Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Effects of Different Disinfectants on

Blue Mussel (Mytilus edulis L.) Embryo
Floriefe Gonzaga Torino
Assistant Professor IV, College of Fisheries, Mindanao State University- Buug Campus
Buug, Zamboanga Sibugay, 7009, Philippines

Abstract:- Occurrence of diseases caused by bacteria It is certainly relevant to have a better understanding
during larval culture is still one of the major constraints on the host-microbe interactions to develop effective
in aquaculture. Understanding the host-microbe solutions of disease control for the aquaculture industry
interactions is certainly relevant to develop disease [44]. A powerful tool to study these host-microbial
control systems for the aquaculture industry. Therefore, relationships is to define the animal functioning in the
obtaining test animals free from microorganisms (germ- absence of all micro-organisms (under germ-free or
free or axenic) is necessary, as the presence of naturally gnotobiotic conditions) and then evaluate the effects of
occurring microorganisms in the host may lead to false adding a single or defined populations of microbes or
conclusions. The aim of this study is to obtain axenic certain compounds [34]. This allows determination of the
blue mussel (Mytilus edulis) embryo using different effects of the tested microbes on the target organisms
disinfectants. The efficacy of chemicals in reducing the without interference from unwanted microbial
bacterial load associated with mussel eggs and embryos contaminants [21]. Moreover, axenic animals provide a
is tested, as well as the resistance of the eggs to these direct means to study the host’s reaction to a single species
chemicals. For that purpose, fertilized eggs are exposed of a pathogenic or parasitic agent [20]. Germ-free culture
to different chemicals at different concentrations. The of animals has also been helpful in defining the nutrient
disinfectants tested include hydrogen peroxide, requirements of the organism [54].
chlorhexidine, and Sanocare HC. All disinfectants are
found to be detrimental for the mussel embryo. The aim of this study is to determine the effects of
different disinfectants on the Blue Mussel (Mytilus edulis)
Keywords:- Mytilus edulis L.; Blue Mussel; broodstock embryo. To date, few studies are performed with
mussels; Axenic; Germ Free; Bacteria Free; Sterile; gnotobiotic aquatic animals. This is the first study that
Disinfectants. attempts to generate bacteria-free mussel larvae (M. edulis)
and will therefore provide baseline information for future
I. INTRODUCTION research.

Aquaculture production is expected to play a crucial II. MATERIALS AND METHODS

role in meeting the growing demand for fishery products
since capture fisheries have markedly stagnated. Currently,  Obtaining Mussel Embryo
it is the fastest growing sector in the food production Adult mussels, Mytilus edulis were obtained from the
industry with an average yearly growth rate of more than hatchery Roem van Yerseke, The Netherlands. They were
six percent over the past two decades [14]. In 2010, one- thoroughly cleaned and stocked dry at 4°C. Thermal shock
third of the world’s farmed fish are coming from bivalve technique was then employed to induce the mussels to
production [25]. Bivalve molluscs are important food spawn by submerging them first in warm filtered
commodity in the world. Natural population cannot meet autoclaved seawater (FASW) at temperatures between 18 to
the increasing demand due to over-exploitation, which led 25°C followed by a cold shock treatment at temperatures
to development of hatcheries [3]. Nevertheless, mollusc between 5 to 10°C. The male and female mussels that
aquaculture growth and sustainability are still hampered by started spawning were placed separately in a sterile plastic
the occurrence of diseases, severely impacting socio- beaker filled with FASW in order to collect the gametes
economic development [12]. Various methods have already separately (Figure 1). The adult mussels were removed
been developed to control proliferation of pathogens and to from their respective spawning beakers when adequate
maintain a healthy microbial environment in aquaculture amount of gametes were released. All the eggs were pooled
systems. Among these are the use of probionts, in a one-litre sterile beaker which was topped-up with
immunostimulants, vaccines, quorum sensing analysis and FASW till one litre. Sperm cells were then added to reach
antimicrobial peptides [41], [68], [40], [46]. However, the an approximate sperm to egg ratio of 10:1. After
implementation of these alternative techniques should be fertilization (15 minutes), the eggs were sieved (30µm) and
based on thorough understanding of the mechanisms washed with FASW to remove the excess of sperm. All
involved and the putative consequences [46]. manipulations were carried out under a laminar flow hood.
Materials used were autoclaved for 20 minutes at 121 oC
and 15 psi.

