Aquaculture 321 (2011) 1–7

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Morphological characteristics of the digestive tract of gnotobiotic Artemiafranciscana nauplii

R.A.Y.S. Asanka Gunasekara a, Anamaria Rekecki a, Pieter Cornillie a, Maria Cornelissen b, Patrick Sorgeloos c,Paul Simoens a, Peter Bossier c, Wim Van den Broeck a,⁎a Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820, Merelbeke, Belgiumb Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, De Pintelaan 185 6B3, Ghent University, 9000 Ghent, Belgiumc Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium

⁎ Corresponding author. Tel.: +32 92647716; fax: +E-mail address: wim.vandenbroeck@UGent.be (W. V

0044-8486/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.aquaculture.2011.07.037

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 April 2011Received in revised form 22 July 2011Accepted 28 July 2011Available online 9 August 2011

Keywords:GutGnotobioticMicroscopyArtemiaMorphology

Cysts of Artemia franciscanawere hatched and nauplii were reared under gnotobiotic conditions (gnotobioticArtemia rearing system). Stereomicroscopy, computer assisted three-dimensional reconstruction, lightmicroscopy, and transmission electron microscopy were used to study the structural and cellular morphologyof their digestive tracts. The alimentary tract of gnotobiotic Artemia nauplii, fed with dead Aeromonashydrophila and wild type strain of Saccharomyces cerevisiae, is a hooked, tubular structure which is composedof three clearly distinguishable parts, i.e. the foregut, midgut and hindgut that are freely suspended inhaemolymph. The epithelium lining of the entire gut consists of a single cell layer. Enterocytes of the foregutand hindgut are cuboidal and lined by a thin cuticle, whereas midgut enterocytes are cuboidal to columnarand possess an apical brush border. The fore- and hindgut mainly display characteristics suggestive formechanical functions, whereas the midgut shows characteristics of absorption, storage and secretion.The gnotobiotic Artemia rearing system is most useful to investigate the effects of micro-organisms on thedevelopment of nauplii. The knowledge acquired in this study potentially facilitates the evaluation of gutmorphology when specific micro-organisms are introduced into the culture system, as compared to thegnotobiotic counterparts.

32 92647790.an den Broeck).

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Successful aquaculture is still hampered by diseases of the larvalphases, leading tomassivemortalities and considerable economic losses(Marques et al., 2005). Antibiotic therapy and disinfectants have onlyhad limited success in the prevention or cure of aquatic diseases(Defoirdt et al., 2004). Moreover, the frequent use of these chemicalsresults in rapid development of resistance (Dias et al., 1995; Molina-Ajaet al., 2002; Vattanaviboon et al., 2003; Vivekanandhan et al., 2002).Therefore, it is of major importance to have alternative disease controltechniques in aquaculture, focusing especially on prevention, which islikely to be more cost-effective than curative treatments (Subasinghe,1997). Managing microbiota in larvae aquaculture is an effectivepreventive strategy. Hence, the study and control of harmful micro-organisms and the promotion of beneficial micro-organisms are ofutmost importance.

A powerful experimental approach to study the function of themicrobiota uses a gnotobiotic rearing system in which animals arereared in germ-free conditions and monitored after introducing

defined microbes (Falk et al., 1998). Although many studies areperformed with gnotobiotic terrestrial animals, studies with gnoto-biotic aquatic organisms are still scarce. Some constraints hamperingthe wide use of these organisms in research are the need fordisinfection methods to produce germ-free organisms and difficultiesin assuring the complete germ-free condition of a culture system(Marques et al., 2006). A successful step forward in the developmentof such an aquatic gnotobiotic model was made more than twentyyears ago when Sorgeloos et al. (1986) and later Marques et al.(2004a) described a gnotobiotic Artemia rearing system to obtainsterile Artemia cysts and nauplii. Future studies of host–microbeinteractions using gnotobiotic aquatic animals should considerpossible parameters, such as survival, growth, immunological re-sponse and histological development (Marques et al., 2006). Thepresent study is aimed especially at themorphology of the gnotobioticArtemia nauplii.

