Đánh giá đa dạng sinh học vi sinh vật liên kết với hải miên tại vùng biển Đà Nẵng Việt Nam thông qua nghiên cứu Metagennomics bằng kỹ thuật 16s rRNA Miseq Sequencing

Header Page of 126 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - NGUYN MNH HNG NH GI A DNG SINH HC VI SINH VT LIấN KT VI HI MIấN TI VNG BIN NNG VIT NAM THễNG QUA NGHIấN CU METAGENOMICS BNG K THUT 16S rRNA MISEQ SEQUENCING LUN VN THC S KHOA HC H Ni Nm 2016 Footer Page of 126 Header Page of 126 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - NGUYN MNH HNG NH GI A DNG SINH HC VI SINH VT LIấN KT VI HI MIấN TI VNG BIN NNG VIT NAM THễNG QUA NGHIấN CU METAGENOMICS BNG K THUT 16S rRNA MISEQ SEQUENCING Chuyờn ngnh: Vi sinh vt hc Mó s: 60420107 LUN VN THC S KHOA HC NGI HNG DN KHOA HC: PGS.TS NGUYN TH KIM CC TS PHM C NGC H Ni Nm 2016 Footer Page of 126 Header Page of 126 Li cam oan Tụi xin cam oan kt qu th hin bn lun ny l cụng trỡnh nghiờn cu thc t Ton b s liu, kt qu nghiờn cu lun l hon ton trung thc, khỏch quan, cha c cụng b cụng khai bi mt khỏc Footer Page of 126 Header Page of 126 Li cm n ti ny l kt qu ca mt quỏ trỡnh hc v nghiờn cu khoa hc ca bn thõn, cựng vi s giỳp sc ca rt nhiu cỏ nhõn v th cú c thnh cụng ny, tụi xin by t lũng kớnh trng v bit n sõu sc n PGS.TS Nguyn Th Kim Cỳc, ngi ó giỳp tụi rt nhiu bng chớnh s tõm huyt v am mờ vi lnh vc nghiờn cu khoa hc Tụi cng xin c t lũng cm n chõn thnh ti cỏc cỏn b thuc phũng Cụng ngh sinh hc, vin Hoỏ sinh bin; Vin nghiờn cu khoa hc Min Trung, Vin Hn lõm khoa hc Vit Nam ó cú nhng h tr, úng gúp, t rt nhit tỡnh giỳp tụi hon thin ti Tụi tht s rt bit n ngi thõn, bn bố v ng nghip ó ginh tỡnh cm, t nim tin, h tr, ng viờn; ng thi cng l ngun ng lc to ln gúp phn giỳp tụi hon thin ti ny Vi s h tr v kin thc; cỏc ngun d liu b sung; cỏc cụng trỡnh nghiờn cu khoa hc cú liờn quan nc v trờn th gii-Tp th cỏc Thy cụ thuc khoa sinh hc; B mụn Vi sinh vt hc v c bit TS Phm c Ngc ó giỳp tụi lm nờn s thnh cụng ca ti ny Tụi xin chõn thnh cm n! H Ni, ngy 27 thỏng 12 nm 2016 Nguyn Mnh Hựng Footer Page of 126 Header Page of 126 MC LC M U CHNG 1: TNG QUAN 1.1 S lc v hi miờn 1.2 Vai trũ v tm quan trng ca vi sinh vt liờn kt vi hi miờn 12 1.3 a dng sinh hc vi sinh vt liờn kt vi hi miờn 18 1.4 S lc v gen 16S rRNA 23 1.5 S lc v MiSeq Sequencing 24 CHNG 2: VT LIU V PHNG PHP NGHIấN CUError! Bookmark not defined 2.1 VT LIU Error! Bookmark not defined 2.2 PHNG PHP NGHIấN CU Error! Bookmark not defined 2.2.1 Tỏch DNA Error! Bookmark not defined 2.2.2 Thit k th vin 16S rRNA cho MiSeq Error! Bookmark not defined 2.2.3 Phõn tớch th vin 16S rRNA Error! Bookmark not defined CHNG 3: KT QU V THO LUN Error! Bookmark not defined 3.1 KT QU Error! Bookmark not defined 3.1.1 Tỏch DNA metagenome ca vi sinh vt liờn kt hi miờnError! Bookmark not defined 3.1.2 Khuch i vựng siờu bin V4 ca gen 16S rRNA v gn barcodes Error! Bookmark not defined 3.1.3 a dng cỏc cng ng vi sinh vt liờn kt hi miờn bin Nng Error! Bookmark not defined 3.2 THO LUN Error! Bookmark not defined Footer Page of 126 Header Page of 126 KT LUN V KIN NGH Error! Bookmark not defined TI LIU THAM KHO 28 DANH MC BNG V HèNH Hỡnh 1.1: Hỡnh thỏi Porifera, (Ruppert