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Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165

Fig 2:- Vials for incubation of fertilized eggs.

Fig 1:- Female (left) and Male (right) Mussels Spawning.
In Experiment 1, the fertilized eggs were incubated for
 Checking Axenity 48 hours in sterile plastic vials with the addition of
Bacterial contamination was checked by plating 100µl Sanocare HC in the following concentrations:
of undiluted culture medium on marine agar (DifcoTM)
plates. Serial dilutions were not done for plating as it was Treatment 1 – Sanocare HC - 10µg ml-1
not necessary to count the colonies. Plates were prepared Treatment 2 – Sanocare HC - 50µg ml-1
by suspending 55.1g of marine agar (DifcoTM) in one litre Treatment 3 – Sanocare HC - 150µg ml-1
of demineralised water. The solution was autoclaved for 20 Treatment 4 – Sanocare HC - 200µg ml-1
minutes at 121oC and 15 psi. Pouring of the solution in the Treatment 5 – control
Petri plates was done under a laminar flow hood.
A stock solution of Sanocare HC (10,000µg ml -1 was
 Checking Larval Survival prepared by mixing 10g of Sanocare HC in one litre of
Larval survival was checked by staining the larvae FASW with electric mixer to obtain an emulsion (100x
with lugol solution inside the culture recipient and then more concentrated).
concentrating the larvae by carefully removing the top layer
of the water in the culture medium, after the larvae have In Experiment 2, fertilized eggs were exposed to
sunk to the bottom. Concentrated larvae were transferred to chlorhexidine in demineralised water (since chlorhexidine
24-micro well plates and placed under an inverted hardly dissolves in seawater), for 1 minute at
microscope for observation. If quantitative data were concentrations of:
needed, the live/dead ratio was counted of exactly 100 Treatment 1 – Chlorhexidine - 100µg ml-1
larvae. Live/good larvae were D-shaped larvae. Treatment 2 – Chlorhexidine - 250µg ml-1
Trochophore larvae (ciliated embryo) were also considered Treatment 3 – Chlorhexidine - 500µg ml-1
live larvae with delayed development. Empty shells and Treatment 4 – control 1 - demineralised water
undeveloped eggs were considered dead. Treatment 5 – control 2 - FASW

 Developing a Sterile Culture Procedure for Mussel Two controls were made to check whether
Embryo Using Different Disinfectants. chlorhexidine or the freshwater has an effect on the eggs:
Different experiments were conducted in order to for Control 1, eggs exposed for one minute in
obtain sterile mussel embryo. The efficacy of chemicals in demineralised water and for Control 2, eggs exposed for
reducing the bacterial load on the mussel eggs and embryos one minute in FASW, to check if the handling has a
were tested, as well as the resistance of the eggs to these negative effect on the embryos. Five beakers were prepared
chemicals. The fertilized eggs were exposed to different containing different concentrations of chlorhexidine and the
chemicals at different concentrations and time exposures, controls. Different concentrations of chlorhexidine (100,
and were incubated in sterile plastic vials at densities 20-50 250 and 500µg ml-1) were prepared by adding 100, 250 and
eggs ml-1 (Figure 2) in 10ml of FASW without aeration or 500µg of chlorhexidine to sterile beakers filled with one
mechanical shaking. Temperature was maintained at 17°C litre water to obtain the desired concentration. Exposure
for all treatments. Axenity, survival and development of was done by submerging the sieve (30µm) containing the
mussel larvae were monitored. All treatments with the eggs fertilized eggs in the beaker for one minute. The eggs were
were replicated thrice and manipulations were done under a then rinsed with FASW and incubated for 72 hours in
laminar flow. sterile plastic vials.