Although the gut morphology of conventional Artemia nauplii wasstudied in detail using transmission electron microscopy a fewdecades ago (Hootman and Conte, 1974), to our knowledge, this isthe first descriptive study illustrating the digestive tract morphologyof gnotobiotic Artemia franciscana nauplii. The present study aims todescribe the histology, the three-dimensional architecture, and thecellular morphology of the gut of gnotobiotic Artemia nauplii during

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the early stages of the life cycle. The knowledge gained from this studywill facilitate future research which aims at comparison of thedigestive tract morphology between gnotobiotic Artemia nauplii andtheir counterparts at same age after introduction of either one ormoredefinite strains of micro-organisms to the culture system.

2. Materials and methods

2.1. Culturing and harvesting of yeast

Wild type (WT) strain of baker's yeast (Saccharomyces cerevisiae)(BY4741 [genotype, Mata his3Δ 1 leu2Δ0 met15Δ0 ura3Δ0]), kindlyprovided by the European Saccharomyces cerevisiae Archive forFunctional Analysis (University of Frankfurt, Frankfurt, Germany),was used as live feed for Artemia. It was cultured in minimal yeastnitrogen based (YNB), with amino acids (histidine HCl: 10 mg/l;methionine: 20 mg/l; tryptophan: 20 mg/l; inositol: 2 mg/l-Sigma,0.67% w/v), supplemented with uracil (Sigma, 0.002% w/v), leucine(Sigma, 0.002% w/v) and D-glucose (Sigma, 0.5% w/v), agar. Harvestedyeast cells were counted following the methodology described byMarques et al. (2006) and Gunasekara et al. (2010b). Yeastsuspensions were stored at 4 °C and used to feed Artemia until theend of the experiment.

2.2. Culturing, harvesting, and killing of bacteria

The Aeromonas hydrophila strain (LVS3) selected for the experi-ment is a rod shaped, motile, gram negative, Aeromonadaceaebacterial strain. LVS3 was cultured and harvested, and the densitywas determined according to the methodology described by Marqueset al. (2005, 2006) and Gunasekara et al. (2010b). As LVS3 cells wereused for feeding, they were killed by autoclaving at 120 °C for 20 min.Bacterial suspensions were stored at 4 °C and used to feed Artemiauntil the end of the experiment.

2.3. Culturing and feeding of gnotobiotic Artemia

The experimentwas carried out usingA. franciscana originating fromtheGreat Salt Lake, Utah. Artemia cystswere hydrated and decapsulatedfollowing the procedures described by Defoirdt et al. (2005) in order toobtain sterile cysts and subsequently sterile nauplii. Sterilization ofnecessary equipment, decapsulation of cysts and setting of culture tubesfor hatching were done according to the procedures described byGunasekara et al. (2010b). After about 20–24 h, 30 nauplii weretransferred to fresh 50 ml sterile tubes containing 30 ml filteredautoclaved sea water made with Instant Ocean (Aquarium systems,Sarrebourg, France). All the culture tubes received living germ-free WTyeast and dead germ-free LVS3 as daily feed and were placed on a rotorturning at 4 rpm. Daily feeding was done ad libitum. As such 140.1 μgWT strain (ash free dryweight content) and 1401 μg dead bacteriawereadministered per culture tube during the six days of the study period asdescribed by Marques et al. (2004a).

Sampling was done at days two, four and six for stereomicroscopy,light microscopy, and transmission electron microscopy and testedwith four replicates for each.

Fig. 1. Stereomicroscopic images of two day-old (a), four day-old (b), and six day-old(c) gnotobiotic Artemia nauplii. 1, gastric caeca; 2, midgut; 3, midgut–hindguttransition; 4, hindgut; 5, anal opening.

2.4. Verification of axenity

The axenity of the decapsulated cysts, the Artemia culture waterand the dead LVS3 suspension (after autoclaving) was verified usingplating. Absence or presence of bacterial growth was monitored afterfive days of incubation at 28 °C of 100 μl of decapsulated cysts, culturemedium or feed plated on Marine agar (n=2).