et al., 2004) Hỡnh 1.2: Cu trỳc c th hi miờn v cỏc t bo chc nng Hỡnh 1.3: Sinh sn hu tớnh hi miờn Hỡnh 1.4 a dng vi khun liờn kt vi hi miờn (Tabares 2011) Hỡnh 1.5: Mụ mesohyl ca (A) Ancorina alata, hi miờn cú vi khun liờn kt phong phỳ Hỡnh 1.6: Gene 16S rRNA cú vựng bin i nm xen gia cỏc vựng bo th Hỡnh 3.1: in di DNA metagenome ca vi sinh vt liờn kt hi miờn bin Nng trờn agarose gel 0,8% Lane 1: DN ; lane 2: DN10; lane Hỡnh 3.2: nh in di PCR khuch i vựng V4 gen 16S rRNA ca vi sinh vt liờn kt hi miờn bin Nng M: Marker DNA kb (GeneRuler); 1, 2, 3: Sn phm PCR vựng V4 ca mu DN9, DN10 v DN12, tng ng Hỡnh 3.3: Sn phm PCR sau gn barcodes M: Marker DNA kb (GeneRuler); 1, 2, 3: Mu DN9, DN10 v DN12, tng ng Hỡnh 3.4: th biu hin t l vi khun v c khun liờn kt vi ba mu hi miờn Hỡnh: 3.5: th biu hin thnh phn cỏc ngnh vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.6: th biu hin thnh phn cỏc lp vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Footer Page of 126 Header Page of 126 Hỡnh: 3.7: th biu hin thnh phn cỏc b vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.8: th biu hin thnh phn cỏc h vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Hỡnh: 3.9: th biu hin thnh phn cỏc chi vi khun liờn kt vi ba mu hi miờn DN9; DN10; DN12 Bng 1: Phn mm, cỏc cụng c da trờn Web v cỏc c s d liu s dng phõn tớch metagenome Bng 3.1: Kt qu kim tra hm lng v tinh sch ca DNA metagenome Bng 3.2: Kt qu nhn dng cỏc mu hi miờn Bng 3.3 Túm tt kt qu phõn loi vi sinh vt liờn kt vi hi miờn Bng 3.4: Kt qu phõn loi vi sinh vt liờn kt vi hi miờn mc gii Bng 3.5: Kt qu phõn loi cỏc ngnh vi sinh vt liờn kt vi hi miờn Bng 3.6 Kt qu phõn loi cỏc lp vi sinh vt liờn kt vi hi miờn Bng 3.7 Kt qu phõn loi cỏc b vi sinh vt liờn kt vi hi miờn Bng 3.8 Kt qu phõn loi cỏc b vi sinh vt liờn kt vi hi miờn Bng 3.9: Kt qu phõn loi cỏc chi vi sinh vt liờn kt vi hi miờn Footer Page of 126 Header Page of 126 Footer Page of 126 Header Page of 126 M U Hi miờn l vt ch ca nhiu vi sinh vt v vai trũ ca nhng vi sinh vt ny thay i theo ngun dinh dng v s cng sinh vi hi miờn Da trờn nhng nghiờn cu cng ng vi sinh vt bng cỏc phng phỏp nh in di gel gradient bin tớnh (Denaturing Gradient Gel Electrophoresis_DGGE), gii trỡnh t gen 16S rRNA v lai hunh quang in-situ (Fluorescense In Situ Hybridization_FISH), ngi ta nhn thy ngoi cỏc thnh viờn ca Archaea cũn cú ti hn 25 ngnh vi khun liờn kt vi hi miờn Trong s ú cú Proteobacteria, Nitrospira, Cyanobacteria, Bacteriodetes, Actinobacteria, Chloroflexi, Planctomycetes, Acidobacteria, Poribacteria v Verrucomicrobia Cha rừ v virus hi miờn, mc dự cỏc ht ging nh virus c phỏt hin nhõn t bo ca Aplysina (Verongia) cavernicola Nhng nghiờn cu v vi sinh vt liờn kt vi hi miờn cho thy vi sinh vt cú th chim n 40% - 50% th tớch hi miờn v cng ng ny c trng cho tng hi miờn [40] Mi liờn h gia vi sinh vt vi hi miờn gn õy ang c quan tõm nghiờn cu do: (i) phỏt hin nhiu hi miờn cú liờn kt vi cng ng vi khun dy c v a dng, (ii) chỳng l mt ngun giu cỏc cht chuyn húa hot tớnh sinh hc cú ý ngha lnh vc dc phm v cụng ngh sinh hc [50] Vic phõn lp, nuụi cy vi sinh vt sinh trng iu kin c bit thng khú khn, c bit vi sinh vt liờn kt