB) must have bacteriostatic properties, and

they could be a sustainable alternative. However, more research needs to be conducted to

make the further applicable in the real production.

The general objective of the present thesis is to test the effect of the combination of PHB

and PHB-degrading bacterial strains isolated from the gastrointestinal tract of some

aquaculture animals fed with PHB as a part in their diet against the challenge from Vibrio

campbellii on Artemia franciscana naupllii in non-sterile conditions.

Detailed goals of this study:

This study was conducted to determine:

o What is the effect of the probioticum with/without PHB on the survival of the

non-challenged non-sterile Artemia?

o What is the effect of the probioticum with/without PHB on the survival of

challenged non-sterile Artemia?

o Can the probioticum develop in the intestinal microbial community and is this

improved by the presence of PHB in non-sterile conditions?

o How do the pathogen and probioticum compete in the intestine and is this effect

changed by the presence of PHB in non-sterile conditions?

o What is the reason for difference in survival when treated with probioticum and/or

PHB: is it the probioticum or the PHB?

o Can molecular fingerprinting be used to determine the interaction in the intestinal

microbial community in non-sterile Artemia?


3

CHAPTER 2. LITERATURE REVIEWS

2.1. Disease as the major problem for aquaculture The aquaculture sector has increased rapidly along with the over-fishing and the

increasing demand for seafood. The production was increased from below 1 million

tonnes in the early 1950s to 51.7 million tonnes in 2006, with a value of US$78.8 billion

(FAO, 2008).

Aquaculture has been expected to fulfill the demand for aquatic food. With this purpose,

new commercialized species and intensification of production have been focused on

vigorously. However, in order to achieve this, the sector has to face significant

challenges. The major constraint that inhibits the productivity is the loss of stock through

disease (examples in Table 2.1).

Table 2.1. Examples of socio-economic and other impacts of diseases in shrimp aquaculture in some Asian and Latin American countries (Bondad-Reantaso, Subasinghe et al. 2005) Country Disease/path

ogen Losses and other impacts Reference

1993 China PR

Shrimp diseases

US$ 420 M in 1993; 60% decline in production from 210,000 to 87,000 tonnes in 1993

Wei (2002) Yulin (2001)

Vietnam Shrimp diseases Monodon baculovirus (MBV), white spot disease (WSD) and yellow head disease (YHD)

US$ 100 M in 1993 Khoa et al. (2001)

Thailand YHD and US$ 650 M in 1994; 12% production declined Chanratcha-


4

WSD from 250,000 tonnes in 1994 to 220,000 tonnes in 1995; shrimp losses for 1997 reached nearly 50% of total farm output value. (Excludes losses in related businesses such as feed production, processing and exporting, ancillary services and lost income for labourers)

kool et al. (2001)

India YHD, WSD Production loss of 10,000–12,000 tonnes during 1994–1995 caused by two viral epizootics (YHD); US$ 17.6 M economic loss in 1994 alone (WSD)

Mohan and Basavaraja-ppa (2001)

Philippines Shrimp diseases (viral and bacterial)

Decline in export from 30,462 to 10,000 tonnes in 1997; great reduction in number of hatcheries

Albaladejo (2001)

Sri Lanka WSD and YHD

Production loss of 1 B Rs. in foreign income during 1996 outbreak; 90% of production units closed (WSD); 68 and 70% drop in shrimp exports in terms of quantity and value in 1998 (mixed infection-WSD and YHD)

Siriwardena (2001)

1999 Ecuador

WSD US$ 280.5 M in 1999 equivalent to 63,000 tonnes; closing of hatchery operations; 13% laying off of labour force (26,000 people); 68% reduction in sales and production of feed mills and packing plants

Alday de Graindorge and Griffith (2001)

Losses in aquaculture by diseases can cause many problems, like monetary losses,

percentage decrease in production, export losses, unemployment, closure

of aquaculture operations and lost consumer confidence (Bondad-Reantaso et al., 2005).

Vibriosis is one of the main diseases for shrimp culture. It has caused critical crop failures

in many countries, especially in South America and Asia (Austin and Zhang, 2006). The

losses due to luminescent vibriosis in Indonesia, 1991, have been reported to be

approximately US$100 million (FAO, 2000). Almost all types of cultured animals can be

affected by these bacteria (examples in Table 2.2).


5

Table 2.2. Mortality caused by luminescent vibrios in different shrimp species (summarized by Defoirdt et al. (2007))

Species Mortality References Kuruma prawn (Penaeus japonicus)

Mass mortality Liu et al. (1996)

Ridgeback prawn (Sicyonia ingentis)

Up to 55% mortality Martin et al. (2004)

Tiger prawn (Penaeus monodon)

Mass mortality Karunasagar et al. (1994), Lavilla-Pitogo et al. (1990)

White shrimp (Litopenaeus vannamei)

Up to 85% mortality Aguirre-Guzman et al. (2001)

Brine shrimp (Artemia franciscana)

Between 45 to 80% mortality

Soto-Rodriguez et al. (2003)

Methods for disease control have been vigorously searched for. However, once a

pathogen or disease agent is introduced and becomes established into the natural

environment, there is almost no possibility for either cure or suppression (Bondad-

Reantaso et al., 2005). The risk of major disease incursions and newly rising diseases will

keep on frightening the sector. More research on pathogen control is needed.

2.2. The use of antibiotics for diseases control

The most common solution for disease problems is the use of antibiotic. It is probably the

most successful family of drugs so far developed for improving animal health (Martinez

2009). Certain antibiotics have been shown to have a positive effect on the survival and

growth of livestock (Campoccia et al., 2010). However, antibiotics have been widely

misused (Aarestrup, 1999). In the United States, 18.000 tonnes of antibiotics are

produced each year for medical and agricultural purposes and 12.600 tonnes are used for

the non-therapeutic treatments of livestock in order to support animal growth (SCAN,

2003). In the European Union, in 1997, 1600 tonnes of antibiotics, representing about

30% of the total use of antibiotics were used for growth promotion purposes (SCAN,

2003). According to the National Office of Animal Health (NOAH, 2001), antibiotic


6

growth promoters could help animals to digest their food more efficiently. Although the

mechanism is still unclear, it is believed that the antibiotics suppress sensitive

populations of bacteria in the intestines. Jensen (1998) reported that as much as 6% of the

net energy in the pig diet could be lost due to microbial fermentation in the intestine. So

if the microbial population could be better controlled by antibiotic, it is possible that the

lost energy could be dedicated to growth.

The use of antibiotics in agriculture and aquaculture has led to the emergence of

antibiotic resistant bacteria. The amounts of redundant antibiotics have put a very strong

selection pressure for resistance among bacteria (Vieira et al., 2010). Resistance

mechanisms can be caused by two ways: chromosomal mutation or acquisition of

plasmids (Lewin, 1992). As a consequence, there was a dramatic drop in the shrimp

industry in some countries where intensified production with excess numbers of animals

and unchecked antibiotic usages led to the antibiotic resistance (Aarestrup, 1999). For

example, the shrimp production in the Philippines dropped by 55% in 2 years; from

90,000 tonnes to only 41,000 tonnes between 1995 and 1997, equal to the loss of $520

million (FAO, 2008). Thai production of shrimp dropped by 40% between 1994 and

1997, due to disease problems (Moriarty, 1999). All over the world, there is a rising

social attitude against the abusiveness of antibiotics (Mudd, 1996; Van Den Bogaard and

Stobberingh, 1996; Millet and Maertens, 2010).

2.3. Most used alternatives for disease management

2.3.1. The use of vaccines

Vaccination is the practice of administering weakened or dead pathogenic bacteria (or

parts of them) in order of triggering specific immune responses to increase resistance to

infectious diseases (Gudding et al., 1999).

Vaccination is only used for fishes, not for crustaceans because of their simple immune

system (Johnson et al., 2008). Vaccines are commonly used by three possible routes;

injection which is labor intensive and costly, immersion which is more applicable and


7

oral vaccination which is an option but still largely in an experimental stage (Huising et

al., 2003).

Efficient vaccines have been developed against many of the major bacterial diseases in

fish aquaculture (Press and Lillehaug, 1995; Buchmann et al., 1997; Zhou et al., 2002).

Many studies focused on luminescent vibriosis in several fish species (Crosbie and

Nowak, 2004; Lin et al., 2006). In experimental trials, adult Atlantic cod responded very

well to vaccination and specific protective immunity was obtained against both classical

vibriosis and cold-water vibriosis after vaccination by intra-peritoneal injection as well as

immersion (Schrøder et al., 2006).

One of the best examples for this successful alteration is the dramatic drop of

antimicrobial drugs consumption in the major producing country Norway. The use of

antimicrobial drugs in Norway has dropped from approximately 50 m3 tonnes per year in

1987 to 746.5 kg in 1997. At the same time, the production of farmed fish in Norway

increased approximately from 5 x 104 to 3.5 x 105 metric tonnes. That success was

mainly caused by the development of effective vaccines (Lunestad, 1998).

Although vaccination methods seem very appropriate, their effects are not always as

expected. The vaccines might be partially degraded by the digestive fluids in the oral

administration method or could not be sufficiently absorbed by the fish body in the

immersion and spray methods (Zhou et al., 2002).

2.3.2. The use of immunostimulants

Immunostimulants are living and dead bacteria, glucans, peptidoglycans, and

lipopolysaccharides… which are able to trigger the immune system (Sakai, 1999). The

use of immunostimulants as dietary supplements, can improve the innate defense of

animals providing resistance to pathogens during periods of high stress. The beneficial

application of immunostimulants has many advantages as they can be implemented at

larval and early fry stages, when mortality is often high due to opportunistic pathogens

(Sakai, 1999). Nutritional factors such as vitamins or growth hormone have also been


8

considered as immunostimulators. The immunostimulants work by facilitating the

function of phagocytic cells to enhance their bactericidal capacity. Some

other immunostimulants can motivate the natural killer cells, complement, lysozyme and

antibody responses of fish (Sakai, 1999). So far, there is no record that those substances

may have bad side effects on the consumers and on the environment (Kumari and Sahoo,

2006).

The use of immunostimulants to control luminescent vibriosis in shrimp is mentioned in

several reports, where it is shown that different immunostimulants increased the survival

significantly when infected experimentally with luminescent Vibrio spp. (Marques et al.,

2006). However, resistance to bacterial pathogens such as Vibrio anguillarum, Vibrio

salmonicida, Aeromonas salmonicida, Yersinia rukeri and Streptococcus spp. and

some parasitic infections such as white spot disease can be increased by the over-use

of immunostimulants (Sakai, 1999).

2.3.3. Bio-control as the best alternative for antibiotic drugs

In recent years, the biological control of diseases by environmental and friendly methods

such as probiotics and prebiotics has become an important subject of investigation

in aquaculture research. Many studies on the use of prebiotics and probiotics in aquatic

cultured species were done (Vinod et al., 2006; Karunasagar et al., 2007; Defoirdt et al.,

2009; Swain and Ray, 2009; Liu et al., 2010). The use of bio-control methods is now

commonly accepted in aquaculture, especially shrimp farming.

2.3.3.1. The use of probiotics in aquaculture

The use of probiotics or beneficial bacteria, which are able to control pathogens through

varied mechanisms, is increasingly used as an alternative for antibiotics. The term

probiotics is generally used to denote bacteria that promote the health of other organisms.

FAO/WHO (2001) defined probiotics as live micro-organisms which can confer a health

benefit on the host by enhancing the host response towards disease, by ensuring improved

use of feed or enhancing its nutritional value, or by improving the quality of its ambient


9

environment or inhibit the growth of pathogens, when they are supplied in adequate

amounts. Lilley and Stillwell (1965) described them as substances secreted by one

microorganism, which stimulated the growth of another.

In fish larviculture, probiotics have been applied in the rearing water, and they may

become integrated in the food chain by allowing the live food organisms, like Artemia

and rotifers, to graze on them (Gatesoupe, 1999). Feeding turbot larvae with rotifers

enriched with Phaeobacter 27-4, brought the mortality to the level of control when

challenged with fish pathogen Listonella anguillarum, demonstrating the effectiveness of

bioencapsulation of the probiotic in rotifers (Planas et al., 2006). As another example, an

increment in survival of turbot larvae (Psetta maxima) was observed when selected

terrestrial lactic acid bacteria were used as probiotics with the axenic rotifer Brachionus

plicatilis as the vector (Gatesoupe, 1991).

The main probiotic bacteria documented for use in shrimp grow-out are gram positive

bacteria, such as Bacillus spp. (Gatesoupe, 1991; Gatesoupe, 2002; Tseng et al., 2009).

Lactic acid bacteria (LABs) also potentially have several probiotic properties: they may

stimulate the growth of preferred micro-organisms, inhibit harmful bacteria, and

strengthen the organism's natural defense mechanisms (Gatesoupe, 1991; Gatesoupe,

2002). In another research on brine shrimp (Artemia) under gnotobiotic conditions,

Marques et al. (2006) showed some defensive effects of some bacterial strains. In

general, Artemia cultured in sub-optimal conditions with medium-quality or good-quality

microalgae were less protected against pathogens than nauplii fed ad libitum. However,

only under poor feeding conditions, as with wild type (WT) yeast cells, the probiotic

effect of a bacterial strain (strain LVS 2 - Bacillus spp.; and strain LVS 3 - Aeromonas

hydrophila) against a weak pathogen such as Vibrio proteolyticus can be demonstrated.

Research on the use of probiotics in aquaculture is at the dawn. So far, it is not yet clear

what are the roles of probiotics in the intestinal tract of aquatic animals. Most of the

understanding about effects of probiotics in fish came from studies on higher vertebrates.

Scientists who are working on this topic should also focus on the use of other molecular


10

methods such as Denaturating gradient gel electrophoresis (DGGE) to investigate the

interactions between probionts and pathogens in the digestive tract of fish. In addition,

endogenous bacteria isolated from aquatic species should be investigated for their

metabolic capabilities such as degradation of anti-nutrients or some indigestible

compounds (Merrifield et al., 2010).

2.3.3.2. The use of prebiotics in aquaculture

The important role of probiotics in Aquaculture sector has been shown by many authors

(Gatesoupe, 1991; Gibson et al., 1998; Gatesoupe, 2002; Balcázar et al., 2008). However,

the use of probiotics may be intricate in aquaculture industry because of the low viability

of the bacteria after processing and during storage and also by the un-solid form of feed

particles in rearing water. Moreover, there are claims that the probiotics are dominant in

the gastrointestinal track only in the treatment period (Merrifield et al., 2010).

Fortunately, prebiotics are able to solve that problem by supporting the healthy gut

microbiota in fish.

Prebiotic is defined by Gibson and Roberfroid (1995) as a non-digestible food ingredient

that can stimulate the growth of healthy bacteria that can improve the health of the host.