2.5. Stereomicroscopy

Immediately after fixation, the specimens were viewed andphotographed using a stereomicroscope (Olympus SZX 7, Olympus,Belgium) to study the gut segments in relation to external structures.

2.6. Light microscopy

Nauplii were fixed, pre-stained and further processed for histologicalsections following the procedures described by Gunasekara et al.

Fig. 2. Three-dimensional reconstruction images of two-day-old (a), four-day-old (b), and six-day-old gnotobiotic nauplii (c), digestive tract segments of four-day-old gnotobioticArtemia nauplius (d). 1, foregut; 2, gastric caeca; 3, midgut; 4, midgut–hindgut transition; 5, hindgut; 6, anal opening.

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(2010b). Per day, six nauplii were cut into serial transverse sections andfour nauplii were cut into longitudinal/sagittal sections of 5 μm thicknesswith a microtome (Microm HM360, Prosan). For general histology, thesections were stained with haematoxylin (Haematoxylin (C.I. 75290),Merck KGaA, Darmstadt, Germany) and eosin (Eosin yellow (C.I. 45380),VWR International bvba/sprl, Leuven, Belgium), and for the detection ofstructures containing high concentrations of carbohydrate macromole-cules, the sections were stained with PAS (periodic-acid-Schiff) reagent(C.I. 42500, Merck KGaA, Darmstadt, Germany). The histological sectionswere examined and photographed using a motorized microscope(Olympus BX 61, Olympus Belgium, Aartselaar, Belgium) linked to adigital camera (Olympus DP 50, Olympus Belgium).

2.7. Three-dimensional (3D) reconstruction

All serial histological sections were photographed using theobjective lens 20× (1.0 μm×1.0 pixel size) or 40× (0.5 μm×0.5 μmpixel size) depending on the size of the tissue section and saved injpeg format in two separate folders according to the magnification.The last image in each folder was obtained from the same section asthe first picture of the next folder.

A plain text file for every folder was created to load the bricks ofpictures in the stacked slice file format into the Amira 4.0.1 (VisageImaging GmbH, Berlin, Germany) application, following the detailedinformation described by Cornillie et al. (2008).

All the slices within a brick were aligned, using an automatedmodule, and by manual correction whenever necessary. Every pixel ofeach picture was assigned a grey-tone value based on the green colourchannel (channel 1). Important structures on these images such as thecontours of the nauplii and the fore-, mid- and hindgut of every fourthsection was labeled manually with the brush tool (for internalstructures) and lasso tool (for external surfaces) in the segmentationeditor. The sections in-between were subsequently labeled throughthe interpolation command.

The voxel size of the two bricks of labeled sections was down-sampled by a factor 2 in each direction (x, y and z) using the resamplingmodule to save memory.

Constrained smoothened surface images of labeled structureswere generated using SurfaceGen module. Two separate 3D imageswere created from two bricks of each nauplius and aligned manuallyusing the transform editor of the Amira program.

2.8. Transmission electron microscopy (TEM)

Samples for TEM were fixed in Karnovsky's fixative. Afterovernight fixation at 4 °C, specimens were washed in sodiumcacodylate buffer (pH 7.4), and postfixed overnight in 1% osmiumtetroxide. Subsequently, the samples were dehydrated using a gradedseries of alcohols (50% to 100%) and finally embedded in SPURR'sresin. After examination of semithin sections to localize the regions ofinterest, ultrathin sections of 60 nmwere made using a Leica EMMZ 6ultramicrotome (Leica Mircosystems GmbH), mounted on formvarcoated single slot copper grids (Laborimpex N.V., Brussels, Belgium).The sections were post-stained with uranyl acetate and lead citrate(Leica EM Stain, LeicaMicrosystems GmbH) and viewed on a Jeol 1200EXII TEM (JEOL Ltd., Tokyo, Japan) at 80 kV accelerating voltage.

3. Results

3.1. Stereomicroscopy

The transparent gut segments (mid- and hindgut) could bevisualized clearly by stereomicroscopy. Gastric caeca and the midgut–hindgut transition zones were also clearly visible (Fig. 1a, b, c).