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Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Experiment 3, fertilized eggs were exposed to the embryo. Artemia cysts have a chorion that protects the
hydrogen peroxide (H2O2) in seawater for 3 minutes at the embryo from the chemicals. In contrast, mussel embryos
following concentrations: only possess a vitelline coat of 0.5-1.0 µm thick [8] which
Treatment 1 – H2O2 - 0.3% makes them very vulnerable to chemicals.
Treatment 2 – H2O2 - 1.5%
Treatment 3 – H2O2 - 3% Experiment Treatment Axenity Development/Survival
Treatment 4 – control (48h after
fertilization)
Exposure of the fertilized eggs with hydrogen
1 1 (10µg - Trochophore
peroxide was done according to the same procedure
ml-1 HC)
described in Experiment 2. The eggs were washed with
2 (50µg - Trochophore,
FASW after exposure and then incubated in the plastic vials
ml-1 HC) Undeveloped eggs
for 72 hours.
3 (100µg - Undeveloped eggs
ml-1 HC)
Axenity, survival and larval development were
checked after 48 hours for Experiment 1 while in 4 (200µg - Undeveloped eggs
Experiments 2, and 3 after 72 hours post fertilization. ml-1 HC)
Schematic diagram of the experiments is presented in 5 (Control) - Trochophore
Figure 3. HC – Sanocare HC, (-) : Bacterial contamination, (+) :
axenic
Table 1:- The effects of different concentrations of
Sanocare HC on mussel embryos

In Experiment 2, different concentrations of the

disinfectant chlorhexidine were evaluated. As shown in
Table 2, none of the concentrations did eliminate microbial
contaminants. Larval survival and development was
checked after 72 hours and D-larvae were observed in the
treatment Control 2 (FASW). Total mortality was observed
in all of the treatments even in the Control 1 (demineralised
water). Chlorhexidine is one of the best and most widely
used antiseptics. It is a strong base and is most stable in the
form of its salt [29]. Dilutions of chlorhexidine were
prepared by mixing in demineralised water since it
precipitates in seawater. Exposure to a freshwater solution
of chlorhexidine caused the eggs to burst because of
HC – Sanocare HC; CHX – Chlorhexidine; H2O2 – osmosis. This in turn leads to leakage of nutrients into the
Hydrogen peroxide water that would enhance microbial growth and deteriorate
Fig 3:- Schematic diagram of the three experiments the water quality [21].

III. RESULTS AND DISCUSSION Experiment Treatment Axenity Development/Survival

(72h after
 Effects of Different Disinfectants on Mussel embryo fertilization)
Sanocare HC® is a product developed by INVE 2 1 (100µg - Total mortality
Aquaculture that reduces the development and transfer of ml-1 CHX)
putative pathogens associated with live food culture. It is a
2 (250µg - Total mortality
self-emulsifying product that ensures maximum bacterial
ml-1 CHX)
suppression during Artemia hatching. Different
concentrations of Sanocare HC were tested in Experiment 3 (500µg - Total mortality
1. Results revealed bacterial contamination at all ml-1 CHX)
concentrations, going from 10 to 200µg ml-1 (Table 1). 4 (Control - Total mortality
After 48 hours of exposure, no D-larvae were observed in 1)
the control treatment, treatments 1 (10µg ml-1) and 5 (Control - D larvae
treatment 2 (50µg ml-1). This is due to the fact that it was 2)
still too early for the larvae to reach this development stage. CHX – Chlorhexidine, (-) - Bacterial contamination, (+) –
Higher concentrations of Sanocare HC, treatments 3 axenic
(100µgml-1) and 4 (200µg ml-1) were found to be lethal Table 2:- The effects of Different Concentrations of
since the eggs had not developed into trochophore larvae. Chlorhexidine on Mussel Embryos
In the study of [69], Sanocare HC significantly reduced the
Vibrio loads in Artemia hatching water without affecting