Gibson et al. (2004) recommended that a prebiotic must be able to:

• Resist gastric acidity, digestive enzymes inside the host

• Be degraded by the intestinal microbiota

• Stimulate selectively the growth and/or the activity of specific intestinal bacteria that

can enhance the growth and/or the disease resistance of the host

Prebiotics such as mannanoligosaccharides (MOS), fructooligosaccharides (FOS) and

inulin, have shown their potential for applications in aquaculture (Grisdale-Helland et al.,

2008; Ibrahem et al., 2010; Refstie et al., 2006).

Ibrahem et al. (2010) conducted an in vivo experiment to investigate the activities of

inulin in the diet for Nile tilapia (Oreochromis niloticus). The results showed that the

body weight gain, the specific growth rate and the survival were significantly increased


11

for fish fed with balanced diet supplemented with inulin (5g kg-1). The nitroblue

tetrazolium and the lysozyme activity were observed to be signigficantly higher for fish

in treatment with inulin (compared to the control) after 1 and 2months. Challenge test

with pathogenic Aeromonas hydrophila showed lower mortality (40%) for fish fed with

inulin as part of their diet compared to control treatment (60%). Refstie et al. (2006) set

up another 3-week trial to investigate the effect of dietary inulin (7.5%), with and without

oxytetracycline addition (0.3%), on Atlantic salmon. The blood plasma analysis revealed

that the concentration of free fatty acids, glucose, cholesterol and triacylglycerides were

not affected by the dietary inulin or the oxytetracyline supplementation. The treatment

with inulin gave higher fish gut weight (relative to total body weight) but relative liver

and stomach weights were not gained with dietary inulin. Additionally, histology study of

the distal intestine showed no morphological change in fish fed with inulin or

oxytetracycline.

The effect of MOS supplementation on on-growing Atlantic salmon was investigated by

Grisdale-Helland et al. (2008). Salmons were fed with the dietary contained of 1% MOS

for 16 weeks. The results showed that the energy digestibility of the MOS supplemented

diet was increased compared with the control treatment. In addition, body composition

analysis showed the increase of the gross energy content with the addition of MOS

although crude protein was reduced. In another research on rainbow trout by Staykov et

al. (2007), 0.2% dietary MOS supplementation could increase the body weight, reduce

the feed conversion rate (FCR) and mortalities compared to the control treatment in both

net cage and raceway reared trout. Moreover, MOS addition was also able to improve the

immune parameters.

Grisdale-Helland et al. (2008) evaluated the effect of 1% dietary FOS supplementation on

on-growing Atlantic salmon for 16 weeks. The results demonstrated some improvement

of feed efficiency ratio (FER) compared to the control group. However, the final body

weight and the nutrient digestibility were not increased by the diet with FOS.


12

Very few studies have evaluated the effects of prebiotics on the immune response of

aquaculture animals (Staykov et al., 2007). Merrifield et al., (2010) suggested that a full

understanding of the uses of prebiotics will only be obtained by more intensive studies on

the effects of prebiotics, in modulating both rapid responses and slower adaptive immune

responses to pathogen for both healthy and pathogen challenged fish models.

2.3.3.2.1. The use of Poly-β-hydroxybutyrate (PHB) in aquaculture as a prebiotic

The short chain fatty acids (SCFAs) have been known widely as anti-microbial

compounds. Its capacity to inhibit pathogenic bacteria was confirmed by many authors

(Bearson et al., 1997; Defoirdt et al., 2006; Cherrinton et al., 1991). Bacteriostatic effect

of short-chain fatty acids was described by Defoirdt et al. (2009). The undissociated and

dissociated forms of fatty acids are in equilibrium, with relative levels of each form

dependent on the pH and the pKa of the fatty acid (according to the Henderson–

Hasselbach equation). In the undissociated form, the SCFAs could pass the cell

membrane. Inside the cell, they dissociate into anions and protons and change the pH in

the cytoplasm. Consequently, bacteria must maintain a more or less constant pH in

the cytoplasm in order to sustain functional macromolecules by exporting of excess

protons. This requires consumption of cellular ATP and may result in depletion of

cellular energy (Figure 2.1).

Figure 2.1. Mechanism of bacteriostatic activity of short-chain fatty acids (taking butyric acid as example). The fatty acids pass the cell membrane in their undissociated form and dissociate in the cytoplasm. As a consequence, the cells have to spend energy to export the excess of protons.


13

However, the use of SCFAs in commercial aquaculture is difficult since it could be lost

easily into water, which causes high cost and also the reduction of pH of the surrounding

environment. A good alternative for SCFAs is the polymers of SCFAs such as

polyhydroxyalkanoats (PHA). And the most well-known PHA is poly-β-hydroxybutyrate

(PHB), containing repeat units of (R)-3 hydroxybutyrate (Lee, 1996).

Poly-β-hydroxybutyrate (PHB) has been shown to increase pathogen resistance and to

beneficially influence the growth of aquatic animals in a number of studies (Defoirdt et

al., 2007b; De Schryver et al., 2009; Dinh et al., 2010; Dang et al., 2009).

According to Defoirdt et al. (2007), in axenic condition, the addition of PHB could

protect brine shrimp from the virulent Vibrio campbellii strain. Artemia could be

protected completely from the pathogen at the highest concentration of PHB particles

(1000 mg/L). At 100 mg/L PHB particles was still be able to inhibit the activity of Vibrio

campbellii but at lower level. Another study of Halet et al. (2007) revealed that the

addition of 107 cells/mL of PHB-containing Brachymonas bacteria (corresponding

to approximately 10 mg/L PHB) also completely protected the brine shrimp from the

vibriosis.

In the same study above of Defoirdt et al. (2007), 1000mg L-1 PHB particles (average

diameter 30µm) were added to the sterile culture water of starved Artemia nauplii. The

result showed the significantly higher survival for the treatment with PHB particles

compared to the control. It suggested that that PHB can also be an energy source for

Artemia when it is decomposed to water-soluble products like β-hydroxybutyrate

monomers and oligomers. De Schryver et al. (2010) showed a similar result in another

study on European sea bass. The supplementation of PHB at 2-5% in the feed diet

significantly enhanced the growth of juvenile European sea bass after 42 days of rearing.

The fact that PHB was intestinally degraded resulted in higher survival of the fishes fed

with only PHB as compared to unfed ones.


14

PHB can be produced fairly easily from some bacterial strains (Lee, 1996).

PHB production on waste streams could make this technique more cost-effective and

sustainable. Finally, it should be possible to produce PHB in situ in the culture water by

adding carbonaceous compounds or by increasing the C:N ratio of the feed. This so-

called biofloc technology is currently getting more consideration as a means to remove

inorganic nitrogen from waste water through conversion into microbial biomass, which

can be used as feed source by the animals (Avnimelech, 1999). Nowadays, with

technology developments, PHB can be commercially produced and its price is

significantly decreasing as the consequence, making it more applicable in aquaculture

industry.

2.3.3.3. The use of synbiotic in aquaculture

The term synbiotics relates to the combination of probiotics and prebiotics in order to

increase the therapeutic effect. Its principle is to support the probiont with its specific

energy source, so that it could out compete with endogenous populations, and thus,

effectively improves its role in the gastrointestinal tract of the host.

The combined application of probiotics and prebiotics could be an interesting prospect

for replacement of growth-promoting chemotherapeutics in the aquaculture industry and

could be a useful tool in the rearing of sensitive early stages of certain marine species.

However, only a limited number of researches on this issue have been done.

Daniels et al. (2010) showed that the dietary combination of Bacillus spp. and Mannan

oligosaccharides (MOS) is cost effective when used to promote survival and provides the

added benefits of improved growth performance of European lobster (Homarus

gammarus L.) larvae, compared to their individual supplementation (Daniels et al., 2010).

In another study, the combination of PHB and PHB-degrading bacteria isolated from the

gastrointestinal tract of some aquatic animals as protective actors against luminescent

vibriosis was tested in sterile condition (Liu et al., 2010). The author was successful to


15

show that when added in combination with PHB, the isolates were able to increase

significantly the survival of Artemia nauplii when compared with the un-treated

challenged treatment.

2.4. Artemia biology

2.4.1. Artemia taxanomy

Phylum: - Arthoropda

Class: - Crustacean

Subclass: - Branchiopoda

Order: - Anostraca

Family: - Artemidae

Genus: - Artemia

2.4.2. Artemia morphology

The brine shrimp Artemia sp. (Branchiopoda, Anostraca) is an inhabitant of hypersaline

waterbodies like lakes or lagoons all over the world. It has some unique characteristics,

which are the efficient osmoregulatory system, the capacity to synthesize very efficient

respiratory pigments to cope with the low O2 levels at high salinities and the ability to

produce dormant cysts at harsh conditions. Artemia therefore, is only found at places with

extremely high salinity where its predators can not manage (Van Stappen, 1996).

Figure 2.2. Adult Artemia (www.novalek.com)


16

It is typical arthropod with segmented body. Its body is sheltered with thin chitinous

exoskeleton to which the entire muscles are attached internally. The Artemia is small with

the body length is usually 8-10 mm for adult male and 10-12 mm for adult female. The

thorax part includes eleven segments, each has a pair of swimming legs. The abdomen

part is composed of 8 segments. The eight and last appendages posses the cercopods, also

called furca and telson (Van Stappen, 1996).

2.4.3. Artemia reproduction

Some Artemia strains are parthenogenetic (only females) but most are cytogenetic

(males and females) (Jan et al., 1980). Most of the time, the Artemia reproduces by

fertilized eggs, which when released by the female, will develop to free-swimming

nauplii (ovoviviparous reproduction). In harsh conditions, inside the mother, the embryo

only develops up to the gastrula stage. Then it will be surrounded by a thick shell

secreted from the brown shell glands located in the uterus and released by the female

(oviparous). That kind of egg is called the cyst. It will be dried up because of the high

salinity water and become inactivated. In good conditions of temperature and salinity, the

cysts will be hydrated and the embryo development is switched on again. The mother

could shift between oviparity and ovoviviparity, depending on the ambient conditions.

2.4.5. Artemia feeding behavior

At the instar-I stage, the Artemia digestive system is still imfunctional, the larvae does

not take up food and develops based on its yolk reserves. Artemia are continuous non-

selective filter feeders. From the stage of instar-II, Artemia becomes a continous non-

selective feeder. It can graze on algae, bacteria yeast, detritus and industrial food pellet

ranging from 1-50 µm in diameter (Dhont et al., 1993).


17

2.4.6. The use of Artemia in aquaculture

Nowaday, Artemia is considered to be the best live food among the live diets used in the

larviculture of fish, estimated to comprise about 85% of the total live feed used in fish

larval aquaculture (Sorgeloos et al., 1986). Artemia of all life stages are used in

aquaculture as a live prey, especially newly hatched nauplii are the most important food

for crustacean and fish larvae (Léger et al., 1986).

Its unique property to form the “cyst” makes Artemia become a convenient and least labor-

intensive livefood source. Annually, over 2000 metric tons of dry Artemia cysts are marketed

worldwide for on-site hatching into 0.4 mm nauplii (Van Stappen, 1996).

Artemia cysts have been harvested all around the world, from coastal lagoons or hypersaline

lakes where high salinity conditions occur. There is an industry of harvesting and processing

Artemia cyst. Cysts could be bought in cans and can be stored for a long time prior use. After

approx 24h incubation in favorable conditions, cysts release small free-swimming nauplii

which can be fed directly to the larvae of a variety of marine as well as freshwater organisms

(Van Stappen, 1996).

According to Van Stappen (1996), Artemia has been known as a food source for

aquaculture since the 1930’s. The demand for Artemia cyst was growing fast in the late

1940’s with the interest for tropical hobby fish, which established the industry of Artemia

cyst production. During the mid-1950’s, commercial attention for brine shrimp was turned to

controlled sources for production in the San Francisco Bay region. Here it was found that

brine shrimp and their cysts could be produced as a by-product of salt production. Since salt

production entails management of the evaporation process, yearly cyst and biomass

productions could be roughly predicted. In the 1960’s, commercial provisions originated

from these few sources in North America and seemed to be unlimited. However, with the

expansion of aquaculture production in the 1970’s, the demand for Artemia cysts soon

exceeded the offer and prices rose exponentially, turning Artemia into a bottleneck for the

expansion of the hatchery aquaculture of marine fishes and crustaceans. In particular, many

developing countries could hardly afford to import the very expensive cysts.


18

Artemia is really useful as a test organism in aquaculture, as it can easily be cultured in

gnotobiotic conditions (Marques et al., 2004). A same batch of cysts could provide

approximately the same animals for different subtests of one experiment. Moreover, it is

possible to bioencapsulate Artemia with probiotic bacteria in different gnotobiotic

environments. Artemia is also the ideal model for genetics experiments. Since cysts can

be stored for a long period, different generations can be cultured at the same time, so that

the genetic variation among generations can be minimized.


19

CHAPTER 3. MATERIALS AND METHODS

3.1. Places of the experiments

● The Artemia cultures and the challenged tests were conducted at the Artemia Reference

Center (ARC), Gent University

● The molecular analyses for the protocol optimization were performed at the Laboratory

of Microbial Ecology and Technology (LabMET), Gent University.

3.2. Protocol optimization experiments

3.2.1. The first protocol optimization experiment

3.2.1.1. Aims of the first protocol optimization experiment

A small experiment was done prior to the main experiment protocol for:

Detecting the differences in the microbial community between externally washed and

non-washed Artemia cultures by means of sterilized seawater to verify the necessity

of a washing step.

Comparing two methods for DNA extraction, CTAB and adjusted DNeasy method, in

order to determine the best method for samples with low concentration of DNA.

Determining to suitability of the primer pairs chosen in a nested PCR approach for

DNA amplification, as to avoid non-specific amplification of the host eukaryotic

DNA.


20

3.2.1.2. Experiment set-up and sampling

The Artemia franciscana originating from the Great Salt Lake, Utah, USA (EG® 404405

Type, INVE Aquaculture, Belgium) were produced as described by Dhont et al. (1993).

One batch was prepared sterile, while the second was prepared non-sterile:

● Batch 1 - Sterile conditions

200 mg Artemia cysts were decapsulated and disinfected in a laminar flow, using

autoclaved equipment and chemicals. The protocol for cyst decapsulation is described in

detailed in the main experiment part. Decapsulated cysts were hatched in filtered and

autoclaved sea water (FASW) for 2 days in closed sterile 50 mL tubes without feeding.

After 2 days, all the Artemia were washed with 500 mL FASW to remove external

bacteria, transferred into 5 mL of new FASW and homogenized for five minutes using a

stomacher. Finally, the sample was stored at - 20°C until further analysis.