3.2. Three-dimensional morphology

The alimentary tract of A. franciscana nauplii is freely suspended inhaemolymph and composed of three clearly distinguishable

Fig. 3. Histological sections of foregut–midgut transition in a longitudinal section (a), midgut–hindgut transition in a longitudinal section (b), foregut–midgut transition in atransverse section (c), and hindgut in a transverse section (d) of gnotobiotic Artemia nauplii. 1, foregut cells; 2, foregut–midgut transition; 3, midgut cells; 4, brush border; 5, midgutlumen; 6, midgut–hindgut transition; 7, hindgut cells; 8, hindgut lumen; 9, gut content.

Fig. 4. Longitudinal section of Artemia nauplius stained with periodic-acid-Schiffreagent. 1, foregut cells; 2, midgut cells; 3, brush border; 4, gut content.

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segments, i.e. the foregut, the midgut and the hindgut. The mid- andthe hindgut are aligned, whereas the foregut is a hooked tube with ashorter part perpendicular to the midgut, while the remaining longerpart is nearly parallel to it. The fore- and hindgut are shorter and havea smaller diameter as compared to the midgut. Two lateral globularprotrusions from the anterior part of the midgut, called gastric orhepatopancreatic caeca, can be identified (Fig. 2a, b, c, d).

3.3. Histology and ultrastructure

3.3.1. ForegutThe foregut is lined cranially with cuboidal cells (Fig. 3a, c) and

where it opens into the midgut a clear transition from cuboidal tocuboidal-columnar lining cells is visible (Fig. 3a, c). The cells of theforegut contain small amounts of cytoplasmic organelles and theirluminal surface is covered by a thin cuticle layer (Fig. 5a). Thebasement membrane separates the epithelium from the underlyingmuscle cells which are circularly arranged at intervals around theforegut and a longitudinal muscle layer.

3.3.2. MidgutThe mucosal lining of the entire midgut, including the two gastric

caeca, is composed of a single epithelium of cuboidal to columnar cells(Fig. 3a, b, c). A brush border which stains PAS positive (Fig. 4) in theepithelial cells of the midgut are visible (Fig. 3a, b, c). The cells arebound to each other with occluding and anchoring junctions (Fig. 5b).Large, central nuclei are observed throughout the whole midgutepithelium (Fig. 5b, c, d). The cytoplasm of the midgut epithelial cellscontains numerous mitochondria and a well developed endoplasmicreticulum. Golgi complexes are seen in some cells. The basal cellmembrane of the midgut epithelial cells, and in particular of the cellslocated in the most caudal region, is folded into narrow channels,called basal infoldings, which protrude into the basal part of thecytoplasm (Fig. 5b, d). The basement membrane separates the

epithelium from the underlying muscle cells which are circularlyarranged at intervals around the midgut (Fig. 5d, f). A longitudinalmuscle layer could not be observed in the midgut. A clear transitionfrom a cuboidal-columnar to a cuboidal epithelium is present at theborder between the midgut and the hindgut (Figs. 3b, 5e).

3.3.3. HindgutThe hindgut is lined with a cuboidal cell layer (Figs. 3b, d, 5f). The

hindgut epithelial cells do not possess apical microvilli, instead theyare covered by a thin cuticle layer (Fig. 5e, f).

The cells of the hindgut contain small amounts of cytoplasmicorganelles. The basement membrane separates the epithelium fromthe underlying muscle cells which are circularly arranged at intervalsaround the hindgut and from a longitudinal muscle layer (Fig. 5f).

During the entire period of experiment (six days) all parts of thealimentary tract increased proportionally in the nauplii (Table 1).

Fig. 5. Transmission electron micrographs of gut segments of gnotobiotic Artemia nauplii.

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Table 1Length of the gut segments (in six replicates) of gnotobiotic brine shrimp nauplii feddaily with dead LVS3 bacteria and wild type yeast.

