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A method for the specific detection of resident bacteria in brine shrimp larvae

A method for the specific detection of resident bacteria in brine shrimp larvae
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  (This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Note A method for the speci fi c detection of resident bacteria in brine shrimp larvae Yufeng Niu  a,d , Tom Defoirdt  a , Anamaria Rekecki  c , Peter De Schryver  a , Wim Van den Broeck  c ,Shuanglin Dong  d , Patrick Sorgeloos  a , Nico Boon  b , Peter Bossier  a, ⁎ a Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium b Laboratory for Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium c Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820, Merelbeke, Belgium d The key laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, China a b s t r a c ta r t i c l e i n f o  Article history: Received 9 February 2012Accepted 10 February 2012Available online 16 February 2012 Keywords: Resident bacteriaBrine shrimpDGGEIntestinal bacteria In this study, we describe an easy but ef  fi cient method to speci fi cally target the intestinal resident microbiotain brine shrimp larvae during DGGE analysis, hereby excluding the interference of both transient (luminal)bacteria and body surface bacteria. This effective technique has several advantages over alternative methods,with respect of ease of use and rapidity.© 2012 Elsevier B.V. All rights reserved. Inthe studyof host – microbeinteractions,theresidentbacteriaareconsidered prime contributors to long-lasting effects. Harris (1993)proposed a conceptual model that differentiates between transientand resident gut micro fl ora associated with invertebrates. Residentbacteria are persistently present in the gut, whereas transient bacteriado not form a stable population. However, separating or speci fi callytargeting resident bacteria is not always easy. Nowadays, DGGE hasbecome a popular molecular technique for the assessment of intestinalmicrobialbiodiversity(Brunvoldetal.,2007;Luoetal.,2006;Merri fi eldetal.,2009).Inlargeranimals,thegutcanbedissectedandthegutcouldbe scraped or washed to remove food or transient bacteria. However,with animals of smaller size, such as  Artemia , dissection is dif  fi cult.Thus, during DGGE analysis, in most cases, whole DNA extracts of theanimal are taken into account and this can result in the detection of only the dominating transient (luminal) intestinal bacteria, whereas,the resident bacteria are undistinguishable from background patters.For this reason, we aimed at developing a simple and ef  fi cient protocolfor screening  Artemia  gut resident bacteria by alleviating the interfer-ence of exteriors and gut transient bacteria during DGGE analysis.The study consists of three major parts. Firstly, a method is pre-sented to wash bacteria adhering to the exterior surface. Secondly, amethod is developed to purge the transient (luminal) bacteria. Finally,the two methods are combined, allowing alleviating the interferenceof exterior and transient bacteria, targeting only resident bacteria inthe gut of brine shrimp nauplii. A schematic view of the method andoutline is presented in Fig. 1.In the  fi rst phase, a washing procedure was developed to washaway the bacteria adhering to the exterior surface of the brineshrimp. The absence of exterior bacteria was con fi rmed by DGGE.Bacteria-free brine shrimp (  Artemia franciscana ) were cultured asdescribed previously (Sorgeloos et al., 1986). 18 h after hatching, in- star I nauplii were collected in 500 mL glass bottles at a density of 1 nauplii/mL. Half of the nauplii were killed by freezing in 10 mL   fi l-tered (0.22  μ  m) and autoclaved seawater (FASW) for 1 h at − 20 °C.After thawing, dead nauplii were exposed to a mixture of seven dif-ferent bacteria for 3 h: LVS3, ISO8, ISO10, ISO11,  Vibrio campbellii LMG 21363 (abbreviated as VC), LT3, LT12 (Defoirdt et al., 2010;Makridis et al., 2005; Marques et al., 2005). As a control, live instar Inauplii were also exposed to the same bacteria. They were all addedat a concentration of 10 6 /mL. After bacterial exposure, the naupliiwere collected by  fi ltering through a sterile 100  μ  m  fi lter and subse-quentlyrinsedfor 30 swith 20 mL1% benzalkonium chloridesolution(Huysetal.,2001),followedbyrinsingtwicefor30 swithFASW.Axe- nically hatched nauplii which were not subjected to bacterial exposurewere used as negative control. From each treatment, 120 nauplii weretaken for DNA extraction. DNA was extracted by using the Blood andTissue Kit (Qiagen). A nested PCR strategy was used. For the externalPCR ampli fi cation we used the primer Eub8F, 5 ′ -agagtttgatcmtggct-cag-3 ′  and the broad coverage primer 984yR, 5 ′ -gtaaggttcytcgcgt-3 ′ .The 338 F-GC/518R primer set was used for the internalPCR ampli fi ca-tion.PCRreactionandDGGEwereperformedasdescribedpreviouslybyBakke et al. (2011).  