● Batch 2 – Non-sterile conditions

200 mg Artemia cysts were decapsulated under non-sterile conditions, using non-

autoclaved equipment and chemicals. Decapsulated cysts were hatched in non-treated sea

water for 2 days in 50 mL tubes, open to the air and supplied with extra aeration. The

animals were also not fed as in the sterile cultures. At harvest, sterile manipulation (in

laminar flow with autoclaved chemicals and equipments) was performed to avoid

additional contamination. The non-sterile batch was separated into 2 groups, equal in

size:

Washed Artemia: the Artemia nauplii were washed with 200 mL FASW to remove

external bacteria, then transferred into 5 mL of new FASW and homogenized for five

minutes using a stomacher. Finally, the sample was stored at -20°C until further analysis.

10 mL of the FASW used for washing was also collected for DGGE analysis.


21

Non-washed Artemia: the Artemia nauplii were concentrated, and homogenized for

five minutes in a stomacher in 5 mL of the culture water without washing. The sample

was then stored at -20°C until further processing.

3.2.1.3 Molecular analysis

Four sample types (sterile Artemia – AA; non-sterile, washed Artemia – NSW; non-

sterile, non-washed Artemia – NSNW; and washing water - WW) were processed

according to the following procedure:

3.2.1.3.1. DNA extraction

1mL of each sample was centrifuged at 6000 rpm for 10 minutes. The supernatant

was discarded and the pellet was further processed.

The DNA from the 4 sample types was extracted using the 2 protocols, CTAB and

adjusted DNeasy. Detailed protocols are described below:

Adjusted DNeasy

1. One mL of PCR water was added then vortexed to bring the pellet back to the

suspension to be transferred to 2 mL eppendorf.

2. The eppendorf was centrifuged again for 10 minutes at 10.000 rpm. The

supernatant was then discharged.

3. Added 180 µL of the enzymatic lysis buffer which was freshly prepared by

mixing 40% lysozyme stock and 60% Tris-HCl buffer, and then stored at -20oC

overnight.

4. Thawed and incubated for 1 h at 37oC in an incubator.

5. Added 40 µL proteinase K and 180 µL ATL buffer, vortex and incubated at 55oC

for 2 h.

6. 200 µL AL buffer was added then incubated at 70oC for 10 min.

7. Added 0.5 gram beads 0.1 mm and beat at 8000 rpm for 1 minute.

8. Centrifuged at 13.000 rpm for 5 minutes. Transferred the supernatant to new 2

mL Eppendorf tube.


22

9. Added 300 µL of 96% ethanol, vortex then transferred to the DNeasy column

placed in a 2-mL tube. Centrifuged at 8000 rpm for 1 min. The filtration was then

discharged.

10. Added 500 µL AW-1 buffer and centrifuged at 8000 rpm for 1 min. Discharged

the filtrate.

11. Added 500 µL AW-2 buffer, followed by centrifugation at 13.000 rpm for 3 min.

Discharged the filtrate.

12. The column was then transferred to a new epppendorf vial. 50 µL of DNA free

water was directly added on the membrane and incubated for 1 min at room

temperature, followed by centrifugation at 8000 rpm for 1 min. The DNA was

eluted once more with 50 µL DNA free water to obtain a total of 100 µL extract.

CTAB

1. CTAB extraction buffer was made by mixing equal volumes of 10% (wt/vol)

CTAB (Sigma, Poole, United Kingdom) in 0.7 M NaCl with 240 mM potassium

phosphate buffer, pH 8.0.

2. Added 0.5 mL of hexadecyltrimethylammonium bromide (CTAB) extraction

buffer and 0.5 mL of phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8.0).

3. Added 0.5 gram beads 0.1 mm and beat for 3 x 30 s at a machine speed setting of

5000 rpm with 10 s cool down between shakings.

4. Centrifuged at 3000 rpm for 5 minutes. Transferred the aqueous phase to a new

Eppendorf tube.

5. Added an equal volume of chloroform-isoamyl alcohol (24:1) and centrifuged at

3000 rpm for 20 seconds for phenol removal. Transferred the aqueous phase to a

new 2 mL Eppendorf tube. Incubated for 2h at room temperature.

6. Added 2 volumes of 30% polyethylene glycol 6000 (Fluka BioChemika) – 1.6M

NaCl and incubated 2h at room temperature then centrifuged at 13.000 rpm at 4oC

for 10 min. Discharged the supernatant.

7. Pelleted nucleic acids was then washed in ice cold 70% ethanol following by

drying under vacuum for 10 minutes.

8. Finally, the nucleic acid pellet was resuspended in 50 µL RNase free water.


23

3.2.1.3.2. Polymerase chain reaction (PCR)

For normal PCR, only one round was performed. The V3 region of the gene was

amplified by PCR using primers 338f (5’-ACTCCTACGGGAGGCAGCAG-3’) and 518r

(5’-ATTACCGCGGCTGCTGG-3’).

For nested PCR, two rounds were carried out. At the first round (external round), a longer

fragment of the gene was amplified using the primer pair EUBF8-27 (5’-

AGAGTTTGATCMTGGCTCAG-3’) and 984R (5’-CCACATGCTCCACCGCTTGTGC-3’),

while in the second round (internal round), like normal PCR, the V3 region of the gene

was amplified by using primers 338f/518r.

A Fermentas PCR kit was used as the ingredients for making master-mix. 100 µL master-

mix was the mixture of 77.25 µL 10X TAQ buffer + KCl-MgCl2, 10 µL MgCl2, 2 µL

dNTP (10mM each), 2 µL of primer 1 (0.01ng/µL), 2 µL primer 2 (0.01ng/µL), 0.25 µL

BSA and 0.5 µL Taq-polymerase. In the PCR vials, 48 µL of master-mix were mixed

with 2 µL of extracted DNA template.

● Normal PCR (single-round) program:

Firstly, 5 minutes of initial denaturation at 94oC, 35 cycles of the following procedure

were applied: 1 minute of denaturation at 95oC, 1 minute of annealing at 53oC, 2 minutes

of primer extension at 72oC; and the final extension at 72oC for 10 minutes, preserved at

4oC.

● Nested PCR program:

1. First round (with primer pair EUB-8f/984r)

After 5 minutes of initial denaturation at 94oC, 20 cycles of the following procedure were

applied: 1 minute of denaturation at 95oC, 1 minute of annealing at 53oC, 2 minutes of

primer extension at 72oC; and the final extension at 72oC for 10 minutes, preserved at

4oC.


24

2. Second round (with primer pair 338f/518r)

After 5 minutes of initial denaturation at 94oC, 20 cycles of the following procedure were

applied: 1 minute of denaturation at 95oC, 1 minute of annealing at 48oC, 1 minute of

primer extension at 72oC; and the final extension at 72oC for 10 minutes, preserved at

4oC.

3.2.1.3.3. DNA presence confirmation by means of electrophoresis

Before DGGE analysis, the PCR products were checked for DNA concentration by

electrophoresis. 5 µL PCR-products were mixed with 2 µL loading dye, and then loaded

into the wells in the 1.2% agarose gel in a 0.5x TAE (Tris-Acetyl-

Ethyleendiaminetetraatcetaat of Tris-Acetyl-EDTA) buffer solution. The nucleic acids

were visualized under UV light by means of ethidium bromide (1 µL/50 µL agarose

solution).

3.2.1.3.4. Denaturation gradient gel electrophoresis (DGGE)

The DGGE analysis was performed with the PCR products to compare for differences in

the microbial community.

The D-code universal mutation detection system (Bio-Rad, Hercules, California, USA)

was used for DGGE analysis. 50 µ PCR products were mixed with 20 µL loading dye

and then 10 µL of that solution were loaded into wells of polyacrylamide gel in 1X TAE

(10 µL each sample). The gels denaturation gradient ranges from 45 - 60%.

Electrophoresis was conducted with a constant voltage of 38V at 60oC for 16 hours. The

gel was then stained with SYBR green for 20 minutes. Finally, the gel was developed by

means of UV- transilluminator.


25

3.2.2 Second protocol optimization experiment

3.2.2.1. Aims of the second protocol optimization experiment

Followed the first optimization protocol, another test was conducted to:

Compare the reproducibility of different batches of Artemia treated in the same way

in non-sterile conditions.

Determine the minimum number of Artemia larvae needed at each sampling for

molecular analysis.

3.2.2.2. Experiment set-up and sampling

The experiments were performed with the Artemia franciscana cysts originating from

Great Salt Lake, Utah, USA (EG® Type, INVE Aquaculture NV, Belgium). 0.5 gram of

cyst was processed under non-sterile conditions. The cysts were decapsulated outside the

laminar flow hood with non-autoclaved equipment and chemicals, and then rinsed with

non-sterile sea water over a 50µm non-sterile filter net. The decapsulated cysts were then

divided into 3 batches, each with 0.01 gram cell-free cysts and hatched in 35 mL of non-

sterile sea water in 50 mL capped Falcon tubes to avoid the extra contamination from the

air. The tubes were placed on a rotor at 4 cycles per minute, exposed to constant

incandescent light at 37°C. 24h after hatching, Artemia nauplii in 3 batches were fed with

autoclaved LVS3 (Aeromonas hydrophila) in laminar flow hood (to avoid extra

contamination) at the density of 107 cells/mL. The bacteria were cultured in autoclaved

Marine Broth 24h before feeding and the bacterial density was obtained by

spectrophotometric measurement. 72h after hatching, the three batches of Artemia were

harvested in laminar flow hood with autoclaved equipments and chemicals.

At harvest, three batches were treated in the same way. Firstly, nauplii were poured

through the sieves then washed 3 times by swirling the sieves with Artemia nauplii for 30

seconds consequently in three Petri plates with 25 mL of FASW, 20 mL of 0.1%

Benzalkoniumchloride and 20 mL of PCR water (recommended by the first optimization


26

experiment). From each batch, five sub-samples with different number of Artemia

naupliii were collected:

1 nauplius – stored in a small tube with 1 mL of PCR water

5 nauplii – stored in a small tube with 1 mL of PCR water

10 nauplii – homogenized in 5 mL of PCR water with a stomacher for 5 minutes



All samples were then stored at -20oC for latter molecular analysis.

3.2.2.3. Molecular analysis

DNA extraction: DNA from the samples of one, five, ten, twenty and fifty Artemia

from three batches were processed with adjusted DNeasy method due to their expectedly

low DNA concentration (suggested by the first optimization experiment). For the samples

of ten, twenty and fifty Artemia, the whole Falcon tubes with sample fluid were

centrifuged at 6000 rpm for 10 mins. The supernatant was discharged and the pellets

were collected for further processing. For the samples of one and five Artemia (non-

homogenized samples), naupllii were brought to new tubes for further steps of DNA

extraction.

PCR: the nested PCR using the general bacterial primers EUBf8-27 and 984yR (5’-

GTAAGGTTCYTCGCGT-3’) for the external PCR round and the primer pair GC-

338F/518R for the internal PCR round were performed for all samples in order to avoid

the amplification of Artemia DNA (protocols were described in detail in the first

optimization experiment).

DGGE: DGGE analysis was performed for the nested PCR products to compare the

differences in the microbial community among three batches (used the same protocol in

the first protocol optimization experiment).


27

3.3. Main Experiment

3.3.1. Overview of the main experiment

The main experiment was done to test the effect of the combination of PHB particles and

two PHB degraders M13 (Ochrobactrum sp.) and B12 (Acinetobacter sp.), isolated from

giant river prawn and European sea bass that had received PHB in their diets (Liu et al.,

2010), on the survival of Artemia naupllii challenged with virulence Vibrio campbellii

LMG 21365. The main trial was divided into three subtests with following treatments:

Subtest 1:

Sterile Artemia

Non-sterile Artemia

Non-sterile Artemia + pathogen

Non-sterile Artemia + 100 mg/L PHB

Non-sterile Artemia + pathogen + 100 mg/L PHB

Subtest 2:

Non-sterile Artemia + probiotic strain M13

Non-sterile Artemia + probiotic strain M13 + 100 mg/L PHB


Non-sterile Artemia + pathogen + probiotic strain M13

Non-sterileArtemia + pathogen + probiotic strain M13 + 100 mg/L PHB

(control 1: Non-sterile Artemia)

(control 2: Non-sterile Artemia + pathogen)

Subtest 3:

Non-sterile Artemia + probiotic strain B12

Non-sterile Artemia + probiotic strain B12 + 100 mg/L PHB


Non-sterile Artemia + pathogen + probiotic strain B12


28

Non-sterile Artemia + pathogen + probiotic strain B12 + 100 mg/L PHB

(control 1: Non-sterile Artemia)

(control 2: Non-sterile Artemia + pathogen)

3.3.1.1. Goals for the main experiment

Three sub-tests were done to answer the below questions:

Subtest 1

Is a challenge test necessary to test the effect of PHB particles when non-sterile

Artemia are used?

What is the effect of 100 mg/L PHB on the survival of non-sterile Artemia?

What happens with the pathogen in/without the presence of 100 mg/L PHB?

What changes occur in microbial community due to presence of PHB and/or the

pathogen?

Is there any link between survival and microbial community?

Subtest 2 & 3

What is the effect of the probiotium with/without PHB on the survival of the non-

challenged Artemia?

What is the effect of the probioticum with/without PHB on the survival of the

challenged Artemia?

Can the probioticum develop in the intestinal microbial community? And is this

improved by the presence of PHB?

How do the pathogen and the probioticum compete in the intestine? And is this

effect changed by the presence of PHB?

What is the reason for the difference in survival when treated with probioticum

and/or PHB: is it the probioticum or the PHB?


29

Figure 3.1. Scheme of the main experiment

3.3.1.1. Parameters to be tested

The main experiment was designed to have two main testing parameters:

Survival

Is there any difference in survival of the Artemia in a sterile and a non-sterile

system without infection?

Can the pathogen induce its effect also in a non-sterile environment?

Can the probiotics protect the Artemia also in a non-sterile environment?

DAY 2: YEAST + CHALLENGE

(36H AFTER STOCKING)

DAY 1: STOCKING (YEAST + PHB)

DAY 1: STOCKING (YEAST + PHB +

PROBIOTICS)

DAY 0: HATCHING

DAY 0: HATCHING

SUBTEST 1 SUBTEST 2 + 3

DAY 3: HARVEST (24H AFTER

CHALLENGING)

DAY 3: HARVEST (24H AFTER

CHALLENGING)

DAY 2: YEAST + CHALLENGE

(36H AFTER STOCKING)


30

Is there any difference in effect of PHB, probiotics and the combination of them

in survival of the non-challenged Artemia?

Is there any difference in effect of PHB, probiotics and the combination of them

in survival of the challenged Artemia?

Structure of microbial community by means of DGGE and plating

How will the probiotic strains integrate in the microbial community (without

pathogen or PHB present)?

How will the pathogen react if other bacteria have already colonized the Artemia?

How will the presence of PHB influence the pathogen: a) no difference in

presence (DGGE), but only difference in activity (survival); b) difference in

presence and thus difference in activity?

What is the effect of the PHB on the proliferation of the probiotic strains in the

Artemia gut?

What is the effect of the PHB on the pathogen in the gut of the Artemia?