Midgut length Hindgut length Total length

Day 2 0.70±0.04 0.25±0.02 0.95±0.05Day 4 0.72±0.02 0.25±0.03 0.97±0.03Day 6 0.73±0.05 0.26±0.02 0.99±0.04

Data are shown as mean±SD.

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4. Discussion

Animals get in contact with complex and dynamic populations ofmicro-organisms during their life cycle (Rawls et al., 2004). Therefore,there is an urgent need to study deleterious and beneficial effects ofsuch microbial communities. An essential part of that understandingcan be provided by studies looking in detail at the host–microbialinteractions (Marques et al., 2006). Furthermore, the need for anefficient model system to test the effects of microbes on the hostemerges. However, when aquatic biota is concerned, difficulties ineliminating the commensal microflora hamper the study of the host–microbial interactions (Marques et al., 2004b).

Gnotobiology is a well established science (Marques et al., 2005),which makes studying of host–microbial interactions effective.Additionally, the use of gnotobiotic organisms allows an increasedcontrol of variables, enhances the reproducibility of results, andenables the more accurate experimental designs (Pleasants, 1973). Akey experimental strategy to study these interactions is to first definethe morphology and functioning of the animal in the absence of allmicro-organisms (i.e. under germ-free or gnotobiotic conditions) andthen to evaluate the effects of adding a single or defined population ofmicrobes (Gordon and Pesti, 1971).

Many studies have been performed in the past, using a wide rangeof terrestrial gnotobiotic animals such as gnotobiotic mice (Abrams etal., 1963; Banasaz et al., 2001, 2002; Uribe et al., 1997), guinea pigs,chickens, dogs, cats, and pigs (Delluva et al., 1968). However, similarstudies on aquatic animals are still scarce. Some studies haveidentified gnotobiotic marine and fresh water fish as a good researchtool to investigate host–microbial interactions, e.g. germ-free turbot(Scophthalmus maximus) larvae by Munro et al. (1995), bacteria-freehalibut (Hippoglossus hippoglossus) larvae by Verner-Jeffreys et al.(2003), gnotobiotic zebra fish (Danio rerio) by Rawls et al. (2004), orgerm-free seabass (Dicentrarchus labrax) larvae by Dierckens et al.(2009) and Rekecki et al. (2009).

In the present study, A. franciscanawas used as an aquatic model todescribe the digestive tract morphology of the gnotobiotic nauplii. A.franciscana is an important live food for commercial production of fishand shellfish larvae (Sorgeloos et al., 1986) and a unique aquaticmodel to elucidate host–microbial interactions under germ-free andgnotobiotic culture conditions using various types of feed sourceswith simple experimental equipment (Verschuere et al., 1999, 2000).

Since it is evident that those gnotobiotic animal models are aprerequisite to study the host–microbe interactions and as morphol-ogy is used as a monitoring tool in our other studies, the study of thegnotobiotic animals and their detailedmorphology before introducingany knownmicrobiota is of high value. Hence, in the present study thedigestive tract of gnotobiotic A. franciscanawas studied in detail usingstereomicroscopy, three-dimensional reconstruction, light microsco-py, and transmission electron microscopy. Stereomicroscopy allowedstudying the general appearance of the Artemia body and the digestivetract. Three-dimensional reconstructions facilitated evaluation ofarchitectural organization and topography of the digestive tract.Histological and transmission electron microscopic examinationallowed evaluating the types of cells, junctions between gut segments,the presence or absence of a cuticle or brush border. Usingtransmission electron microscopy, the cells could be studied for the

organization of organelles, transition zones and presence of microvilli.As such, the whole study enabled evaluating the digestive tract ofgnotobiotic Artemia nauplii in detail.

The alimentary tract of gnotobiotic Artemia nauplii is a hooked tubeconsisting of three major segments, which is in agreement withprevious reports of Hootman and Conte (1974)whomade a descriptiveultrastructural account on conventional second instar Artemia nauplii,Schrehardt (1987) who illustrated the morphology of adult conven-tional Artemia, and Criel (1991) who described conventional Artemianauplii and adults. The mucosal cell types and the arrangement oforganelles such as nuclei and mitochondria in the cells of the entire gutof conventional Artemia nauplii described by the above-mentionedauthors are similar to the present findings. However, the abundance ofGolgi apparatus was little less as seen in the current study. According tothedescription of Criel (1991), the fore- and the hindgut are surroundedbycircularly arranged anda longitudinalmuscle layerswhile themidgutis only surrounded by a circularly arranged muscle layer, as was alsoobserved in the present study in gnotobiotic Artemia nauplii.