Journal of Microbiological Methods 89 (2012) 33 – 37 ⁎  Corresponding author. E-mail address: (P. Bossier).0167-7012/$  –  see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2012.02.004 Contents lists available at SciVerse ScienceDirect  Journal of Microbiological Methods  journal homepage:  Author's personal copy As can be observed from the DGGE  fi ngerprinting patterns, thebacterial bands that were present in the treatments without washing(Fig.2C andE)wereabsentinthewashing treatments(Fig.2B andD). Instar I nauplii has an axenic intestinal tract, because it does not takeup food as its digestive system is not functional yet; it thrivescompletely on its yolk reserves (Benesch, 1969). Thus, all the bacteriadetected by DGGE should be attached to the exterior surface of the  Artemia  body. This result con fi rmed that the washing procedure cansuccessfully wash away the bacteria attaching to the exterior surfaceof   Artemia nauplii. ThebandsindicatedbythearrowsinFig.2arealsopresent when DNA is ampli fi ed from axenically hatched  Artemia (Fig. 2F and G). They are absent in the ampli fi cation of pure bacterialDNA (Fig. 2A). Therefore, it is suggested that these bands are unspe-ci fi c  Artemia  ampli fi cation products of the PCR approach. Becausethese unspeci fi c bands share no overlap with the targeted bacterialbands, they are not interfering with the overall interpretation of the result.In the second phase, a method was developed to purge transientbacteriafromthe intestinaltract usingcelluloseparticles. Thepurgingeffect of cellulose was con fi rmed by  fl uorescence microscope.Brine shrimp larvae were axenically hatched. 28 h after hatchinginstar II nauplii were collected and subsequently enriched withLVS3 or VC (Marques et al., 2006) for 12 h. Both bacterial strains were labelled with the green  fl uorescent protein (GFP) by conjuga-tion with plasmid as described earlier (Andersen et al., 1998; Boonet al., 2001; Rekecki et al., in press). After enrichment with these bac-teria, the nauplii were cultured in a cellulose suspension (1.5 g/L)(Sigma, type 20) for 4 h. Brine shrimp are non-selective particle  fi lterfeeders grazing on particles smaller than 50  μ  m (Dobbeleir et al.,1980). By feeding the instar II nauplii with sterile cellulose particles,the transient bacteria were purged by means of the cellulose. Naupliiloaded with bacteria but receiving no cellulose were used as controlgroup.Thesamewashingprocedurewasthenappliedtoallthenauplii.The whole larval body was individually mounted on glass slides with a Fig. 1.  Schematic view of experiment outline. FASW: Filtered and autoclaved sea water. Fig. 2.  Removal of exterior bacteria by the washing procedure. Lane A, mixture of PCR ampli fi cation products from the seven bacteria; Lane B, dead nauplii subjected to the washingprocedure; Lane C, dead nauplii not subjected to the washing procedure; Lane D, live nauplii subjected to the washing procedure; Lane E, live nauplii not subjected to the washingprocedure; Lane F, axenic nauplii subjected to the washing procedure as negative control for the washing procedure; Lane G, axenic nauplii not subjected to the washing procedureas negative control for axenity of instar I nauplii. *The bands indicated by the arrows in Fig. 2 are also present when DNA is ampli fi ed from axenically hatched  Artemia  (F, G). Theyare absent in the ampli fi cation of pure bacterial DNA (Lane A). Therefore, it is suggested that these bands are unspeci fi c  Artemia  ampli fi cation products of the PCR approach. Becausethese unspeci fi c bands share no overlap with the targeted bacterial bands, they are not interfering with the overall interpretation of the result.34  Y. Niu et al. / Journal of Microbiological Methods 89 (2012) 33 –  37   Author's personal copy solution of glycerine and 1, 4-diaza-bicyclo [2, 2, 2]-octane (DABCO)(ACROS Organics, Geel, Belgium), and then examined, using a  fl uores-cence microscope (Olympus BX61 Fluorescence Microscope; OlympusBelgium N.V., Aartselaar, Belgium) with the  fi lter cube U-MWIB2. Thisallowed verifying for the presence of the GFP-labelled bacteria in thelarval intestinal tract (Rekecki et al., in