What is the effect of the probiotic strain combined with PHB on the gut of the

Artemia?

How does the presence of the pathogen/probiotic strain/PHB or a combination of

these influences the community structure and can this be related to the differences

in survival?

3.3.2. Preparation

3.3.2.1. Feed preparation

The wild type (WT) yeast strain Saccharomyces cerevisiae was used as the food source

for the Artemia naupllii in this experiment. The same stock was stored at -80oC for all

three subtests. The yeast must be prepared 48h before the each subtest following below

steps:


31

1. Inoculate 500 uL of yeast stock into an autoclaved Elenmayer glass with 100 mL

of fresh YPD Medium, a blend of peptone, yeast extract and dextrose.

2. Incubate for 48h at 37 – 38oC on the shaker at 120 rpm.

3. Wash 2 times with FASW.

4. Check the cell density by counting 20 squares of the Buerker counting chamber

four times then use the below formula:

Cells/ml = (Average value of 4 times counting / 20) x 250 x 1000 x dilution time

5. Check the sterility on Marine Agar plate before use.

6. Artemia nauplli were fed ad libitum with yeast on day 1, day 2 and day 3 followed

the modified feeding schedule of Coutteau et al. (1990).

3.3.2.2. Probiotic preparation

Two probiotics were tested in the subtest 2 and the subtest 3:

M13 (Ochrobactrum sp.) isolated from giant river prawn that had received PHB

in their diets (Liu et al., 2010).

B12 (Acinetobacter sp.) isolated from European sea bass that had received PHB

in their diets (Liu et al., 2010).

Preparation protocols

1. Added into 30 mL fresh Marine Broth with 300 µL probiotic stock from -80°C.

2. Incubated overnight or for 24h at 28°C on 120 rpm shaker.

3. Washed 2 times with FASW.

4. Set at OD550 of approx. 1 (diluted with FASW) using the Thermo spectronic

Genesis 20 approx. 109 cells per mL

5. Add 200 µL for 20 mL of culture water approx.107 cells per mL Artemia

culture water.

6. Added at stocking (day 1).


32

3.3.2.3. Pathogen preparation

Vibrio campbellii LMG 21365 culture was set up at day 1 to be used on day 2 (36h after

stocking). One stock of Vibrio campbellii was stored at minor 800C for all subtests. To

reach the virulence peak of the pathogen, the following procedure was used:

1. Grown on Marine Agar for 24h.

2. Five colonies from Marine Agar were inoculated into 20 mL fresh Marine Broth

and keep in the incubator under the condition of shaking speed 120 rpm and 28oC

for 7 – 8h.

3. Washed 2 times with FASW.

4. Set at OD550 of approx. 1 (dilute in FASW) using the thermo spectronic Genesis

20 approx. 109 cells per mL.

5. Added 20 µL of pathogen suspension to 20 mL of Artemia culture water

approx. 106 cells per mL of Artemia culture water.

3.3.2.4. Microbial community background

This experiment was conducted in the non-axenic conditions. To ensure the similarity

among three subtests which were conducted at different times, the microbial background

was prepared.

1. 0.01 g non-decapsulated Artemia cyst was hatched in a Falcon tube with 30 mL

normal seawater for 24h at 37oC on a rotor running at 4 rpm.

2. After 24h, all nauplii was sieved through 100 um filter, and the hatching water

was kept and stored in sterile Eppendorf tubes in autoclaved Glycerol 20%

solution at – 800C for later use.

3.3.2.5. Decapsulation and hatching of cysts (Day 0)

By decapsulation, the cyst choryon (outer hard shell) is removed, giving the sterile

decapsulated cysts for axenic hatching. Equipments and chemicals were autoclaved at

120oC prior to use. For this experiment, Artemia franciscana cysts, originating from the

Great Salt Lake, Utah, USA (EG® Type INVE Aquaculture, Belgium) were used.


33

Hydration (non-sterile)

1. 300 mg cysts were brought in a glass con with 98 mL tap water

2. Aerated for 1h

Decapsulation (sterile condition – in laminar flow with autoclaved chemicals and

equipments)

1. Brought the con with hydrated cysts into the laminar flow.

2. Disinfected tubing for sterile aeration and installed (sterile) aeration (see

figure 3.2).

3. Added 3.3 mL NaOH + 50 mL NaOCl to the cyst suspension, aerated for 2

minutes.

4. Added 70 - 100 mL Na2S2O3 to stop reaction.

5. Washed over 100 µm sieve with FASW.

6. Distributed the decapsulated cysts equally over several falcons with 30 mL

FASW.

7. For non-sterile hatching, added 20 uL from the stock of microbial community

background into hatching Falcon tubes. One falcon tube was not added to

serve as the control.

8. Hatched on the rotor at 4 rpm with constant light and 37 – 38oC for 22-24h.

9. For axenic Artemia, the sterility verification was done by plating 100 uL

hatching water on Marine Agar. 48h later, if any colony was found the

experiment had to be stopped.


34

Figure 3.2. Sterile aeration of the suspension of cysts

3.3.3. Experiment set-up

Five replicates were prepared for all treatments of the three subtests. For each replicate,

50 Artemia nauplii were cultured in glass tube with 20 mL of FASW in order to have

enough 50 living Artemia nauplii for each treatment at final sampling (concluded by

protocol optimization experiment).

Stocking was done 24h after hatching. For the sterile treatment (control treatment for the

subtest 1), the Artemia nauplii from axenic hatching were used while the nauplii from

non-axenic hatching were taken for the non-sterile treatments. For minimizing the

difference of the bacteria concentrations due to the difference in the amount of water

transferred during the counting step by using a micro-pipette, firstly the naupllii were

poured through a 100 µm sieve, and then washed with 100 mL FASW. 50 µL of hatching

water was added to tubes of non-sterile treatments, equivalent to ~ 105 cells/mL of

culture water. Probiotic and PHB particles were also added only one time during the

experimental time at stocking. All the tubes were placed randomly on the rotor at 4 rpm,

exposed to incandescent light at 28oC.

Sterile tubing (disinfected inside and outside)

Millipore filter

Sterile 1 mL pipette

Airpump

Tubing

Con


35

The daily feeding schedule was adjusted from Coutteau et al. (1990), aimed to provide ad

libitum ratios but avoiding excessive feeding which could affect the water quality in the

test tubes. Challenging with Vibrio campbellii was performed 36h after stocking,

followed with final harvest 24h later.

3.3.4. Sampling

3.3.4.1. Plating

The bacterial concentration in the culture water of different treatments for the three

subtests was verified by plating the water samples on Marine Agar and TCBS plates

using the Spiral Plater (Led techno Company). Plating was conducted two times during

the experimental time:

36h after stocking (just before challenging): water samples from one tube taken

randomly from each treatment were plated on Marine Agar plates.

60h after stocking (final sampling): water samples from 3 tubes taken randomly

from each treatment were plated on Marine Agar plates (for total bacterial

concentration) and TCBS plates (for Vibrio campbellii concentration).

3.3.4.2. Counting survival

Survival is one of the parameter of this experiment. It was measured only one time i.e. at

the final harvest. The counting step was done in sterile conditions since the samples were

needed for molecular analysis. Alive nauplii were counted one by one using a micro-

pipette.

3.3.4.3. Sampling for molecular analysis

For each treatment of three sub-tests, 50 survived Artemia nauplii were kept for

molecular analysis. Samples were washed by swirling the sieves with Artemia nauplii for

90 seconds in three Petri plates with 20 mL of 0.1% benzalkoniumchloride, 25 mL of

FASW and 5 mL of PCR water respectively. 50 nauplii were then transferred into 5 mL


36

of new FASW and homogenized for five minutes using a stomacher. Finally, all samples

were stored at – 20oC for further analysis.

3.3.5. Molecular analysis

3.3.5.1. DNA extraction

The modified QIAGENT DNA extraction protocols were used to extract the DNA from

Artemia using DNeasy kit (concluded to be efficient by optimization experiment). The

tube with 5 mL of homogenized sample was centrifuged for 10 minutes at 6000 rpm. The

supernatant was then poured off while the pellet was kept for further steps.

3.3.5.2. PCR amplification

On the extracted DNA template, a polymerase chain reaction (PCR) was performed to the

amplification of the DNA on the extracted DNA. Suggested by the result of the protocol

optimization experiment, nested PCR was used to avoid the interference of the DNA

from the host – the Artemia.

3.3.5.3. DGGE

The D-code universal mutation detection system (Bio-Rad, Hercules, California, USA)

was used for DGGE analysis. 50 µ PCR products were mixed with 20 µL loading dye

and then 10 µL of that solution were loaded into wells of polyacrylamide gel in 1X TAE

(10 µL each sample). The gels denaturation gradient ranges from 45 - 60%.

Electrophoresis was conducted with a constant voltage of 38V at 60oC for 16 hours. The

gel was then stained with SYBR green for 20 minutes. Finally, the gel was developed by

means of UV- transilluminator.


37

3.4. Data analysis

3.4.1. Survival

Within each subtest, the survival number for each treatment was converted to percentage.

The data were then analyzed by one-way ANOVA using the Statistical Analysis Software

(SAS) 9.2. The means were compared by a Turkey’s post hoc test at a 5% confidence

level.

3.4.2. Molecular fingerprints

DGGE outputs were processed with Bionumerics software version 5.1 (Applied Maths,

Sint-Martens-Latem, Belgium). The bands on the patterns were marked manually, then

the similarities between the PCR-DGGE banding patterns were analyzed using the

Pearson correlation coefficient and displayed graphically as a dendrogram.


38

CHAPTER 4. RESULTS

4.1. Main experiment

4.1.1. Subtest 1

4.1.1.1. Survival

The survival was the main parameter in this experiment. In the three subtests we tested

different treatments based on their capacity of protecting the Artemia nauplii from the

challenge with Vibrio campbellii strain LMG 21365. There were 5 replicates (5 tubes) for

each treatment. In each tube, 50 Artemia nauplii were fed daily with WT yeast. The

challenge with Vibrio campbellii was performed 36h after stocking, followed by the

survival counting 24h later.

Subtest 1 was aimed to test the effect of 100 mg/L PHB on survival of the non-challenged

and the challenged Artemia.

Subtest 1

0102030405060708090

100

Sterile Artemia NS Artemia NS Artemia +Challenge

NS Artemia +PHB

NS Artemia +PHB + Challenge

Treatments

Surv

ival

(%)

Figure 4.1. Survival of Artemia larvae in different treatments of subtest 1. 36h after stocking, the challenge was performed with Vibrio campbellii LMG 21365 at the concentration of 106 cells/mL. 24h later, the survival was determined. Bars indicate standard mean survival ± standard deviation. Error bars with different letters indicate significant difference (p < 0.05)

a

a

a

b

c


39

Results of the first challenge study (figure 4.1) showed that the significant difference in

the Artemia nauplii survival among the challenged and non-challenged treatmens (p <

0.05).

When not exposed to Vibrio campbellii, the survival of Artemia franciscana after 60h

culture fed with WT yeast Saccharomyces cerevisiae was really high, approximately

90%. The peak of survival was recorded for the control treatment of the non-treated non-

sterile Artemia (91.6 ± 2.61%), and it was found to be not significantly different from the

survival of the non-challenged sterile Artemia (90.8 ± 3.15%).

The survival for the challenged non-sterile Artemia was rather low (25.2 ± 4.82%), and it

was shown to be significantly lower than the non-challenged non-sterile Artemia. With

the addition of 100 mg/L PHB particles, the survival of the challenged non-sterile

Artemia was significantly improved (48.4 ± 5.18%) (p < 0.05).

High survival was also observed for the non-challenged non-sterile Artemia added with

PHB particles at the concentration of 100 mg/L (90.8 ± 3.03%). It was also shown to be

statistically similar to the survival of non-challenged non-sterile Artemia (p > 0.05).

4.1.1.2. Bacterial density

The three subtests were operated in the non-sterile conditions, starting with a total

bacterial density ~105 CFU/mL for all treatments by adding a certain amount of the

hatching water to all experimental tubes. During the experiment, the bacterial

concentration in the Artemia culture water was checked by plating the water samples

taken from experimental tubes on MA plates (for total bacteria density) and on TCBS

plates (for Vibrio campbellii density) plates.

Table 4.1 shows the plating counting results on MA plates for the total bacterial density

of the treatments in subtest 1. It was illustrated that the bacterial density in the culture

water was increased around 2 log units in all trials from the stocking to the harvest 60h


40

later, and there was not much difference in the total bacteria density among all treatments

(treated and non-treated with PHB particles).

Table 4.1. Growth of bacteria (CFU/mL) on Marine Agar plates during the subtest 1

Treatments CFU at 36h

(x106)

CFU at 60h

(x107)

NS Artemia 3.00 ± 0.14 1.01 ± 0.27

NS Artemia + Challenge 3.05 ± 0.35 0.89 ± 0.13

NS Artemia + PHB (100mg/L) 4.55 ± 0.07 0.73 ± 0.15

NS Artemia + PHB (100mg/L)+ Challenge 4.50 ± 0.28 0.62 ± 0.12

Table 4.2 indicates the Vibrio campbellii concentration in the culture water in different

treatments of subtest 1. No colony was observed for the non-challenged treatments. For

the challenged group, at harvest the Vibrio campbellii concentration was decreased 1 log

unit compared to the initial density (106 cells/mL). No considerable difference was found

for the Vibrio campbellii density between the two challenged treatments.

Table 4.2. Growth of bacteria (CFU/mL) on TCBS plates during the subtest 1

Treatments CFU at 60h (x105)

NS Artemia 0

NS Artemia + Challenge 2.79 ± 0.27

NS Artemia + PHB (100mg/L) 0

NS Artemia + PHB (100mg/L)+ Challenge 3.57 ± 0.16

4.1.1.3. Molecular analysis

The molecular analyses for the samples collected from the three subtests of the main

experiment were processed at the same time. Unfortunately, the molecular results for the

all three subtests could not be obtained due to the failure of DNA extraction step,


41

possibly caused by the expiry of the DNA extraction kit. Therefore, the molecular

analysis will not be mentioned again in the result parts of the subtest 2 and 3.

4.1.2. Subtest 2

4.1.2.1. Survival

In the subtest 2, the effects on challenging Artemia supported by the probioticum M13

alone and in combination with 100 mg/L PHB particles were tested.

Figure 4.2 shows the significant difference (p < 0.05) in survival among the non-sterile

treatments of the subtest 2.

Without the presence of Vibrio campbellii, like in the subtest 1, the survival for all non-

challenged treatments was high, about 90%. With the addition of M13, the survivals of

the non-sterile Artemia in the treatments with and without 100 mg/L PHB particles were

still high (91.6 ± 3.58% and 85.6 ± 4.77% respectively). No significant difference in

survival between them was found (p > 0.05).