In the present study,microvilli of themidgut epithelial cells could beobserved in gnotobiotic Artemia nauplii which are suggestive for anabsorptive function as described by Kikuchi (1971) in conventionaladult Artemia. The microvilli are coated with a fine fibrous material,probably mucopolysaccharides as mentioned by Criel (1991). In thecurrent study, the brush borderwaspositively stainedwith PAS reagent.This finding suggests that the microvilli are surrounded by structurescontaining high concentrations of carbohydrate macromolecules (e.g.glycogen, glycoprotein, proteoglycan), which is in accordance with theresults of Hauber and Zabel (2009). In contrast, Hootman and Conte(1974) did not find any muco-polysaccharide glycocalyx, but they didobserve numerous vesicles filling the spaces among microvilli. As noepithelial goblet cells were detected using PAS staining in nauplii at thisstage, further studies are necessary to confirm the origin andbiochemical nature of these compounds. The presence of infoldings ofthe basal cellular surfaces and associated mitochondria in the epithelialcells lining the caudal part of themidgut support the absorptive functionof these cells (Hootman and Conte, 1974).

Gnotobiotic techniques have been utilised to assess the ability ofspecific bacteria to protect live food cultures against pathogenicbacterial strains (Marques et al., 2006). Therefore in future researchusing such gnotobiotic systems, beneficial effects such as probiotic(single or in mixtures) or deleterious effects of micro-organisms can bescreened by comparing to the gnotobiotic basic model. Similarly sucheffects can be compared with conventional Artemia in which theresident microbial community is more poorly defined. With stereo-microscopy, the effects of micro-organisms on individual naupliarlength can be measured (Gunasekara et al., 2010a). Furthermore,histology and morphometrical analysis can facilitate comparison of thegross morphology of the various gut segments (Gunasekara et al.,2010b), while ultrastructural changes such as site specific colonization,adhesion or translocation can be verified by transmission electronmicroscopy. Further studies usingpathogenic bacteriawill eventually beof importance for investigating preventive and / or curing measures.

Acknowledgements

This study was supported by a Special Research Grant (BijzonderOnderzoeksfonds, BOF, grant numbers B/07289/02 and 05B01906) ofGhent University (Belgium) awarded to the first author and by theFoundation for Scientific Research-Flanders (FWO) (Project: Probiontinduced functional responses in aquatic organisms, G.0491.08).

We are indebted to Prof. Godelieve Criel for the guidance duringthe experiment and comments for the manuscript. Thanks are also toProf. Annemie Decostere and Dr. Christophe Casteleyn for criticallyreading the manuscript. Excellent technical assistance of Lobke DeBels, Bart De Pauw, Liliana Standaert and Leen Pieters is alsothankfully acknowledged.

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References

Abrams, G.D., Bauer, H., Sprinz, H., 1963. Influence of the normal flora on mucosalmorphology and cellular renewal in the ileum. A comparison of germ-free andconventional mice. Lab. Invest. 12, 355–364.

Banasaz, M., Norin, E., Midtvedt, T., 2001. The role of gender, age andmicrobial status oncell kinetics in the gastrointestinal tract of mice. Microb. Ecol. Health Dis. 13,135–142.

Banasaz, M., Norin, E., Holma, R., Midtvedt, T., 2002. Increased enterocyte production ingnotobiotic rats mono-associated with Lactobacillus rhamnosus GG. Appl. Environ.Microbiol. 68, 3031–3034.

Cornillie, P., Van den Broeck, W., Simoens, P., 2008. Three-dimensional reconstructionof the remodeling of the systemic vasculature in early pig embryos. Microsc. Res.Techniq. 71, 105–111.