press).After cellulose treatment, LVS3 was purged from the shrimp gut(Fig. 3E and F). In contrast, a signi fi cant level of VC was still detectedafter cellulose purging(Fig.3G and H). Thisobservationwas expectedasit wasshownbeforethatthisstrainattachestothebrineshrimpin-testinal epithelium (Gunasekara, 2011). This result con fi rmed thatthe cellulose treatment can purge transient bacteria.In summary, the exterior bacteria and gut transient bacteria couldbe subsequently removed from  Artemia  nauplii by a washing proce-dure and cellulose purging treatment.Finally, thewashingprocedure combined with the cellulose purgingtreatmentwas applied on  Artemia  to investigate the capability of differ-ent bacteria to become resident in brine shrimp larvae as veri fi ed byPCR-DGGE.Axenically hatched instar II nauplii were collected and kept in 2 L glass bottle with 1.5 L FASW at a density of 1 nauplii/mL. Sterile aer-ation was provided throughout the experiment. Then a mixture of bacteria (the same as in washing experiment, see also Fig. 2) wasadded to the nauplii, each at a concentration of 10 6 /mL. Samples of 240 nauplii were taken 2, 4, 8, 16 and 24 h, respectively, after thestart of the bacteria exposure. Firstly, cellulose treatment was appliedto half of the nauplii for purging the gut transient bacteria. The otherhalf of the nauplii were kept as control group which did not receivethe cellulose treatment. Then, the washing procedure was appliedto the nauplii for both the cellulose-treated group and the controlgroup to remove bacteria attached to the exterior surface of the nau-plii. The washed nauplii were then subjected to DGGE analysis.The DGGE banding patterns of the pure strains used in this exper-iment are presented in Fig. 4 (A to G). Lane H is the mixture of PCR products from these bacteria and it indicates that ampli fi cationproducts of the strains LT3 and LT12 have the same electrophoreticmobility.From2to8 h,thecellulosetreatmentgroupandthecontrolgroup showed the same pattern (Fig. 4L, M, N, Q, R, and S): VC wasthemostdominantbacteriaandotherbacteriawerehardlydetected.After 16 h, more bacteria were detected in both cellulose treatmentgroup and control group and VC, ISO10 and ISO11 were shown asdominant (Fig. 4O and T). Interestingly, after 24 h, VC was shownas the only dominant bacteria in the control group while ISO 10and ISO11 were not detected (Fig. 4P); whereas, in cellulose treat-ment group, VC, ISO 10 and ISO11 were still shown as dominant(Fig. 4U). The bands indicated by the arrows represent some DNAampli fi cation products only present in  Artemia  samples and controlampli fi cation (see also Fig. 2). It isabsent when amplifying pure bac-terial DNA. Therefore, it is suggested that these bands are unspeci fi campli fi cations of the PCR approach. Because the pattern of thesebands shares no overlap with the targeted bands, they are not infer-ring with the interpretation of the results.In general, the results indicate that VC, ISO10 and ISO11 have astronger ability to become resident in the  Artemia  gut than theother bacteria in this experiment. From 2 to 8 h, VC was shown asthe onlydominantspecies, whichmightsuggestan insuf  fi cient devel-opment of adhesion sites in the gut during such an early stage and/ora stronger ability of VC than other bacteria to become part of the res-ident microbiota. After 16 and 24 h, more bacteria besides VC wereable to become resident in the gut. However, after 24 h, VC was theonly dominant bacteria in the non-purged control group whereas,in cellulose-purged treatment group, ISO10 and ISO11 were alsoshown as dominant. This could be explained by the fact that VC con-stituted a very large part of the luminal microbial community Fig. 3.  Removal of transient bacteria by cellulose. Pictures A and B, untreated nauplii enriched with LVS3; Pictures C and D, untreated nauplii enriched with VC; Pictures E and F,cellulose treated nauplii enriched with LVS3; Pictures G and H, cellulose treated nauplii enriched with VC. *Picture H was taken with lower magni fi cation than picture D, whichcan provide a larger visual scale. This allowed better illustrating that after cellulose treatment a considerate amount of VC still resident in the gut.35 Y. Niu et al. / Journal of Microbiological Methods 89 (2012) 33 –  37   Author's personal copy masking the presence of ISO10 and ISO11. This interpretation sug-gests again that transient bacteria might bias the detection of resi-dent bacteria. As in this case, if unjusti fi ably interpreted, VC wouldbe regarded as the only dominant