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 Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Hydrogen peroxide is a highly reactive, strong [3]. Avendaño, R. E., and Riquelme, C. E. (1999).
oxidizing and bleaching agent [73]. It has long been used as Establishment of mixed-culture probiotics and
a disinfectant for different species and life stages of fish microalgae as food for bivalve larvae. Aquaculture
against organisms that cause diseases such as external Research, 30(11-12), 893–900. doi:10.1046/j.1365-
parasites, bacteria and fungi [73]. [21] obtained bacteria- 2109.1999.00420.x
free red drum (Sciaenops ocellatus L.) larvae by exposing [4]. Baker, D. E. (1966). The commercial production of
the eggs to hydrogen peroxide (3%) for five minutes. This mice with a specified flora.National Cancer Institute
disinfectant was used in Experiment 3 at different monograph, 20, 161–166.
concentrations. Treatments 2 (1.5%) and 3 (3%) showed no [5]. Baker, J. A., Ferguson, M. S., and TenBroeck, C.
evidence of bacterial contamination while treatment 1 (1942). Growth of Platyfish (Platypoecilus maculatus)
(0.3%) and treatment control had bacterial colonies Free from Bacteria and Other Microorganisms.
growing on the plates (Table 3). The disinfectant however Proceedings of the Society for Experimental Biology
adversely affected the larval development causing total and Medicine. Society for Experimental Biology and
mortality. This is in contrast to the results of [21] where no Medicine (New York, N.Y.), 51(1), 116–119.
adverse effects on the larval survival of red drum doi:10.3181/00379727-51-13854
(Sciaenops ocellatus L.) were observed. However, when [6]. Bayne, B. L. (1964). The Responses of the Larvae of
the disinfectant was tested on eggs of two other marine Mytilus edulis L. to Light and to
fishes (yellowtail snapper, Ocyurus chrysurus, and spotted Gravity. Oikos, 15(1), 162. doi:10.2307/3564753
seatrout, Cynoscion nebulosus Cuvier), the exposure to [7]. Bayne, B. L. (1965). Growth and the delay of
different concentrations showed differential toxicity [21]. metamorphosis of the larvae of Mytilus edulis
L.. Ophelia, 2(1), 1-
Experiment Treatment Axenity Development/Survival 47.doi:10.1080/00785326.1965.10409596
(72h after [8]. Bayne, B. L. (1976). Marine mussels: their ecology
fertilization) and physiology. Cambridge University Press.
[9]. Bayne, B. L., Bubel, A., Gabott, P. A., Livingstone,
3 1 (0.3% - Total mortality D. R., Lowe, D. M., and Moore, M. N.
H2O2) (1982). Glycogen utilisation and gametogenesis in
2 (1.5% + Total mortality Mytilus edulis L.
H2O2) [10]. Bayne, B. L., Holland, D. L., Moore, M. N., Lowe, D.
3 (3% + Total mortality M., and Widdows, J. (1978). Further studies on the
H2O2) effects of stress in the adult on the eggs of Mytilus
4 (Control) - D larvae, empty shell edulis L. Journal of the Marine Biological Association
of the United Kingdom, 58(04), 825–841.
H2O2 – Hydrogen peroxide, (-) : Bacterial contamination, doi:10.1017/S0025315400056794
(+) : axenic [11]. Berthe, F. (2004). Report about mollusc diseases. In:
Table 3:- The Effects of Different Concentrations of Alvarez-Pellitero P. (ed.), Barja, L. (ed.), Basurco, B.
Hydrogen Peroxide on Mussel Embryos (ed.), Berthe, F (ed.), Toranzo A. E., (ed.).
Mediterranean Aquaculture diagnostic laboratories.
IV. CONCLUSION Zaragoza: CIHEAM. 33-48.
[12]. Berthe, F. (2005). Diseases in mollusc hatcheries and
All disinfectants (hydrogen peroxide, chlorhexidine their paradox in health management. Presented at the
and Sanocare HC) seriously damaged the embryos resulting Diseases in Asian Aquaculture. Retrieved
in delayed development and high mortalities. Further from http://archimer.ifremer.fr/doc/00000/3289/
research and verification are needed, such as finding [13]. Bouchet, P., and Gofas, S. (2013). Mytilus
disinfectants that are not harmful to the mussel embryos edulis Linnaeus, 1758. World Register of Marine
and use of antibiotics. Species. Retrieved from
http://www.marinespecies.org/aphia.php?p=taxdetails
REFERENCES &id=138228 on 2013-03-06
[14]. Brugère, C. D., and Ridler, N. B. (2004). Global
[1]. Anguiano-Beltrán, C., Lizárraga-Partida, M. L., and aquaculture outlook in the next decades: an analysis
Searcy-Bernal, R. (2004). Effect of Vibrio of national aquaculture production forecasts to 2030.
alginolyticus on larval survival of the blue mussel Rome: Food and Agriculture Organization of the
Mytilus galloprovincialis. Diseases of aquatic United Nations. Retrieved
organisms, 59(2), 119–123. doi:10.3354/dao059119 from ftp://ftp.fao.org/docrep/fao/007/y5648e/y5648e0
[2]. Arzul, I., Renault, T., and Lipart, C. (2001). 0.pdf
Experimental herpes-like viral infections in marine [15]. Chernin, E. (1957). A Method of Securing
bivalves: demonstration of interspecies transmission. Bacteriologically Sterile Snails (Australorbis
Diseases of aquatic organisms, 46(1), 1–6. Retrieved glabratus). Experimental Biology and
from http://archimer.ifremer.fr/doc/00000/819/ Medicine, 96(1), 204–210. doi:10.3181/00379727-96-
23433