Criel, G.R.J., 1991. Morphology of artemia. In: Browne, R.A., Sorgeloos, P., Trotman,C.A.N. (Eds.), Artemia Biology. CRC Press, Inc., USA, pp. 119–154.

Defoirdt, T., Boon, N., Bossier, P., Verstraete, W., 2004. Disruption of bacterial quorumsensing: an unexplored strategy to fight infections in aquaculture. Aquaculture 240,69–88.

Defoirdt, T., Bossier, P., Sorgeloos, P., Verstraete, W., 2005. The impact of mutations inthe quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and Vibrioharveyi on their virulence towards gnotobiotically cultured Artemia franciscana.Environ. Microbiol. 7, 1239–1247.

Delluva, A.M., Markley, K., Davies, R.E., 1968. The absence of gastric urease in germ-freeanimals. Biochim. Biophys. Acta (BBA) Enzymology 151, 646–650.

Dias, J.C.D.R., Ernandez, D., Silva, A.A.D.E., de Oliveira, L.M.R.R., da Silva, S.E.A., 1995.Antimicrobial andmercury chloride resistance in Vibrio isolates frommarine fish ofthe southeastern Brazilian region. Rev. Microbiol. 26, 253–259.

Dierckens, K., Rekecki, A., Laureau, S., Sorgeloos, P., Boon, N., Van den Broeck, W.,Bossier, P., 2009. Development of a bacterial challenge test for gnotobiotic sea bass(Dicentrarchus labrax) larvae. Environ. Microbiol. 11, 526–533.

Falk, P.G., Hooper, L.V., Midtvedt, T., Gordon, J.I., 1998. Creating and maintaining thegastrointestinal ecosystem: what we know and need to know from gnotobiology.Microbiol. Mol. Biol. Rev. 62, 1157–1170.

Gordon, H., Pesti, L., 1971. The gnotobiotic animal as a tool in the study of host–microbial relationships. Bacteriol. Rev. 35, 390–429.

Gunasekara, R.A.Y.S.A., Cornillie, P., Casteleyn, C., De Spiegelaere, W., Sorgeloos, P.,Simoens, P., Bossier, P., Van den Broeck, W., 2010a. Stereology and computerassisted three-dimensional reconstruction as tools to study probiotic effects ofAeromonas hydrophila on the digestive tract of germ-free Artemia franciscananauplii. J. Appl. Microbiol. 110, 98–105.

Gunasekara, R.A.Y.S.A., Rekecki, A., Baruah, K., Bossier, P., Van den Broeck, W., 2010b.Evaluation of probiotic effect of Aeromonas hydrophila on the development of thedigestive tract of germ-freeArtemia franciscananauplii. J. Exp.Mar. Biol. Ecol. 393, 78–82.

Hauber, H.P., Zabel, P., 2009. PAS staining of bronchoalveolar lavage cells for differentialdiagnosis of interstital lung disease. Diagn. Pathol. 4, 13.

Hootman, S.R., Conte, F.P., 1974. Fine structure and function of the alimentaryepithelium in Artemia salina nauplii. Cell Tiss. Res. 155, 423–436.

Kikuchi, S., 1971. The fine structure of the alimentary canal of the brine shrimp. Artemiasaline: the midgut. Ann. Rept. Iwate. Medical Univ. 6, 17–47.

Marques, A., Dhont, J., Sorgeloos, P., Bossier, P., 2004a. Evaluation of different yeast cellwall mutants and microalgae strains as feed for gnotobiotically-grown brineshrimp Artemia franciscana. J. Exp. Mar. Biol. Ecol. 312, 115–136.

Marques, A., François, J., Dhont, J., Bossier, P., Sorgeloos, P., 2004b. Influenceof yeast quality onperformance of gnotobiotically-grown Artemia. J. Exp. Mar. Biol. Ecol. 310, 247–264.

Marques, A., Dinh, T., Ioakeimidis, C., Huys, G., Swings, J., Verstraete, W., Dhont, J.,Sorgeloos, P., Bossier, P., 2005. Effects of bacteria on Artemia franciscana cultured indifferent gnotobiotic environments. Appl. Environ. Microbiol. 71, 4307–4317.