resident bacteria in the gut after24 h and the diversity of resident bacteria in  Artemia  gut wouldhave been underestimated.In conclusion, in this study, we have developed a simple and ef-fective method to speci fi cally target the intestinal resident micro-biota in brine shrimp larvae during DGGE analysis. The interferenceof exteriors and gut transient bacteria could be alleviated by sequen-tially removing bacteria attached to the external surfaces (washingprocedure) and transient (luminal) intestinal bacteria (purging ef-fect of cellulose). Although laser-capture-micro-dissection (LCM) isfeasible to isolate gut specimen from small animal, considering itscomplexity of handling when dealing with multiple samples, it isstill not a routine when studying general microbiota in the gut of aquatic animals.This approach is of high importance as the brine shrimp model isfrequently used in studies on host – microbe interactions. Throughthis method, the composition of the resident bacterial communitycould be revealed, which consequently contributes to a better under-standing of host – microbe interactions.  Acknowledgements Thiswork was funded by China Scholarship Council, Project  “ Pro-biont induced functional responses in aquatic organisms ”  by theFoundationforScienti fi cResearch-Flanders(FWO)(projectnumber:G.0491.08)andthe EuropeanCommunity'sSeventhFrameworkPro-gramme (FP7/2007 – 2013) under grant agreement no. 227197 Pro-microbe  “ Microbes as positive actors for more sustainableaquaculture ” . 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Genetic diversity among3-chloroaniline- andaniline-degradingstrainsofthe Comamonadaceae .Appl.Environ.Microbiol. 67, 1107 – 1115.Brunvold,L.,Sandaa,R.A.,Mikkelsen,H.,Welde,E.,Bleie,H.,Bergh,2007.Characterisationof bacterial communities associated with early stages of intensively reared cod(Gadusmorhua)usingDenaturingGradientGelElectrophoresis(DGGE).Aquaculture272, 319 – 327.Defoirdt, T., Thanh, L.D., Van Delsen, B., De Schryver, P., Sorgeloos, P., Boon, N., Bossier,P., 2010. Short communication: N-acylhomoserine lactone-degrading Bacillusstrains isolated from aquaculture animals. Aquaculture 311, 258 – 260.Dobbeleir, J., Adam, N., Bossuyt, E., Bruggeman, E., Sorgeloos, P., 1980. New aspects of the use of inert diets for high density culturing of brine shrimp. In:Persoone, G. (Ed.), The brine shrimp  Artemia : Proceedings of the InternationalSymposium on the brine shrimp  Artemia Salina , Corpus Christi, Texas, USA,pp. 165 – 171.Gunasekara, R.A.Y.S.A., 2011. Morphological analysis of the digestive tract of gnotobioticbrine shrimp (  Artemia franciscana ) inthe presenceof de fi ned microbiota. PhD thesis,Ghent University, Belgium.Harris, J.M., 1993. Thepresence, nature, and role of gut micro fl ora in aquatic invertebrates:a synthesis. Microb. Ecol. 25, 195 – 231.Huys, L., Dhert, P., Robles, R., Ollevier, F., Sorgeloos, P., Swings, J., 2001. Search forbene fi cial bacterial strains for turbot ( Scophthalmus maximus  L.) larviculture.Aquaculture 193, 25 – 37.Luo, P., Hu, C., Xie, Z., Zhang, L., Ren, C., Xu, Y., 2006. PCR-DGGE analysis of bacterialcommunity composition in brackish water  Litopenaeus vannamei  culture system. J. Trop. Oceanogr. 2, 51 – 55.Makridis, P., Martins, S., Vercauteren, T., Van Driessche, K., Decamp, O., Dinis, M., 2005.Evaluationofcandidateprobioticstrainsforgiltheadseabreamlarvae( Sparusaurata )using an in vivo approach. Lett. Appl. Microbiol. 40, 274 – 277.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  culturedin different gnotobiotic environments. Appl. Environ. Microbiol. 71, 4307. Fig. 4.  The ability of different bacteria to become resident in the gut of   Artemia . Lane A to G, seven pure bacteria culture; Lane H, mixture of PCR ampli fi cation products from theseven bacteria; Lane I, Marker; Lane J, negative control of   fi ltered autoclaved sea water; Lane K, negative control of cellulose; Lane L to P, control group from 2 to 24 h; Lane Q to U, cellulose purging treatment group from 2 to 24 h. *The bands indicated by the arrows represent some DNA ampli fi cation product only present in  Artemia  samples and controlampli fi cation (See also Fig. 2). They are absent when amplifying pure bacterial DNA. Therefore, it is suggested that these bands are either arti fi cial or unspeci fi c ampli fi cation of thenested PCR approach and they are not interfering with the interpretation of the experiment.36  Y. Niu et al. / Journal of Microbiological Methods 89 (2012) 33 –  37 
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