IJISRT20AUG824 www.ijisrt.com 1427

Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
[16]. Coates, M. E. (1975). Gnotobiotic animals in research: [29]. Foulkes, D. M. (1973). Some toxicological
their uses and limitations. Laboratory animals, 9(4), observations on chlorhexidine. Journal of periodontal
275–282. Retrieved from research. Supplement, 12, 55–60.
http://lan.sagepub.com/content/9/4/275.full.pdf [30]. Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S.,
[17]. Coates, M. E., and O’donoghue, P. N. (1967). Milk Wilson, B. A., and Olsen, G. J. (2008). Critical
Allergy in Infant Germ-free Evaluation of Two Primers Commonly Used for
Rabbits.Nature, 213(5073), 307–308. Amplification of Bacterial 16S rRNA Genes. Applied
doi:10.1038/213307a0 and Environmental Microbiology, 74(8), 2461–2470.
[18]. Delahaut, Vyshal. (2012). Development of a doi:10.1128/AEM.02272-07
Challenge Test for the Blue Mussel, Mytilus edulis. [31]. Franklin, T. J., and Snow, G. A. (2005). Biochemistry
Gent University, Gent, Belgium. Retrieved from and Molecular Biology of Antimicrobial Drug Action.
http://lib.ugent.be/fulltxt/RUG01/001/894/276/RUG0 Springer.
1-001894276_2012_0001_AC.pdf [32]. Galley, T. H., Batista, F. M., Braithwaite, R., King, J.,
[19]. Dierckens, K., Rekecki, A., Laureau, S., Sorgeloos, P., and Beaumont, A. R. (2010). Optimisation of larval
Boon, N., Van den Broeck, W., and Bossier, P. culture of the mussel Mytilus edulis (L.).Aquaculture
(2009). Development of a bacterial challenge test for International, 18(3), 315–325. doi:10.1007/s10499-
gnotobiotic sea bass (Dicentrarchus labrax) 009-9245-7
larvae. Environmental microbiology, 11(2), 526–533. [33]. Gee, L. L., and Sarles, W. B. (1942). The Disinfection
doi:10.1111/j.1462-2920.2008.01794.x of Trout Eggs Contaminated with Bacterium
[20]. Douillet, P. (1998). Disinfection of rotifer cysts Salmonicida. Journal of Bacteriology, 44(1), 111–
leading to bacteria-free populations. Journal of 126. Retrieved from
Experimental Marine Biology and Ecology,224(2), http://www.ncbi.nlm.nih.gov/pmc/articles/PMC37365
183–192. doi:10.1016/S0022-0981(97)00200-1 4/
[21]. Douillet, P. A., and Holt, G. J. (1994). Surface [34]. Gordon, H. A., and Pesti, L. (1971). The gnotobiotic
disinfection of red drum (Sciaenops ocellatus L.) eggs animal as a tool in the study of host microbial
leading to bacteria-free larvae. Journal of relationships. Bacteriological Reviews, 35(4), 390–
Experimental Marine Biology and Ecology, 179(2), 429. Retrieved from
253–266. doi:10.1016/0022-0981(94)90118-X http://www.ncbi.nlm.nih.gov/pmc/articles/PMC37840
[22]. Elmolla, E. S., and Chaudhuri, M. (2010). 8/
Degradation of amoxicillin, ampicillin and cloxacillin [35]. Guillard, R. R. L. (1959). Further Evidence of the