Considering the challenged treatments, the survival was notably poorer (Figure 4.2). The

lowest survival was recorded for the control treatment of non-sterile Artemia (26.4 ±

2.61%), obviously lower than that of the non-challenged non-sterile Artemia (p < 0.05).

Moreover, it was shown to be statistically lower than the other challenged treatments also

(p < 0.05).

The survivals for the two treatments of challenged non-sterile Artemia with the addition

of PHB particles (41.2 ± 4.82%) and with M13 (44.8 ± 4.15%) were significantly

enhanced (compared to the challenged non-sterile Artemia) (Figure 4.2). Statistic analysis

showed no significant difference in survival between these two treatments (p > 0.05).


42

The survival for the treatment of challenged non-sterile Artemia supported by the

combination of 100 mg/L PHB particles and M13 was 58.0 ± 4.0%, significantly higher

than the rest three challenged treatments (p < 0.05) (Figure 4.2).

Subtest 2

0102030405060708090

100

NS Artemia NS Artemia+ Challenge

NS Artemia+ PHB +Challenge

NS Artemia+ PHB +

M13

NS Artemia+ M13

NS Artemia+ M13 +

Challenge

NS Artemia+ PHB +M13 +

Challenge

Treatments

Sur

viva

l (%

)

Figure 4.2. Survival of Artemia larvae in different treatments of subtest 2. 36h after stocking, the challenge was performed with Vibrio campbellii LMG 21365 at the concentration of 106 cells/ml. 24h later, the survival was determined. Bars indicate standard mean survival ± standard deviation. Errors bars with different letters indicate significant difference (p < 0.05)


Looking at table 4.3 below, like in the subtest 1, the increasing trend of the total bacterial

concentration in the water samples could also be observed. In addition, at both 36h (just

before challenging) and 60h (24h after challenging), there was a difference in the total

bacterial concentration between with (higher) and without the presence of M13.

However, within the treatments treated with M13, there was no notable difference in the

total bacterial density between with and without the PHB addition.

b

e d

a ab

d c


43



CFU at 60h (x107)

NS Artemia 3.05 ± 0.21 0.66 ± 0.04 NS Artemia + Challenge 3.45 ± 0.07 0.63 ± 0.15 NS Artemia + PHB + Challenge 4.75 ± 0.21 0.79 ± 0.06 NS Artemia + PHB + M13 12.20 ± 0.57 1.50 ± 0.13 NS Artemia + M13 8.60 ± 0.28 1.52 ± 0.30 NS Artemia + M13 + Challenge 8.20 ± 0.14 1.43 ± 0.12 NS Artemia + PHB + M13 + Challenge 11.95 ± 0.35 1.79 ± 0.17

The pattern of the TCBS plate counting from the subtest 1 was also observed in the

subtest 2 with the decreasing trend for the Vibrio campbellii concentration. No difference

in the Vibrio campbellii density among the challenged treatments was found (Table 4.4).


Treatments CFU at harvest (x105)

NS Artemia 0 NS Artemia + Challenge 5.39 ± 0.24 NS Artemia + PHB + Challenge 5.17 ± 0.64 NS Artemia + PHB + M13 0 NS Artemia + M13 0 NS Artemia + M13 + Challenge 5.32 ± 0.30 NS Artemia + PHB + M13 + Challenge 5.11 ± 0.18

4.1.3. Subtest 3

4.1.3.1. Survival

The subtest 3 was designed equally to the subtest 2 but analyzed the effect of the

probioticum B12. Figure 4.7 shows very good survivals for the non-challenged

treatments. They were 86.4 ± 3.85%, 85.6 ± 4.77% and 86.0 ± 6.63% for non-sterile

Artemia, non-sterile Artemia with PHB and B12 and non-sterile Artemia with only B12

respectively. In addition, the Turkey’s post hoc test at a 5% probability level found no

statical difference among the three above treatments (p > 0.05) (Figure 4.3).


44

As challenged with Vibrio campbellii, like in the subtest 1 and 2, the poorest survival was

for the treatment of non-sterile Artemia (19.6 ± 7.80%), statistically lower than the non-

challenged non-sterile Artemia (p < 0.05) (Figure 4.3). Vibrio campbellii also caused the

low survival in the treatment of non-sterile Artemia with the probiotic strain B12 (26.8 ±

5.4%). The survival for the treatment of non-sterile Artemia with PHB particles (53.6 ±

6.23%) was significantly improved compared to the challenged non-sterile Artemia and

the challenged non-sterile Artemia with B12 (p < 0.05).

Subtest 3

0102030405060708090

100

NS Artemia NS Artemia+ Challenge

NS Artemia+ PHB +Challenge

NS Artemia+ PHB +

B12

NS Artemia+ B12

NS Artemia+ B12 +

Challenge

NS Artemia+ PHB +

B12 +Challenge

Treatments

Surv

ival

(%)

Figure 4.3. Survival of Artemia larvae in different treatments of subtest 3. 36h after stocking, the challenge was performed with Vibrio campbellii LMG 21365 at the concentration of 106 cells/ml. 24h later, the survival was determined. Bars indicate standard mean survival ± standard deviation. Error bars with different letters indicate significant difference (p < 0.05)

When exposing to virulent Vibrio campbellii, the best survival was found for the

treatment of non-sterile Artemia treated with the combination of 100 mg/L PHB particles

and probioticum B12 (69.2 ± 8.20%), and it was significantly higher than all other

challenged treatments (Figure 4.3).


Table 4.5 illustrates the results for MA plate counting for the subtest 3. Like in the two

previous subtests, the total bacterial concentration in culture water increased with 2 log

a a a b c

d d


45

units after 60h. It can be seen that before adding the challenge, the treatments treated with

B12 had a higher bacterial density compared to the treatments without B12. Nevertheless,

at harvest, that difference was not observed anymore. For the both treatments of treated

and non-treated with B12, there was no difference in the total bacterial density between

with and without 100 mg/L PHB particles.



CFU at 60h (x107)

NS Artemia 4.15 ± 0.21 1.01 ± 0.11 NS Artemia + Challenge 4.30 ± 0.28 0.79 ± 0.04 NS Artemia + PHB + Challenge 4.40 ± 0.42 0.81 ± 0.32 NS Artemia + PHB + B12 8.60 ± 0.42 1.01 ± 0.12 NS Artemia + B12 8.50 ± 0.28 1.01 ± 0.07 NS Artemia + B12 + Challenge 7.70 ± 0.28 0.95 ± 0.09 NS Artemia + PHB + B12 + Challenge 8.95 ± 0.35 1.11 ± 0.07

At harvest, no colony was seen on TCBS plates for water samples from the non-

challenged treatments of the subtest 3 (Table 4.6). For the group with the addition of

challenge, the Vibrio campbellii density in the culture water was almost the same for all

treatments. It was decreased with 1 log unit compared to the initial concentration (106

CFU).



NS Artemia 0 NS Artemia + Challenge 1.60 ± 0.12 NS Artemia + PHB + Challenge 3.02 ± 0.61 NS Artemia + PHB + B12 0 NS Artemia + B12 0 NS Artemia + B12 + Challenge 1.58 ± 0.59 NS Artemia + PHB + B12 + Challenge 3.33 ± 0.16


46

4.2. Protocol optimization experiments

The analysis of the microbial community associated with Artemia larvae treated with

probiotics and PHB particles may be important for this kind of research. Due to the small

size of the experimental animals, it is not possible to separate the bacteria from the host

before analysis. It has been shown in experiments with larvae of cod/sea bass/... that a

matrix containing both the bacterial and the host genome can result in problems during

PCR amplification. The main problem is the amplification of eukaryotic DNA originating

from the host, in addition to the 16S rRNA genes from the bacteria, due to the non-

specific activity of the used primer pair. In addition, this is the first investigation targeting

the intestinal microbial community in the live feed used for aquaculture rather than the

microbial community prevailing on both the in- and outside of the live feed. Therefore,

both the sampling protocol to obtain mainly the intestinal microbial community and the

molecular protocol for bacteria specific amplification needed to be optimized.

4.2.1 First protocol optimization experiments

4.2.1.1 Single round PCR versus nested PCR

In a first instance, the PCR protocol for the amplification of the bacterial 16S rRNA

genes from an Artemia DNA containing matrix was optimized. As DNA template, the

DNA extracts from the sterile Artemia, non-sterile and non-washed Artemia, non-sterile

and washed Artemia, and washing water were used in both the single round PCR (primer

pair GC-338F/518R) and the nested PCR (primer pairs EUBF8-27/984R and GC-

338F/518R).

When verifying the PCR fragment length on agarose gel by running it next to a DNA mix

ladder, it was observed that the samples of the sterile Artemia showed a band when

amplified in the single round PCR. This was not so in the case of the nested PCR (data

not shown).


47

To verify the absence of amplified PCR products in the nested approach, all PCR

amplified samples were loaded for DGGE (Figure 4.4 and 4.5).

Figure 4.4. DGGE comparison of the PCR amplified DNA extracts from sterile Artemia. PCR was performed in a single round PCR (primer pair GC-338F/518R) and the nested PCR (primer pairs EUB8F-27/984R and GC-338F/518R). The DGGE analysis showed that the PCR product of the sterile Artemia amplified with

the primer pair GC-338F/518R resulted in clear band, whereas the product amplified in

the nested PCR approach showed no band at all (Figure 4.4).

Figure 4.5. DGGE comparison of the PCR amplified DNA extracts from non-sterile and non-washed Artemia, non-sterile and washed Artemia, and washing water. PCR was performed in a single round PCR (primer pair GC-338F/518R) and the nested PCR (primer pairs EUB8F-27/984R and GC-338F/518R). Upon comparison of the two PCR approaches for amplification of the non-sterile, washed

and non-sterile, non-washed Artemia DNA extracts, it could be determined that the

patterns showed a similarity value of 92.1% and 79.7%, respectively (Figure 4.5). The

nested PCR approach seemed to results in slightly lower diversity than the single round

Nested PCR

Single-round

Non-washed Nested PCR

Washed Nested PCR

Washed Single-round PCR

Non-washed Single-round PCR

Washing water Nested PCR

Washing water Single-round PCR

Reference 1

Reference 2


48

PCR as illustrated by the lower number of bands in the patterns. Finally, the amplification

of the bacterial DNA extracted from the washing water did not seem to result in

detectable bands when using the single round PCR. When using the nested approach,

several bands also occurring on the patterns of the non-sterile Artemia were observed.

4.2.1.2. DNA extraction methods: CTAB versus adjusted DNeasy

A DGGE was performed to compare the patterns obtained with the CTAB and adjusted

DNeasy DNA-extracts from sterile, non-sterile and non-washed, non-sterile and washed,

and washing water amplified by the nested PCR approach (Figure 4.6).

Figure 4.6. DGGE comparison of the CTAB and adjusted DNeasy extraction method. The bacterial community patterns from non-sterile and non-washed Artemia, non-sterile and washed Artemia, and washing water are visualized by means of DGGE. In Figure 4.6, it can be seen that the band patterns obtained with the CTAB and adjusted

DNeasy were clearly different from each other for the non-sterile and non-washed

Artemia (20.6% similarity) and the non-sterile and washed Artemia (13.6% similarity).

The band patterns for the washing water were almost the same (80.5% similarity) but

more intense for the adjusted DNeasy method.

Washing water CTAB

Washing water DNeasy

Non-washed CTAB

Washed CTAb

Non-washed DNeasy

Washed DNeasy

Reference 1


49

4.2.2. Second protocol optimization experiments

4.2.2.1. Minimal amount of Artemia nauplii needed for sampling

From the second protocol optimization experiment, the samples of one, five, ten, twenty

and fifty Artemia were collected to determine the lowest amount of Artemia naupllii that

should be collected for molecular analysis.

DNAs from those samples were extracted by the adjusted DNeasy method, followed by

nested PCR with primer pairs EUBF8-27/984yR and GC-338F/518R. By verifying on

agarose gel, it could be seen that there was no band for the samples of one, five and ten

Artemia, while clear bands were obtained for samples of twenty and fifty Artemia. A

DGGE was also performed with PCR products from those samples (Figure 4.7).

Figure 4.7. Comparison of the band pattern intensification of the PCR samples of one, five, ten, twenty and fifty Artemia, DNA extracted with adjusted DNeasy method, visualized by means of DGGE. PCR was performed with the nested PCR (primer pairs EUBF8-27/984YR and GC-338F/518R).

Looking at the DDGE output, only the banding patterns for the samples of twenty and

fifty Artemia could be observed. Those two patterns looked almost the same (74%

similarity), but the one for the sample of fifty nauplii was much more intense.

1 nauplius

Reference 1

20 nauplii

50 nauplii

5 nauplii

10 nauplii


50

4.2.2.1. The reproducibility of three batches of Artemia treated in the same

way in non-sterile conditions

To test the reproducibility for molecular analysis of batches treated in non-sterile

conditions but in the same way, the samples of fifty nauplii from three batches were

compared.

Those samples were DNA-extracted with adjusted DNeasy and then developed with

nested PCR (primer pairs EUBF8-27/984yR and GC-338F/518R). At last, a DGGE was

conducted for PCR amplified samples (Figure 4.8).

Figure 4.8. DNA extracted with adjusted DNeasy method, visualized by means of DGGE. PCR was performed with the nested PCR (primer pairs EUBF8-27/984YR and GC-338F/518R).

Figure 4.8 shows the clear patterns for the three samples of fifty Artemia nauplii from

three batches. It could also been indicated the similarity values of 90.8%, 76.4% and

89.7% respectively. We also could not find major differences among the three patterns in

terms of band intensity.

Batch 1

Batch 2

Batch 3

Reference 1

Reference 2


51

CHAPTER 5. DISCUSSSION

5.1. The main experiment

5.1.1. Subtest 1

The subtest 1 was aimed to test the difference in survival of Artemia nauplii cultured in

sterile and non-sterile conditions, and the effect of PHB at the concentration of 100 mg/L

on the survival of the non-sterile Artemia challenged with virulent Vibrio campbellii

LMG 21365.

Figure 4.1 shows the high survival of the Artemia franciscana nauplii in all non-

challenged treatments. After 60h culture with the WT yeast strain Saccharomyces

cerevisiae as the food source, the survival of the non-sterile Artemia (91.6 ± 2.61%) was

a bit higher than the sterile Artemia (90.8 ± 3.15%), but there was no statistical difference

between them. In addition, no colony was found on TCBS plates for the non-challenged

treatments, means that there was no presence of Vibrio strains in the seawater used in our

experiment. From above results, it can be assumed that without the presence of

pathogens, the non-sterile conditions with normal bacteria has no negative impact on the

Artemia naupllii during the experimental time (60h).