vi cỏc dng sng khỏc nh hi miờn bi mi tng tỏc gia chỳng khỏ phc Cng khụng th khai thỏc hi miờn tỏch chit cỏc hot cht vỡ ngun nguyờn liu ny cú hn, khú phc hi v gõy hy hoi mụi trng ti ny c thc hin vi mc ớch nghiờn cu s a dng ca vi sinh vt liờn kt vi hi miờn bin trờn c s phõn tớch d liu metagenome ca vi sinh vt Kt qu nghiờn cu s cung cp ngun c s d liu cú giỏ tr cho cỏc nghiờn cu tip theo v mi liờn h gia vi sinh vt-hi miờn bin Footer Page of 126 Header Page 10 of 126 CHNG 1: TNG QUAN 1.1 S lc v hi miờn Hi miờn (bt bin) l ng vt n lc, a bo, c xa thuc ngnh ng vt thõn l (Porifera) Ngnh Porifera l mt nhúm a ngnh bao gm ba lp chớnh, c c trng bi c cu gai ca chỳng: (1) Hexactinellida (glass sponges -bt bin thy tinh) cha gai silic cu trỳc hexactine; (2) Calcarea (calcareous sponges -bt bin vụi) cha gai vụi (calcareous spicules); (3) Demospongiae (demosponges) cú khong 85% cỏc loi c cụng nhn hin nay, thng cú mt b khung khoỏng cht bao gm gai silic (siliceous spicules) Tuy nhiờn, mt s loi Demospongiae khụng cha b khung [21] Hi miờn cú cu trỳc c th n gin nht tt c cỏc loi ng vt a bo: thiu c quan v mụ thc; khụng cú c, h thn kinh, h tiờu húa, h tun hon, c quan bờn trong, v kh nng ng Cỏc t bo ca hi miờn sp xp cú th t gm nhiu lp t bo chuyờn bit (Hỡnh 1.2) phự hp vi li sng n lc Hu ht hi miờn cú b khung cng c hỡnh thnh t canxi cacbonat v cỏc gai silic dioxide Cỏc si Collagen v spongin cú tỏc dng h tr thờm s cng chc v n hi cho b khung [21] Ngoi cỏc loi t bo xp khỏc nhau, mt cao ca cỏc vi sinh vt cng c tỡm thy cỏc mesohyl ca mt s loi hi miờn sõu ph bin tỡm thy hi miờn t 5-10m ng vt thõn l, ú cú hi miờn thng n bng cỏch hỳt nc qua cỏc l chõn lụng Mt dũng chy c trỡ liờn tc qua c th ly thc n, ụxy cng nh loi b cht thi Khi nc ó c lc qua cỏc khoang choanocyte, nú b thi c th qua cỏc osculum, v c bn l vụ trựng [43, 44] Hu ht hi miờn cú th bm mt lng nc tng ng vi lng c th ca nú mi giõy [47], iu ny cú th bng vi ngn lớt mi ngy [55] Mi liờn h gia vi sinh vt vi hi miờn gn õy ang c quan tõm nghiờn cu do: (i) phỏt hin nhiu hi miờn cú liờn kt vi cng ng vi 10 Footer Page 10 of 126 Header Page 24 of 126 mt s loi hi miờn v vi sinh vt cng sinh thuc bin Vit Nam vi i tỏc Phỏp v Bo tng lch s thiờn nhiờn quc gia Phỏp Cỏc nghiờn cu v hi miờn ti Vit Nam cũn hn ch v cha cú nghiờn cu no v h vi sinh vt liờn i vi hi miờn cng nh phỏt hin cỏc cht cú hot tớnh sinh hc t h vi sinh vt ny, c bit l vic s dng k thut metagenomics phỏt hin v sng lc cỏc gen cú hot tớnh sinh hc t ngun mụi trng tim nng ny 1.4 S lc v gen 16S rRNA L on trỡnh t tng i ngn, bo th v c trng cho mi loi Nhng kho sỏt v gen trờn rRNA c thc hin trc tip t DNA mụi trng ó cho thy, s lng loi vi khun v vi khun c ó tỡm thy trc õy bng phng phỏp gii trỡnh t truyn thng ch tng ng khong 1% s lng thc ca chỳng mụi trng Gen 16S rRNA l thnh phn ca b mỏy tng hp protein cú t lõu i Hn na, cỏc rRNA l nhng phõn t cú chc nng khụng thay i, phõn b hu ht cỏc h thng sng v cú bo tn va phi cho phộp phn ỏnh s liờn quan tin hoỏ gia cỏc loi sinh vt Zuckerkand v Pauling (1967) cho rng 16S rRNA cú cỏc c im phự hp lm thc o tin hoỏ vỡ: (i) phõn t cú mt tt c cỏc sinh vt kho sỏt; (ii) phõn t khụng truyn qua li gia