antibiotics in aqueous solution by the UV/ZnO Destruction of Bivalve Larvae by Bacteria. The
photocatalytic process. Journal of Hazardous Biological Bulletin, 117(2), 258–266. Retrieved from
Materials,173(1–3), 445–449. http://www.biolbull.org/content/117/2/258
doi:10.1016/j.jhazmat.2009.08.104 [36]. Kesarcodi-Watson, A., Kaspar, H., Lategan, M. J., and
[23]. Elston, R., and Leibovitz, L. (1980). Pathogenesis of Gibson, L. (2009). Two pathogens of
Experimental Vibriosis in Larval American Oysters, GreenshellTMmussel larvae, Perna canaliculus: Vibrio
Crassostrea virginica.Canadian Journal of Fisheries splendidus and a V. coralliilyticus/neptunius-like
and Aquatic Sciences, 37(6), 964–978. isolate. Journal of Fish Diseases, 32(6), 499–507.
doi:10.1139/f80-126 doi:10.1111/j.1365-2761.2009.01006.x
[24]. Erasmus, J. H., Cook, P. A., and Coyne, V. E. (1997). [37]. Lambert, C., Nicolas, J. L., Cilia, V., and Corre, S.
The role of bacteria in the digestion of seaweed by the (1998). Vibrio pectenicida sp. nov., a pathogen of
abalone Haliotis midae. Aquaculture, 155(1–4), 377– scallop (Pecten maximus) larvae. International
386. doi:10.1016/S0044-8486(97)00112-9 journal of systematic bacteriology, 48 Pt 2, 481–487.
[25]. FAO (2012). Fishery Statistics of Cultured Aquatic [38]. Langdon, C. J. (1983). Growth Studies with Bacteria-
Species Information Programme. Retrieved from Free Oyster (Crassostrea gigas) Larvae Fed on Semi-
http://www.fao.org/fishery/species/2688/en Defined Artificial Diets. The Biological
[26]. FAO (2012). The State of World Fisheries and Bulletin, 164(2), 227–235. Retrieved from
Aquaculture. Rome: Food and Agriculture http://www.biolbull.org/content/164/2/227
Organization of the United Nations. [39]. Le Deuff, R. M., Nicolas, J. L., Renault, T., and
[27]. Faraji, R., Parsa, A., Torabi, B., and Withrow, T. Cochennec, N. (1994). Experimental transmission of a
(2006). Effects of kanamycin on the macromolecular Herpes-like virus to axenic larvae of Pacific oyster,
composition of kanamycin sensitive Escherichia coli Crassostrea gigas. Bulletin Of The European
DH5α strain. Journal of Experimental Microbiology Association Of Fish Pathologists, 14(2), 69–72.
and Immunology, 9, 31–38. Retrieved from Retrieved from
http://www.microbiology.ubc.ca/sites/default/files/rol http://archimer.ifremer.fr/doc/00000/2887/
es/drupal_ungrad/JEMI/9/9-31.pdf [40]. Li, C.-H., Zhao, J.-M., and Song, L.-S. (2009). A
[28]. Forberg, T., Arukwe, A., and Vadstein, O. (2011). A review of advances in research on marine molluscan
protocol and cultivation system for gnotobiotic antimicrobial peptides and their potential application
Atlantic cod larvae (Gadus morhua L.) as a tool to in aquaculture . Molluscan Research, 29(1), 17–26.
study host microbe interactions. Aquaculture, 315(3–
4), 222–227. doi:10.1016/j.aquaculture.2011.02.047