The survival for the treatment of the non-sterile Artemia with 100 mg/L PHB particles

was also statistically shown to be the same with the two above non-challenged

treatments. It is suggested that in non-sterile conditions, the presence of PHB particles at

the concentration of 100 mg/L did not have any impact on the survival of the non-

challenged Artemia franciscana nauplii. Moreover, data from Table 4.1, which

represented the counting result on Marine Agar plates, showed that the bacteria were able

to grow in the non-sterile conditions of our Artemia cultures, from ~105 CFU/mL at the

stocking to ~ 107 CFU/mL at the harvest for all treatments. At both 30h and 60h after the

stocking, the total bacterial concentration was almost the same for the both treated and

non-treated with PHB particles. It demonstrated that in non-sterile conditions of the


52

Artemia cultures, PHB particles at the concentration of 100 mg/L did not have a

remarkable effect on the growth of the whole bacterial community in the culture water.

Additionally, it can be also supposed that the PHB-degrading bacteria seems not exist in

the normal cultures or at too low concentration to be highly active.

In previous research, Defoirdt et al. (2007) showed that the addition of 1000 mg/L PHB

particles to the Artemia culture water protected completely the host from the virulent

Vibrio campbellii strain in the sterile conditions. The same study revealed that at a lower

concentration (100 mg/L) and also in the sterile conditions, PHB particles still had

partially protective effect. In the first challenge test, we aimed at investigating whether

the PHB at the concentration 100 mg/L could be used to protect the Artemia from the

pathogenic Vibrio campbellii in the non-sterile conditions.

Figure 4.1 shows that the challenged treatment with PHB particles had significantly

higher survival than the treatment of challenged non-sterile Artemia (48.4 ± 5.18%

relative to 25.2 ± 4.82%) (p < 0.05). It appears that even at a concentration of 100 mg/L

and in the non-sterile conditions, PHB particles still have a protective effect but not

complete on Artemia franciscana infected with Vibrio campbellii. This result is the same

to a previous research (Defoirdt et al., 2007), in which this PHB concentration was also

shown to be only suboptimal in protecting Artemia against infection, so that more

protective effect with the addition of a PHB-degrading bacterium could be shown. It also

can be observed in our study that the normal microbial community was not able to

increase the effectiveness of PHB. It suggests that the addition of PHB degraders is

needed.

Table 4.2 shows no difference in the Vibrio campbellii concentration between the two

challenged treatments treated and non-treated with 100 mg/L PHB particles. When

related to the better survival data of the PHB-treated treatment, it could be assumed that

in the non-sterile conditions, 100 mg/L PHB particles do not kill off the challenge (Vibrio

campbellii) but decrease the Vibrio campbellii pathogenicity and/or growth by forcing it

to spend energy to maintain the intracellular pH (Defoirdt et al., 2009). The decreasing


53

trend of Vibrio campbellii was found in all three subtests of this experiment. That was not

caused by the activity of PHB particles but the poor food source and the grazing impact

from Artemia cultures.

A noticeable negative impact of the non-sterile conditions on PHB particles came from

the growth of bacteria on the particle surface. This could cause the clustering of the PHB

particles, which could decrease the protective effect of PHB particles in longer term

experiments.

5.1.2. Subtest 2

The subtest 2 was aimed to test the protective effect of different treatments of

probioticum M13 on the non-sterile Artemia nauplii challenged by Vibrio campbellii

LMG 21365. In the subtest 2, the protective effect of PHB particles at the concentration

of 100 mg/L against Vibrio campbellii was again confirmed. The survival of the non-

sterile Artemia with PHB treatment was 41.2 ± 4.82%. This was significantly higher than

the control treatment of the non-sterile Artemia with challenge, which was only 26.4 ±

2.61%.

For the non-challenged group, Figure 4.2 indicates that the treatments with M13 and M13

+ PHB particles did not have a negative impact on the Artemia survival. The survival of

the treatment of M13 and PHB particles was even higher than that of the control

treatment (non-sterile Artemia fed with wild type (WT) yeast). The reason for this

difference might be the quality of the food sources. Since WT is a poor food source for

Artemia (Marques et al., 2006) and M13 was shown to have no function as a food source

(Liu et al., 2010), that difference could be caused by the presence of β-hydroxybutyrate

(degraded from PHB particles by the probiotic M13), which could act as the extra energy

source for Artemia (Defoirdt et al., 2009).

The protecting effect against Vibrio campbellii was also observed for the treatment of

M13 alone. The survival recorded from this treatment was 44.8 ± 4.15%, even


54

statistically higher than that of the challenged treatment treated with only PHB particles.

This outcome is different from a previous study (Liu et al., 2010), which indicated that in

the sterile conditions, M13 had no effect on the challenged Artemia. From this result,

M13 could be considered to be a probioticum in the non-sterile conditions, since it could

decrease the bad impact of the pathogenic Vibrio campbellii. It is possible that in the non-

sterile Artemia cultures of our experiment, M13 could grow better, so that it was able to

play a more prominent role against the challenger. This assumption is confirmed by Table

4.3, which showed the higher total bacterial concentration for the treatments supplied

with the probioticum M13 (added 107 cells/mL of culture water at stocking) compared to

the treatments without M13 at both 36h and 60h. That result proclaimed that M13 was

able to develop in the non-sterile conditions of our experiment. Another possible reason

may be the different Vibrio campbellii stock that we used, PMG 21365, which is stronger

than PMG 21363 (used in previous study of Liu et al., 2010). It is likely that when

exposed to a stronger pathogen, we could see more effect from the probiotic M13.

Moreover, concerning the challenged Artemia, figure 4.2 shows the best survival for the

treatment of M13 with PHB particles at 100 mg L-1 (58.0 ± 4.0%, i.e. statistically higher

than that of the treatments with only PHB or M13). This result was alike to a previous

study (Liu et al., 2010), in which M13 was shown to be able to degrade PHB particle to

the form of β-hydroxybutyrate, which was explained to defend Artemia from bacterial

infections by providing energy to the intestinal mucosa, thereby increasing intestinal

health and resistance to infections, or by diminishing the growth and/or the virulence of

the pathogens (Defoirdt et al., 2009). In the non-sterile conditions of our experiment, that

explanation seems still valid. The protective effect of this treatment (PHB + M13) could

be enhanced if with the addition of PHB particles, M13 could grow better to create more

competing impact on Vibrio campbellii. However, when considering all treatments

containing M13, no remarkable dissimilarity in the total bacterial density between with

and without PHB particles was found (Table 4.3). It is likely that in the non-sterile

conditions, PHB particles at 100 mg/L do not have an effect on the growth of the isolate

M13 in the culture water.


55

The Vibrio campbellii density in the water samples is shown in Table 4.4 to be not

different between the non-treated challenged treatment and the treated challenged ones, in

which a difference in survival Artemia naupllii was recorded. It means that in all

challenged treatments, the growth of Vibrio campbellii in the culture water was not

prohibited. If we could accept that there is a correlation between the Vibrio campbellii

densities inside and outside the Artemia then we could say that all the therapeutics in the

subtest 2 could only restrain the activity of Vibrio campbellii but at different levels.

5.1.3. Subtest 3

Looking at Figure 4.3, a high survival was obtained for the all tree treatments in the non-

challenged group of the subtest 3 (the non-sterile Artemia, the non-sterile Artemia with

B12 and the non-sterile Artemia with B12 and PHB particles at 100 mg/L). Moreover, no

significant difference was found among them. It demonstrated that in the non-axenic

conditions, the presence of B12 or the combination of B12 and PHB particles did not

have an impact on the survival of the non-challenged brine shrimp.

Similar to the two previous subtests, the challenged treatment treated with 100 mg/L PHB

particles gave significantly better survival than that of the control with the challenged

non-sterile Artemia (53.6 ± 6.23% compared with 19.6 ± 7.80%). Higher survival relative

to the non-treated treatment was also recorded for the treatment of B12 alone (26.8 ±

5.4%), but it was not significant. This result is different from that in a previous study (Liu

et al., 2010), which showed that in the non-sterile conditions, the treatment of B12

without PHB resulted in statistically higher survival than the non-treated challenged

treatment (control).

It could be assumed that the probiotic B12 is effective in protecting Artemia against the

pathogen (Vibrio campbellii) only under the sterile conditions. While in the non-sterile

conditions, it shows a lower shielding effect, possibly caused by the interference of other

bacteria, especially from Vibrio campbellii. That assumption was proven by the results of

plate counting. It is shown by Table 4.5 that the difference in the total bacterial

concentration between treated and non-treated with B12 could not be observed anymore


56

after the addition of Vibrio campbellii. Moreover, for both groups of treated and non-

treated with B12, there was no difference between with and without the addition of PHB

particles. It revealed that in the non-sterile conditions, B12 could not dominate the other

bacteria, even when combined with 100 mg/L PHB particles.

Figure 4.3 shows the best survival for the challenged non-sterile Artemia nauplii obtained

by the treatment of B12 with PHB particles (69.2 ± 8.20%). This optimal defensive effect

on challenged Artemia of the combination treatment could be again explained by the

capacity of microbial degradation of PHB by the isolate B12, resulted in the main end

product β-hydroxybutyrate, which could protect brine shrimp from the pathogen

(Defoirdt et al., 2009). The above results proclaimed that in the non-sterile conditions,

B12 alone could be less effective on enhancing survival for challenged Artemia than in a

the sterile context, but with the addition of PHB particles at 100 mg L-1 it could show the

protective effect again.

The Vibrio campbellii concentrations in the water samples are shown (Table 4.6) to be

not different between the treated challenged treatments and the non-treated challenged

one (control). It could be implied that all the therapeutic methods in subtest 3 were not

able to eliminate the Vibrio campbellii outside the challenged Artemia and somehow also

inside the Artemia gut. It is highly possible that the difference in survival of the Artemia

naupllii recorded in the challenged treatments was not caused by the Vibrio campbellii

elimination capacities of different treatments. In other words, all the treatments in the

subtest 2 were only able to decrease the pathogenicity of Vibrio campbellii but at

different levels.


57

5.2. Sampling protocol and molecular protocol optimization

5.2.1. Single round PCR versus nested PCR

Single round PCR and nested PCR were performed on the samples of axenic and non-

axenic Artemia. It was observed on the DGGE pattern that in the case of single round

PCR using the GC-338F/518R primer pair, non-specific amplification occurred (figure

4.4). This should be avoided as it could result in mis-interpretation of the bacterial DGGE

band patterns. By using a nested PCR approach with the primer pair EUBF8-27/984R for

the external round and primer pair GC-338F/518R for the internal round, no

amplification of eukaryotic DNA was observed (Figure 4.4). However, the use of the

nested approach seemed to result in a lower band richness indicating that the external

primer pair left out some bacterial strains during amplification (Figure 4.5). It was

attempted to decrease this problem by the use of the reverse primer 984yR rather then

984R, as it is known to target more species (Wang and Qian, 2009).

5.2.2. Method for DNA extraction: CTAB versus adjusted DNeasy

When comparing the DGGE gel for the adjusted DNeasy and the CTAB method, two

different lay-outs of bands were observed (figure 4.6). The fact that the patterns for the

washing water were highly similar for the two extraction methods indicates a similar

extraction efficiency.

The differences in the patterns for the non-sterile Artemia between the two extraction

methods might be resulted from differences in extraction efficiencies for the dominating

DNA. The pattern from the washing water was almost the same to the patterns of the

non-sterile Artemia when extracted with the CTAB method. It is likely that the adjusted

DNeasy method is more efficient in extracting the DNA from the Artemia matrix,

resulting in a lower signal from the washing water after PCR. This was also enhanced by

using an adjusted washing protocol. The nauplii will be washed 3 times by swirling the

sieves with Artemia nauplii for 30 seconds consequently in three Petri plates with 25 mL

of FASW, 20 mL of 0.1% benzalkoniumchloride and 20 mL of PCR water.


58

5.2.3. Minimal amount of Artemia needed for sampling

Minimizing the amount of Artemia required for sampling is very important for this

research as it can provide many benefits. Fewer number of Artemia collected at sampling

means less effort for Artemia culturing, counting, feed preparing and so on.

Using the adjusted DNeasy method, five DNA-extracts originating from one, five, ten,

twenty and fifty nauplii were tested with nested PCR and DGGE. The output showed on

agarose gel indicated that twenty nauplii is the minimum number that is needed to

provide enough materials for nested PCR with 20 cycles for the first round and also 20

cycles more for the second round. When looking to the DGGE gel (Figure 4.7), it can be

seen that the band patterns for the samples of twenty and fifty Artemia are almost the

same, but fifty Artemia offered a more intensive lay-out. From those results, it is highly

recommended that fifty Artemia nauplii should be collected at each sampling for

molecular analysis.

5.2.4. The reproducibility of three batches of Artemia treated in the same way in non-sterile conditions In the main experiment, the three subtests with different treatments were cultured in non-

sterile conditions. As a result, there were some uncontrollable exogenous factors, which

may have resulted in shifts in the microbial patterns.

In the second protocol optimization experiment, three batches were performed in the non-

sterile conditions but treated in the same way. Three samples of 50 Artemia nauplii taken

from each batch were molecularly analyzed to verify the reproducibility.

The DGGE output showed only few differences among the lay-outs of three samples

from three batches (Figure 4.8). The similarity value was high enough to make a

conclusion that different batches of non-sterile Artemia cultures treated in the same way

could give a high reproducibility in the cases of molecular analysis.


59

5.3. The failure of the molecular analysis for the main experiment

By one part of this study, we aimed to investigate the interaction among normal bacteria,

probiotics and pathogen with/without the presence of PHB particles in the gut of non-

sterile Artemia by using the genetic fingerprint method based on polymerase chain

reaction (PCR) amplification of 16S rDNA and denaturing gradient gel electrophoresis

(DGGE). This method could provide information about all the dominant bacteria that

present in the Artemia intestinal microbial community and how they interact with each

others. By relating the molecular results with the survival data, the anti-pathogenic

mechanism of two probioticums with/without PHB particles could be observed.

Unfortunately, the molecular results could not be obtained by this study since all the

samples for molecular analysis of the three subtests were ruined due to an unexpected

failure of DNA extraction step. As the consequence, the question that how the two

isolates M13 and B12 when combined with PHB particles could protect the non-sterile

Artemia from the pathogenic Vibrio campbellii could not be answer. Further research on

this matter is recommended.


60

CHAPTER 6. CONCLUSIONS AND FUTURE

PERSPECTIVES

6.1. Conclusions

The following conclusions can be drawn from the obtained results:

Also under non-axenic conditions, the addition of either two PHB-degraders M13

and B12, together with PHB particles at 100 mg/L significantly increased the

survival of brine shrimp larvae infected with the virulent pathogen Vibrio

campbellii LMG 21365.

In non-sterile conditions, the isolate M13 could play a probiotic role by providing

a considerable protection for Artemia against the virulent Vibrio campbellii LMG

21365.

In non-sterile conditions, the administration of either two PHB-degrading isolates

M13 and B12, with or without PHB particles at 100 mg/L did not have a negative

impact on the survival of the non-challenged Artemia.