Marques, A., Ollevier, F., Verstraete, W., Sorgeloos, P., Bossier, P., 2006. Gnotobioticallygrown aquatic animals: opportunities to investigate host–microbe interactions. J.Appl. Microbiol. 100, 903–918.

Molina-Aja, A., Garcia-Gasca, A., Abreu-Grobois, A., Bolan-Mejia, C., Roque, A., Gomez-Gil, B.,2002. Plasmid profiling and antibiotic resistance of Vibrio strains isolated from culturedpenaeid shrimp. FEMS Microbiol. Lett. 213, 7–12.

Munro, P., Barbour, A., Birkbeck, T., 1995. Comparison of the growth and survival oflarval turbot in the absence of culturable bacteria with those in the presence ofVibrio anguillarum, Vibrio alginolyticus, or a marine Aeromonas sp. Appl. Environ.Microbiol. 61, 4425–4428.

Pleasants, J., 1973. Germ-free animals and their significance. Endeavour 117, 112–116.Rawls, J.F., Samuel, B.S., Gordon, J.I., 2004. Gnotobiotic zebrafish reveal evolutionarily

conserved responses to the gut microbiota. Proc. Natl. Acad. Sci. USA 101,4596–4601.

Rekecki, A., Dierckens, K., Laureau, S., Boon, N., Bossier, P., Van den Broeck, W., 2009.Effect of germ-free environment on survival and gut development of larval sea bass(Dicentrarchus labrax L.). Aquaculture 293, 8–15.

Schrehardt, A., 1987. Ultrastructural investigations of the filter-feeding apparatus andthe alimentary canal of Artemia. In: Sorgeloos, P., Bengtson, D.A., Decleir, W.,Jaspers, E. (Eds.), Artemia Research and its Applications. Universa Press, Wetteren,Belgium, p. 33.

Sorgeloos, P., Lavens, P., Léger, P., Tackaert, W., Versichele, D., 1986. Manual for theculture and use of brine shrimp Artemia in aquaculture. Artemia Reference Center,Faculty of Agriculture. State University of Ghent, Ghent, Belgium, p. 318.

Subasinghe, R., 1997. Fish health and quarantine, Review of the State of WorldAquaculture. Food and Agriculture Organization of the United Nations, Rome, Italy:FAO Fisheries Circular, 886, pp. 45–49.

Uribe, A., Alam, M., Midtvedt, T., Smedfors, B., Theodorsson, E., 1997. Endogenousprostaglandins and microflora modulate DNA synthesis and neuroendocrinepeptides in the rat gastrointestinal tract. Scand. J. Gastroenterol. 32, 691–699.

Vattanaviboon, P., Panmanee, W., Mongkolsuk, S., 2003. Induction of peroxide andsuperoxide protective enzymes and physiological cross-protection against peroxidekilling by a superoxide generator in Vibrio harveyi. FEMSMicrobiol. Lett. 221, 89–95.

Verner-Jeffreys, D., Shields, R., Birkbeck, T., 2003. Bacterial influences on Atlantic halibutHippoglossus hippoglossus yolk-sac larval survival and start-feed response. Dis.Aquat. Organ. 56, 105–113.

Verschuere, L., Rombaut, G., Huys, G., Dhont, J., Sorgeloos, P., Verstraete, W., 1999.Microbial control of the culture of Artemia juveniles through preemptivecolonization by selected bacterial strains. Appl. Environ. Microbiol. 65, 2527–2533.

Verschuere, L., Heang, H., Criel, G., Sorgeloos, P., Verstraete, W., 2000. Selected bacterialstrains protect Artemia spp. from pathogenic effects of Vibrio proteolyticus CW8T2.Appl. Environ. Microbiol. 66, 1139–1146.

Vivekanandhan, G., Savithamani, K., Hatha, A.A.M., Lakshmanaperumalsamy, P., 2002.Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish andprawn of South India. Int. J. Food Microbiol. 76, 165–168.