IJISRT20AUG824 www.ijisrt.com 1428

Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
[41]. Macey, B. M., and Coyne, V. E. (2006). Colonization [51]. Paillard, C., Le Roux, F., and Borrego, J. J. (2004).
of the Gastrointestinal Tract of the Farmed South Bacterial disease in marine bivalves, a review of
African Abalone Haliotis midae by the Probionts recent studies: Trends and evolution. Aquatic Living
Vibrio midae SY9, Cryptococcus sp. SS1, and Resources, 17(04), 477–498. doi:10.1051/alr:200405
Debaryomyces hansenii AY1. Marine [52]. Pronker, A. E., Nevejan, N. M., Peene, F., Geijsen, P.,
Biotechnology, 8(3), 246–259. doi:10.1007/s10126- and Sorgeloos, P. (2008). Hatchery broodstock
005-0113-9 conditioning of the blue mussel Mytilus edulis
[42]. Manahan, D. T. (1989). Amino acid fluxes to and (Linnaeus 1758). Part I. Impact of different micro-
from seawater in axenic veliger larvae of the bivalve algae mixtures on broodstock performance.
(Crassostrea gigas). Marine Ecology-progress Series Aquaculture International,16(4), 297–307.
- MAR ECOL-PROGR SER, 53, 247–255. doi:10.1007/s10499-007-9143-9
doi:10.3354/meps053247 [53]. Provasoli, L., and Shiraishi, K. (1959). Axenic
[43]. Marques, A., Dhont, J., Sorgeloos, P., and Bossier, P. Cultivation of the Brine Shrimp Artemia
(2006a). Immunostimulatory nature of β-glucans and salina. Biological Bulletin,117(2), 347-355.
baker’s yeast in gnotobiotic Artemia challenge doi:10.2307/1538914
tests. Fish and Shellfish Immunology, 20(5), 682–692. [54]. Provasoli, L., Shiraishi, K., and Lance, J. R. (1959).
doi:10.1016/j.fsi.2005.08.008 Nutritional Idiosyncrasies of Artemia and Tigriopus in
[44]. Marques, A., Dinh, T., Ioakeimidis, C., Huys, G., Monoxenic Culture*. Annals of the New York
Swings, J., Verstraete, W., and Bossier, P. (2005b). Academy of Sciences, 77(2), 250–261.
Effects of bacteria on Artemia franciscana cultured in doi:10.1111/j.1749-6632.1959.tb36905.x
different gnotobiotic environments. Applied and [55]. Rawls, J. F., Samuel, B. S., and Gordon, J. I. (2004).
environmental microbiology, 71(8), 4307–4317. Gnotobiotic zebrafish reveal evolutionarily conserved
doi:10.1128/AEM.71.8.4307-4317.2005 responses to the gut microbiota. Proceedings of the
[45]. Marques, A., François, J.-M., Dhont, J., Bossier, P., National Academy of Sciences of the United States of
and Sorgeloos, P. (2004). Influence of yeast quality on America, 101(13), 4596–4601.
performance of gnotobiotically grown doi:10.1073/pnas.0400706101
Artemia. Journal of Experimental Marine Biology and [56]. Rekecki, A., Dierckens, K., Laureau, S., Boon, N.,
Ecology, 310(2), 247–264. Bossier, P., and Van den Broeck, W. (2009). Effect of
doi:10.1016/j.jembe.2004.04.009 germ-free rearing environment on gut development of
[46]. Marques, A., Ollevier, F., Verstraete, W., Sorgeloos, larval sea bass (Dicentrarchus labrax
P., and Bossier, P. (2005a). Gnotobiotically grown L.). Aquaculture,293(1–2), 8–15.
aquatic animals: opportunities to investigate host- doi:10.1016/j.aquaculture.2009.04.001
microbe interactions. Journal of applied [57]. Reyniers, J. A. (1932). The Use of Germ-Free Guinea
microbiology, 100(5), 903–918. doi:10.1111/j.1365- Pigs in Bacteriology. Proceedings of the Indiana
2672.2006.02961.x Academy of Science, 42(0), 35–36. Retrieved
[47]. Marques, A., Thanh, T., Sorgeloos, P., and Bossier, P. from https://journals.iupui.edu/index.php/ias/article/vi
(2006b). Use of microalgae and bacteria to enhance ew/4464
protection of gnotobiotic Artemia against different [58]. Robert, R., Miner, P., and Nicolas, J. L. (1996).
pathogens. AQUACULTURE, 258(1-4), 116–126. Mortality control of scallop larvae in the
Retrieved from http://hdl.handle.net/1854/LU-367250 hatchery. Aquaculture International, 4(4), 305–313.
[48]. Munro, P. D., Barbour, A., and Birkbeck, T. H. doi:10.1007/BF00120947
(1995). Comparison of the Growth and Survival of [59]. Salvesen, I., and Vadstein, O. (1995). Surface
Larval Turbot in the Absence of Culturable Bacteria disinfection of eggs from marine fish: evaluation of
with Those in the Presence of Vibrio anguillarum, four chemicals. Aquaculture International,3(3), 155–
Vibrio alginolyticus, or a Marine Aeromonas 171. doi:10.1007/BF00118098
sp. Applied and Environmental Microbiology, 61(12), [60]. Sedlacek, R. S., and Mason, K. A. (1977). A simple
4425–4428. Retrieved from and inexpensive method for maintaining a defined
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC13886 flora mouse colony. Laboratory animal science, 27(5
61/ Pt 1), 667–670.
[49]. Newell, R.I.E. and Thompson, R.J. (1984). Reduced [61]. Seed, R. (1976). Ecology. p13-65 in Bayne, B.L., ed.
Clearance Rates Associated with Spawning in the Marine mussels: their ecology and physiology.
Mussel, Mytilus edulis L. (Bivalvia, Mytilidae). Cambridge University Press, New York.
Marine Biology Letters 5(1), 21-33 [62]. Shaw, E. (1957). Potentially Simple Technique for
[50]. Newell, R.I.E. (1989). Species profiles: life histories Rearing “Germ-Free” Fish. Science,125(3255), 987–
and environmental requirements of coastal fishes and 988. doi:10.1126/science.125.3255.987
invertebrates (North and Mid-Atlantic)-blue mussel. [63]. Thompson, R. J. (1979). Fecundity and Reproductive
U.S. Fish. Wildl. Serv. Biol. Rep. 82(11. 102 ). U.S. Effort in the Blue Mussel (Mytilus edulis L.), the Sea
Army Corps of Engineers, TR El-82-4. 25 pp. Urchin (Strongylocentrotus droebachiensis), and the
Snow Crab (Chionoecetes opilio) from Populations in
Nova Scotia and Newfoundland. Journal of the