Under conditions of normal non-sterile cultures, at the concentration of 100 mg/L,

PHB particles were able to protect partially Artemia from the virulent Vibrio

campbelli LMG 21365. When not exposed to a challenger, PHB particles at the

same concentration had no negative impact on the survival of Artemia.


61

In addition, some conclusions can be made about the molecular research using Artemia as

an experimental model:

Amplification of eukaryotic DNA in general and Artemia DNA in particular can

be avoided by using nested PCR approach with the primer pair EUBF8-27/984yR

for the external round and the primer pair GC-338F/518R for the internal round.

It is likely that the adjusted DNeasy method is more efficient over CTAB in

extracting the DNA from the Artemia matrix, resulting in a lower signal from the

washing water after PCR.

For molecular analysis, samples of 50 Artemia nauplii are recommended.

Batches of Artemia cultured under non-sterile conditions treated in the same way

can give a high reproducibility of results using molecular analysis.

6.2. Perspectives

The experiment was conducted under non-sterile conditions and showed really good

results with major increase in survival for challenged brine shrimp. The dose of the PHB

particles (100 mg L-1) is economical reasonable and possibly decreased. Moreover, the

involved isolates are easily developed. The both aspects make this research vastly

practical. These treatments should be studied on other aquaculture animals for expanding

this line of applications.

However, the link between the survival and the change of microbial community in the gut

of the host could not be investigated by the molecular techniques in this work. Further

research is recommended to yield more information on this matter.


62

REFERENCES Aarestrup, F. M., Seyfarth, A. M., Emborg, H. D., Pedersen, K., Hendriksen, R. S. and Bager, R. S., (2001). Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in Denmark. Antimicrobial Agents and Chemotherapy 45(7), pp. 2054-2059. Aguirre-Guzmán et al., (2001). Differences in the susceptibility of American white shrimp larval substages (Litopenaeus vannamei) to four Vibrio species. J. Invertebr. Pathol. 78, pp. 215–219. Albaladejo, J.D., (2001). Philippines: national review on management strategies for major diseases in shrimp aquaculture, pp. 67–73. In: Subasinghe, R., Arthur, R., Phillips, M.J., Reantaso, M. (Eds.). WB/NACA/WWF/FAO. Thematic Review on Management Strategies for Major Diseases in Shrimp Aquaculture. Proceedings of a Workshop held in Cebu, Philippines, 28–30 November 1999. Alday de Graindorge, V. and Griffith, D., (2001). Ecuador: national review on management strategies for major diseases in shrimp aquaculture,pp. 17–19. In: Subasinghe, R., Arthur, R., Phillips, M.J., Reantaso, M. (Eds.). WB/NACA/WWF/FAO. Thematic Review on Management Strategies for Major Diseases in Shrimp Aquaculture. Proceedings of a Workshop held in Cebu, Philippines on 28–30 November 1999. Austin, B. and Zhang, X. H., (2006). Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett. Appl. Microbiol. 43, pp. 119–124. Avnimelech, Y., (1999). Carbon/nitrogen ratio as a control element in aquaculture systems. Aquaculture 176, pp. 227–235. Balcázar, J. L., Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Muzquiz, J. L. and Girones, O., (2008). Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture 278(1-4), pp. 188-191. Bearson, S., Bearson, B., Foster, J.W., (1997). Acid stress responses in enterobacteria. FEMS Microbiol Lett 147, pp. 173-180.


63

Bondad-Reantaso, M. G., Subasinghe, R. P., Arthur, J. R., Ogawa, K., Chinabut, S., Adlard, R., Tan, Z. and Shariff, M., (2005). Disease and health management in Asian aquaculture. Veterinary Parasitology 132(3-4), pp. 249-272. Buchmann, K., Dalsgaard, I., Nielsen, M. E., Pedersen, K., Uldal, A., Garcia, J. A. and Larsen, J. L., (1997). Vaccination improves survival of Baltic salmon (Salmo solar) smolts in delayed release sea ranching (net-pen period). Aquaculture 156(3-4), pp. 335-348. Cabello, F.C., (2006). Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment, Environ. Microbiol. 8, pp. 1137–1144. Campoccia, D., Montanaro, L., Speziale, P. and Arciola, C. R., (2010). Antibiotic-loaded biomaterials and the risks for the spread of antibiotic resistance following their prophylactic and therapeutic clinical use. Biomaterials 31(25), pp. 6363-6377. Chanratchakool, P., Fegan, D.F., Phillips, M.J., 2001. Thailand: national review on management strategies for major diseases in shrimp aquaculture,pp. 85–90. In: Subasinghe, R., Arthur, R., Phillips, M.J., Reantaso, M. (Eds.). WB/NACA/WWF/FAO. Thematic Review on Management Strategies for Major Diseases in Shrimp Aquaculture. Proceedings of a Workshop held in Cebu, Philippines on 28–30 November 1999. Cherrinton, C.A., Hinton, M., Pearson, G.R., and Chopra, I., (1991). Short-chain organic acids at pH 5.0 kill Escherichia coli and Salmonella spp. without causing membrane pertubation. Journal of Applied Bacteriology 70, pp. 161-165. Coutteau, L. B., Lavens, P., Sorgeloos, P., (1990). The use of manipulated baker's yeast as an algal substitute for the laboratory culture of Anostrata. Hydrobiologa 234, pp. 25-32. Coutteau, P., Hadley, N. H., Manzi, J. J., and Sorgeloos, P. (1994). Effect of algal ration and substitution of algae by manipulated yeast diets on the growth of juvenile Mercenaria mercenaria. Aquaculture 120(1-2), pp. 135-150. Crosbie, P.B.B., Nowak, B.F., (2004). Immune responses of barramundi, Lates calcarifer (Bloch), after administration of an experimental Vibrio harveyi References


64

bacterin by intraperitoneal injection, anal incubation, and immersion. J Fish Dis 27, pp. 623–632. Daniels, C. L., Merrifield, D. L., Boothroyd, D. P., Davies, S. J., Factor, J. R. and Arnold, K. E., (2010). Effect of dietary Bacillus spp. and mannan oligosaccharides (MOS) on European lobster (Homarus gammarus L.) larvae growth performance, gut morphology and gut microbiota. Aquaculture 304(1-4), pp. 49-57. Dang, T. V. C., Nguyen, V. H., Dierckens, K., Defoirdt, T., Boon, N., Sorgeloos, P. and Bossier, P. (2009). Novel approach of using homoserine lactone-degrading and poly-[beta]-hydroxybutyrate-accumulating bacteria to protect Artemia from the pathogenic effects of Vibrio harveyi. Aquaculture 291(1-2), pp. 23-30. De Schryver, P., A. K. S., Kunwar, P. S., Baruah, K., Verstraete, W., Boon, N., De Boeck, G. and Bossier, P., (2010). Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax. Applied Microbiology and Biotechnology 86 (May, 2010), pp. 1535-1541. Defoirdt, T., Halet, D., Vervaeren, H., Boon, N., Van de Wiele, T., Sorgeloos, P., Bossier, P. & Verstraete, W., (2007). The bacterial storage compound poly-beta-hydroxybutyrate protects Artemia franciscana from pathogenic Vibrio campbellii. Environ Microbiol 9, pp. 445–452. Defoirdt, T., Boon, N. & Bossier, P. (2009). Short-chain fatty acids and poly-β-hydroxyalkanoates: (New) biocontrol agents for a sustainable animal production. Biotechnol Adv 27, pp. 680–685. Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W. and Bossier, P., (2009). Short-chain fatty acids and poly-[beta]-hydroxyalkanoates: (New) Biocontrol agents for a sustainable animal production. Biotechnology Advances 27(6), pp. 680-685. Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P. (2007). Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example. Trends Biotechnol 25, pp. 472–479. Defoirdt, T., Halet, D., Sorgeloos, P., Bossier, P. and Verstraete, W., (2006). Short-chain fatty acids protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii. Aquaculture 261(2), pp. 804-808.


65

Dhont, J. and Sorgeloos, P., (2002). Applications of Artemia. In: Artemia: Basic and applied biology. Eds: Abatzopoulos, Th. J., Beardmore, J. A., Clegg, J. S., Sorgeloos, P., Dordrecht: Kluwer Academic Publisher, pp. 251- 271. Dhont, J., Lavens, P. and Sorgeloos, P., (1993). Preparation and use of Artemia as food for shrimp and prawn larvae. In CRC Handbook of mariculture ed.Mcveg, J.CRC press, Boca Rtaton, FL, pp. 61-103. Dinh, T. N., Wille, M., De Schryver, P., Defoirdt, T., Bossier, P. and Sorgeloos, P., (2009). The effect of poly [beta]-hydroxybutyrate on larviculture of the giant freshwater prawn Macrobrachium rosenbergii. Aquaculture 302(1-2), pp. 76-81. FAO/APEC/NACA/SEMARNAP, (2000). Report of a Joint APEC/FAO/NACA/SEMARNAP ad-hoc expert consultation on trans-boundary aquatic animal pathogen transfer and development of harmonised standards on aquaculture health management, 24–28 July 2000, Puerto Vallarta, Jalisco, Mexico. FAO/WHO, (2001). Report of a joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Co ´rdoba, Argentina. FAO, (2004). The state of world fisheries and aquaculture 2004. FAO Fisheries Department, Rome, Italy. FAO, (2008). The state of world fisheries and aquaculture 2008. FAO Fisheries Department, Rome, Italy. Gatesoupe, F.-J. (1991). The effect of three strains of lactic bacteria on the production rate of rotifers, Brachionus plicatilis, and their dietary value for larval turbot, Scophthalmus maximus. Aquaculture 96(3-4), pp. 335-342. Gatesoupe, F.-J. (2002). Probiotic and formaldehyde treatments of Artemia nauplii as food for larval pollack, Pollachius pollachius. Aquaculture 212(1-4), pp. 347-360. Gibson, L. F., Woodworth, J., and George, A. M., (1998). Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigas, when challenged with Vibrio tubiashii. Aquaculture 169(1-2), pp. 111-120.


66

Gibson, G.R. and Roberfroid, M.B., (1995). Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125, pp. 1401–1412. Gibson, G.R., Probert, H.M., Loo, J.V., Rastall, R.A. and Roberfroid, M.B., (2004). Dietary modulation of the human colonic microbiota: updating the concept of prebiotics. J. Nutr. Res. Rev. 17, pp. 259–275. Grisdale-Helland, Helland, S.J. and Gatlin III, D.M., (2008). The effects of dietary supplementation with mannanoligosaccharide, fructooligosaccharide or galactooligosaccharide on the growth and feed utilization of Atlantic salmon (Salmo salar), Aquacult. 283, pp. 163–167. Gudding, R., Lillehaug, A., Evensen, O. (1999). Recent developments in fish vaccinology. Vet Immunol Immunopathol 72, pp. 203–212 Halet, D., Defoirdt, T., Damme, P. V.,Vervaeren H., Forrez, I., Van de Wiele, T., Boon, N., Sorgeloos, P., Bossier, P. & Verstraete, W., (2007). Poly-β-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii, FEMS Microbiol. Ecol. 60, pp. 363–369. Huising, M. O., T. Guichelaar, C. Hoek, B. M. L. Verburg-van Kemenade, G. Flik, H. F. J. Savelkoul and J. H. W. M. Rombout (2003). Increased efficacy of immersion vaccination in fish with hyperosmotic pretreatment. Vaccine 21(27-30), pp. 4178-4193. Ibrahem, Mai D., Fathi, M., Mesalhy, S., Abd El-Aty, A.M., (2010). Effect of dietary supplementation of inulin and vitamin C on the growth, hematology, innate immunity, and resistance of Nile tilapia (Oreochromis niloticus). Fish & Shellfish Immunology 29, pp. 241 – 246. Jan, V. and Nympha L. D. E., ROSA, L. A., (1980). Manual on Artemia production in salt ponds in the Philippines, fao/undp-bfar brackish water aquaculture demonstration and training project phi/75/005. Jensen, B.B., (1998). The impact of feed additives on the microbial ecology of the gut in young pigs. Journal of Animal and Feed Sciences, 7: 45-64, Suppl. 1. Johnson, K. N., Van Hulten, M. C. W. and Barnes, A. C., (2008). “Vaccination" of shrimp against viral pathogens: Phenomenology and underlying mechanisms. Vaccine 26(38), pp. 4885-4892.


67

Karunasagar, I., R. Pai, G. R. Malathi and I. Karunasagar (1994). Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture 128(3-4), pp. 203-209. Karunasagar, I., Shivu, M. M., Girisha, S. K., Krohne, G. and Karunasagar, I., (2007). Biocontrol of pathogens in shrimp hatcheries using bacteriophages. Aquaculture 268(1-4), pp. 288-292. Khoa, L.V., Hao, N.V., Huong, L.T.L., (2001). Vietnam: national review on management strategies for major diseases in shrimp aquaculture, pp. 91–94. Kumari, J. and Sahoo, P. K., (2006). Non-specific immune response of healthy and immunocompromised Asian catfish (Clarias batrachus) to several immunostimulants. Aquaculture 255(1-4), pp. 133-141. Lavilla-Pitogo et al., (1990). Occurrence of luminous bacterial disease of Penaeus monodon larvae in the Philippines, Aquaculture 91, pp. 1–13. Lee, S.Y., (1996). Plastic bacteria? Progress and propects for polyhydroxyalkanoates production in bacteria. Trends in Biotechnology 14, pp. 431-438. Lewin, C.S., (1992). Mechanisms of resistance development in aquatic microorganisms. In: Michel, C., Alderman, D.J.(Eds.), ChemotherapyinAquaculture: from Theoryto Reality. Office International des Epizooties, Paris, France, pp. 288–301. Li, J.Q., Tan, B.P. and Mai, K.S., (2009). Dietary probiotic Bacillus OJ and isomaltooligosaccharides influence the intestine microbial populations, immune responses and resistance to white spot syndrome virus in shrimp (Litopenaeus vannamei), Aquaculture 291 (2009), pp. 35–40. Lilley, D.M., Stillwell, R.J., (1965). Probiotics: growth promoting factors produced by micro-organisms. Science 147, pp. 747–748. Lin, J.H.Y., Chen, T.Y., Chen, M.S., Chen, H.E., Chou, R.L., Chen, T.Y., Su, M.S., Yang, H.L. (2006). Vaccination with three inactivated pathogens of cobia (Rachycentron canadum) stimulates protective immunity. Aquaculture 255, pp. 125–132 Liu et al., (1996). Isolation of Vibrio harveyi from diseased kuruma prawns Penaeus japonicus, Curr. Microbiol. 33, pp. 129–132.


68

Liu, K.-F., Chiu, C.-H., Shiu, Y.-L., Cheng, W. and Liu, C.-H. Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae. Fish & Shellfish Immunology 28(5-6), pp. 837-844. Liu, Y., De Schryver, P., Van Delsen, B., Maignien, L., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P. and Defoirdt, T., (2010). PHB-degrading bacteria isolated from the gastrointestinal tract of aquatic animals as protective actors against luminescent vibriosis. FEMS Microbiol Ecology, pp. 1-9. Lunestad, B. T., (1998). Impact of farmed fish on the environment, presentation by representative of Norway. In Notes of the Workshop on Aquaculture/Environment Interactions: impacts on microbial ecology. Canadian Aquaculture Society, Halifax, Nova Scotia, Canada. Mahious, A.S., Gatesoupe, F.J., Hervi, M., Metailler, R., and Ollevier, F., (2006). Effect of dietary inulin and oligosaccharides as prebiotics for weaning turbot, Psetta maxima (Linnaeus, C. 1758). Aquaculture International 14, pp. 219-229. Marques, A., Huynh Thanh, T., Verstraete, W., Dhont, J., Sorgeloos, P. and Bossier, P., (2006). Use of selected bacteria and yeast to protect gnotobiotic Artemia against different pathogens. Journal of Experimental Marine Biology and Ecology 334(1), pp. 20-30. Martin et al., (2004). Vibrio parahaemolyticus and Vibrio harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis, Dis. Aquat. Org. 60, pp. 21–29. Martinez, J. L., (2009). Environmental pollution by antibiotics and by antibiotic resistance determinants. Environmental Pollution 157(11), pp. 2893-2902. Marzorati, M., Wittebollen, L., Boon, N., Daddonchio, D., and Verstraete, W., (2008). How to get more out of molecular fingerprintints: practical tools for microbial ecology. Environmental Microbiology. Merrifield, D. L., Dimitroglou, A., Foey, A., Davies, S. J., Baker, R. T. M., Bøgwald, J., Castex, M. and Ringø, E., (2010). The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302(1-2), pp. 1-18.


69

Millet, S. and Maertens, L., (2010). The European ban on antibiotic growth promoters in animal feed: From challenges to opportunities. The Veterinary Journal In Press, Corrected Proof. Mohan, C.V., Basavarajappa, H.N., (2001). India: national review on management strategies formajor diseases in shrimp aquaculture, pp. 51–58. Moriarty, D.J.W., (1999). Disease control in shrimp aquaculture with probiotic bacteria. In: C.R. Bell et al., Editors, Microbial Biosystems: New Frontiers. Proceedings of the 8th International Symposium on Microbial Ecology, Atlantic Canada Society for Microbial Ecology. Mudd, A. J., (1996). Is it time to ban all antibiotics as animal growth-promoting agents? The Lancet 348(9039), pp. 1454-1454. National Office of Animal Health (NOAH). Antibiotics for animals. http://www.noah.co.uk/issues/antibiotics.htm. Last accessed 28 October 2001. Planas, M., Pérez-Lorenzo, M., Hjelm, M., Gram, L., Uglenes Fiksdal, I., Bergh, Ø. and Pintado, J., (2006). Probiotic effect in vivo of Roseobacter strain 27-4 against Vibrio (Listonella) anguillarum infections in turbot (Scophthalmus maximus L.) larvae. Aquaculture 255(1-4), pp. 323-333. Press, C. M. and Lillehaug, A., (1995). Vaccination in European salmonid aquaculture: A review of practices and prospects. British Veterinary Journal 151(1), pp. 45-69. Refstie, S., Bakke-McKellep, A.-M., Penn, M.H., Sundby, A., Shearer, K.D. and Krogdahl, Å., (2006). Capacity for digestive hydrolysis and amino acid absorption in Atlantic salmon (Salmo salar) fed diets with soybean meal or inulin with or without addition of antibiotics, Aquacult. 261, pp. 392–406. Ringø, E., Sperstad, S., Myklebust, R., Mayhew, T.M., and Olsen, R.E., (2006). The effect of dietary inulin on aerobic bacteria associated with hindgut of Arctic charr (Salvelinus alpinus L.). Aquaculture Research 37, pp. 891-897. Sakai, M., (1997). G4 11:30 Fish immunostimulants; the application for aquaculture. Developmental & Comparative Immunology 21(2), pp. 136-136.


70

Sakai, M., (1999). Current research status of fish immunostimulants. Aquaculture 172(1-2), pp. 63-92. SCAN, (2003). Opinion of the Scientific Committee on Animal Nutrition on the criteria for assessing the safety of microorgan-isms resistant to antibiotics of human clinical and veterinary importance. European Commission Health and Consumer Protection Directorate-General. Schrøder, M. B., Mikkelsen, H., Børdal, S., Gravningen, K. and Lund, V., (2006). Early vaccination and protection of Atlantic cod (Gadus morhua L.) juveniles against classical vibriosis. Aquaculture 254(1-4), pp. 46-53. Siriwardena, P.P.G.S.N., (2001). Sri Lanka: national review on management strategies for major diseases in shrimp aquaculture,pp. 79–84. In: Subasinghe, R., Arthur, R., Phillips, M.J., Reantaso, M. (Eds.). WB/NACA/WWF/FAO. Thematic Review on Management Strategies for Major Diseases in Shrimp Aquaculture. Proceedings of a Workshop held in Cebu, Philippines on 28–30 November 1999. Soltanian, S., Dhont, J., Sorgeloos, P. AND Bossier, P., (2007). Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia. Fish Shellfish Immunol, 23, pp. 141-153. Soto-Rodriguez, S. A., Roque, A., Lizarraga-Partida1, M. L., Guerra-Flores, A. L., Gomez-Gil, A. L., (2003). Virulence of luminous vibrios to Artemia franciscana nauplii, Dis. Aquat. Org. 53, pp. 231–240. Staykov, Y., Spring, P., Denev, S. and Sweetman, J., (2007). Effect of mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss), Aquacult. Int. 15, pp. 153–161. Subasinghe, R.P. et al., (2001). Aquaculture development, health and wealth. In: R.P. Subasinghe et al., Editors, Aquaculture in the Third Millennium. Technical Proceedings of the Conference on Aquaculture in the Third Millennium, NACA, Bangkok and FAO, pp. 167–191. Swain, M. R. and Ray, R. C., (2009). Biocontrol and other beneficial activities of Bacillus subtilis isolated from cowdung microflora. Microbiological Research 164(2), pp. 121-130.


71

Tseng, D.-Y., Ho, P.-L., Huang, S.-Y., Cheng, S.-C., Shiu, Y.-L., Chiu, C.-S. and Liu, C.-H., (2009). Enhancement of immunity and disease resistance in the white shrimp, Litopenaeus vannamei, by the probiotic, Bacillus subtilis E20. Fish & Shellfish Immunology 26(2), pp. 339-344. Van Den Bogaard, A. E. and Stobberingh, E. E., (1996). Time to ban all antibiotics as animal growth-promoting agents? The Lancet 348(9027), pp. 619-619. Van Stappen, G., (1996). Artemia: Use of cysts, pp. 107-137. In: Manual on the production and use of live feed for the Aquaculture. FAO Fisheries Technical Paper No. 361. Lavens, P. & Sorgeloos, P. (Eds.), 295pp. Van Stappen, G., (1996). Artemia: Introduction, biology and ecology of Artemia, pp. 79-106. In: Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper No. 361. Lavens, P. & Sorgeloos, P. (Eds.), 295pp. Vieira, R., Carvalho, E. M. R., Carvalho, F. C. T., Silva, C. M., Sousa, O. V. and Rodrigues, D. P., (2010). Antimicrobial susceptibility of Escherichia coli isolated from shrimp (Litopenaeus vannamei) and pond environment in northeastern Brazil. Journal of Environmental Science and Health Part B-Pesticides Food Contaminants and Agricultural Wastes 45(3), pp. 198-203. Vinod, M. G., Shivu, M. M., Umesha, K. R., Rajeeva, B. C., Krohne, G., Karunasagar, I. and Karunasagar, I., (2006). Isolation of Vibrio harveyi bacteriophage with a potential for biocontrol of luminous vibriosis in hatchery environments. Aquaculture 255(1-4), pp. 117-124. Wang, Y., and Qian, P-Y., (2009). Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies. PLoS ONE 4(10): e7401. doi:10.1371/journal.pone.0007401. Wei, Q., (2002). Social and economic impacts of aquatic animal health problems in aquaculture in China, pp. 55–61. In: Arthur, J.R., Phillips, M.J., Subasinghe, R.P., Reantaso, M.B., MacRae, I.H. (Eds.). Primary Aquatic Animal Health Care in Rural, Small-Scale, Aquaculture Development. FAO Fish. Tech. Pap. No. 406. Yulin, J., (2001). China: national review on management strategies for major diseases in shrimp aquaculture,pp. 74–78. In: Subasinghe, R., Arthur, R., Phillips, M.J., Reantaso, M. (Eds.). WB/NACA/WWF/FAO. Thematic Review on Management Strategies for


72

Major Diseases in Shrimp Aquaculture. Proceedings of a Workshop held in Cebu, Philippines on 28–30 November 1999. Zhou, X.-x., Wang, Y.-b. and Li, W.-f., (2009). Effect of probiotic on larvae shrimp (Penaeus vannamei) based on water quality, survival rate and digestive enzyme activities. Aquaculture 287(3-4), pp. 349-353. Zhou, Y.-C., Huang, H., Wang, J., Zhang, B. and Su, Y.-Q., (2002). Vaccination of the grouper, Epinephalus awoara, against vibriosis using the ultrasonic technique. Aquaculture 203(3-4), pp. 229-238.


73

APPENDICES

Appendix 1. Subtest 1 - Survival rate of Artemia larvae after 24h of challenge with

V.campbellii. Data with different alphabet letters indicate significant difference (P<0.05)

Treatment Average of

survival rate (%) Std deviation

Sterile Artemia 90.8a 4.15

NS Artemia 91.6a 2.61

NS Artemia + Challenge 25.2c 4.82

NS Artemia + PHB (100mg/L) 90.8a 3.03

NS Artemia + PHB (100mg/L)+ Challenge 48.4b 5.18

Appendix 2. Pair-wise comparisons of survival rate among treatments of subtest 1. p-values

were calculated by Turkey’s post hoc test at a 5% probability level.

Treatment pair p-value

Sterile Artemia & NS Artemia 0.9978

Sterile Artemia & NS Artemia + Challenge < 0.0001

Sterile Artemia & NS Artemia + PHB (100mg/L) 1.000

Sterile Artemia & NS Artemia + PHB (100mg/L) + Challenge < 0.0001

NS Artemia & NS Artemia + PHB (100mg/L) 0.9978

NS Artemia & NS Artemia + Challenge < 0.0001

NS Artemia & NS Artemia + PHB (100mg/L) + Challenge < 0.0001

NS Artemia + Challenge & NS Artemia + PHB (100mg/L) < 0.0001

NS Artemia + Challenge & NS Artemia + PHB (100mg/L) +

Challenge < 0.0001

NS Artemia + PHB (100mg/L) & NS Artemia + PHB (100mg/L) +

Challenge < 0.0001


74



Treatment Average of

survival rate (%) Std variation

NS Artemia 81.6 2.97

NS Artemia + Challenge 26.4 2.61

NS Artemia + PHB + Challenge 41.2 4.82

NS Artemia + PHB + M13 91.6 3.58

NS Artemia + M13 85.6 4.77

NS Artemia + M13 + Challenge 44.8 4.15

NS Artemia + PHB + M13 + Challenge 58 4.00


were calculated by Turkey’s post hoc test at a 5% probability level.



Sterile Artemia & NS Artemia + PHB + Challenge < 0.0001

Sterile Artemia & NS Artemia + PHB + M13 0.0063

Sterile Artemia & NS Artemia + M13 0.6754

Sterile Artemia & NS Artemia + M13 + Challenge < 0.0001

Sterile Artemia & NS Artemia + PHB + M13 + Challenge < 0.0001

NS Artemia + Challenge & NS Artemia + PHB + Challenge < 0.0001

NS Artemia + Challenge & NS Artemia + PHB + M13 < 0.0001

NS Artemia + Challenge & NS Artemia + M13 < 0.0001

NS Artemia + Challenge & NS Artemia + M13 + Challenge < 0.0001

NS Artemia + Challenge & NS Artemia + PHB + M13 + Challenge < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + PHB + M13 < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + M13 < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + M13 + Challenge 0.7695

NS Artemia + PHB + Challenge & NS Artemia + PHB + M13 + Challenge < 0.0001


75

NS Artemia + PHB + M13 & NS Artemia + M13 0.2279

NS Artemia + PHB + M13 & NS Artemia + M13 + Challenge < 0.0001

NS Artemia + PHB + M13 & NS Artemia + PHB + M13 + Challenge < 0.0001

NS Artemia + M13 & NS Artemia + M13 + Challenge < 0.0001

NS Artemia + M13 & NS Artemia + PHB + M13 + Challenge < 0.0001

NS Artemia + M13 + Challenge & NS Artemia + PHB + M13 + Challenge 0.0002



Treatment Average Std variation

NS Artemia 86.4 3.85

NS Artemia + Challenge 19.6 7.80

NS Artemia + PHB + Challenge 53.6 6.23

NS Artemia + PHB + B12 86.8 4.15

NS Artemia + B12 86 6.63

NS Artemia + B12 + Challenge 26.8 5.40

NS Artemia + PHB + B12 + Challenge 69.2 8.20


were calculated by Turkey’s post hoc test at a 5% probability level



Sterile Artemia & NS Artemia + PHB + Challenge < 0.0001

Sterile Artemia & NS Artemia + PHB + B12 1.000

Sterile Artemia & NS Artemia + B12 1.000

Sterile Artemia & NS Artemia + B12 + Challenge < 0.0001

Sterile Artemia & NS Artemia + PHB + B12 + Challenge 0.0027

NS Artemia + Challenge & NS Artemia + PHB + Challenge < 0.0001

NS Artemia + Challenge & NS Artemia + PHB + B12 < 0.0001

NS Artemia + Challenge & NS Artemia + B12 < 0.0001


76

NS Artemia + Challenge & NS Artemia + B12 + Challenge 0.5424

NS Artemia + Challenge & NS Artemia + PHB + B12 + Challenge < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + PHB + B12 < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + B12 < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + B12 + Challenge < 0.0001

NS Artemia + PHB + Challenge & NS Artemia + PHB + B12 + Challenge 0.0076

NS Artemia + PHB + B12 & NS Artemia + B12 1.000

NS Artemia + PHB + B12 & NS Artemia + B12 + Challenge < 0.0001

NS Artemia + PHB + B12 & NS Artemia + PHB + B12 + Challenge 0.0021

NS Artemia + B12 & NS Artemia + B12 + Challenge < 0.0001

NS Artemia + B12 & NS Artemia + PHB + B12 + Challenge 0.0035

NS Artemia + B12 + Challenge & NS Artemia + PHB + B12 + Challenge < 0.0001

Documents

Practical application of PHB degrading probiotics