IJISRT20AUG824 www.ijisrt.com 1429

Volume 5, Issue 8, August – 2020 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Fisheries Research Board of Canada, 36(8), 955–964.
doi:10.1139/f79-133
[64]. Tinh, N. T. N., Nguyen Ngoc Phuoc, Dierckens, K.,
Sorgeloos, P., and Bossier, P. (2006). Gnotobiotically
grown rotifer Brachionus plicatilis sensu strictu as a
tool for evaluation of microbial functions and
nutritional value of different food
types. Aquaculture, 253(1–4), 421–432.
doi:10.1016/j.aquaculture.2005.09.006
[65]. Trexler P.C., and Orcutt R. P. (1999). Development of
Gnotobiotics and Contamination Control in
Laboratory Animal Science, 16:121-127.
[66]. Trust, T. J. (1974). Sterility of Salmonid Roe and
Practicality of Hatching Gnotobiotic Salmonid
Fish. Applied Microbiology, 28(3), 340–341.
Retrieved from http://aem.asm.org/content/28/3/340
[67]. Tubiash, H. S., Chanley, P. E., and Leifson, E. (1965).
Bacillary Necrosis, a Disease of Larval and Juvenile
Bivalve Mollusks I. Etiology and
Epizootiology. Journal of Bacteriology, 90(4), 1036–
1044. Retrieved
from http://jb.asm.org/content/90/4/1036
[68]. Vadstein, O. (1997). The use of immunostimulation in
marine larviculture: possibilities and
challenges. Aquaculture,155(1–4), 401–417.
doi:10.1016/S0044-8486(97)00114-2
[69]. Van De Braak, K., Decamp, O., and Lavens, P.
(2004). Integrated Health Management Combines
Hygiene, Targeted Treatments in Shrimp
Hatcheries. Global Aquaculture Advocate, 82–84.
Retrieved from http://pdf.gaalliance.org/pdf/GAA-
Braak-Oct04.pdf
[70]. Verner-Jeffreys, D. W., Shields, R. J., and Birkbeck,
T. H. (2003). Bacterial influences on Atlantic halibut
Hippoglossus hippoglossus yolk-sac larval survival
and start-feed response. Diseases of aquatic
organisms, 56(2), 105–113. doi:10.3354/dao056105
[71]. Wehrli, W. (1983). Rifampin: mechanisms of action
and resistance. Reviews of infectious diseases, 5 Suppl
3, S407–411.
[72]. Widdows, J. (1991). Physiological ecology of mussel
larvae. Aquaculture, 94(2–3), 147–163.
doi:10.1016/0044-8486(91)90115-N
[73]. Yanong, R. P. E. (2013, June 25). Use of Hydrogen
Peroxide in Finfish Aquaculture. Retrieved from
http://edis.ifas.ufl.edu/fa157

IJISRT20AUG824 www.ijisrt.com 1430