cỏc loi; (iii) trỡnh t ca phõn t cú bo tn v bin ng thớch hp khong cỏch tin hoỏ kho sỏt; (iv) phõn t cú kớch thc ln cha nhiu thụng tin Mc dự cỏc tRNA cú mt hu ht cỏc vi sinh vt nhng chỳng khụng c chn lm thc o tin hoỏ kớch thc quỏ nh (75 ribonucleotides) Ba loi rRNA l thnh phn ca ribosome u cú mt s tớnh cht cú th c chn lm thc o tin hoỏ Tuy nhiờn ch cú 16S rRNA l s la chn phự hp cho cỏc nghiờn cu hin cú kớch thc va phi (khong 1500 ribonucleotides) Cũn li 5S cú kớch thc quỏ nh (khong 120 ribonucleotides), mang ớt thụng tin ca quỏ trỡnh tin hoỏ nờn ớt c chn 23S rRNA l phõn t cú kớch thc ln (khong 2900 24 Footer Page 24 of 126 Header Page 25 of 126 ribonucleotides), nờn hin khụng my thun li cho vic nghiờn cu Phõn t 16S rRNA c coi nh tiờu chun vng cho phõn loi hc Tuy nhiờn, RNA d b phõn hu nờn cn phi rt cn trng thao tỏc, vỡ vy khuynh hng hin l s dng gene 16S rDNA mó hoỏ cho RNA cỏc nghiờn cu Gene 16S rRNA ca cỏc vi khun thuc nhúm Eubacteria cú mt c im rt c bit l cú nhng vựng bo tn cao cp ca nhúm xen k nhng vựng bin ng khỏc gia cỏc loi cựng nhúm ny Tuy l nhng vựng bin ng, nhng nhng vựng ny li l nhng vựng bo tn cp loi S bo tn v bin ng cỏc cp khỏc ó giỳp cho gen 16S rRNA luụn l s la chn u tiờn ca cỏc nh nghiờn cu tin hnh nh danh vi khun hay xỏc nh mi quan h h hng gia hai hay nhiu loi vi khun prokaryote, gene 16S RNA ribosome (rRNA 16S) di khong 1.500 bp v cha chớn vựng bin i ri rỏc gia cỏc vựng bo th Vựng bin i 16S rRNA thng c s dng phõn loi phỏt sinh loi nh chi hoc loi a dng qun th vi sinh vt Hỡnh 1.6: Gene 16S rRNA cú vựng bin i nm xen gia cỏc vựng bo th 1.5 S lc v MiSeq Sequencing Platforms Illumina l h sn phm c s dng rng rói nht K thut c a vo nm 2006 v nhanh chúng c cỏc nh khoa hc s dng bi cú th to lng ln s liu vi chi phớ hiu qu hn Theo thi gian, chiu di read c tng lờn v nhng ngi thng s dng 454 ó chuyn sang Illumina platform hiu qu kinh t ca k thut ny Mc dự phng phỏp gii trỡnh t bng tng hp nh ca 454, nhng phng phỏp Illumina cú im ni bt khỏc vi 454: (1) s dng flow cell vi 25 Footer Page 25 of 126 Header Page 26 of 126 vựng ớnh oligo, thay cho chip cha cỏc vi ging (microwell) riờng l cú ht v (2) nú khụng liờn quan n pyrosequencing, m kt thỳc bng thuc nhum thun nghch Phng phỏp kt thỳc bng thuc nhum ging nh gii trỡnh t Sanger, nhng khỏc l chỳng thun nghch vỡ vy chỳng c g b sau mi chu k tip tc kt thỳc cho nucleotide tip theo Chun b gii trỡnh t c bt u vi cỏc on DNA cú cỏc b chuyn i (adapter) c hiu mt u, chỳng i qua qua flow cell cú cỏc oligonucleotides c th v lai vi u cui ca on DNA Mi on DNA sau ú tỏi to (replicate) to thnh cm cỏc on ging Cỏc nucleotides mu kt thỳc thun nghch sau ú chy qua flow cell v gn vo ú Nucleotides tha b trụi, flow cell c to hỡnh nh v cỏc cht kt thỳc thun nghch, vỡ vy quỏ trỡnh cú th nhc li v nucleotides cú th tip tc c thờm vo cỏc chu k tip theo Hin nay, read di nht m platform Illumina to l trờn MiSeq, cỏc read cú uụi kt ụi (paired-end) l 300 bp Hiu bit ca chỳng ta v vi sinh vt cỏc mụi trng t nhiờn rt hn ch v vic nghiờn cu a dng sinh hc t nhiờn khụng phi nhim v d dng Trong cỏc h sinh thỏi t nhiờn, vi sinh vt tn ti vi mt cao mc dự rt nhiu loi hin cha c mụ t Vi sinh vt thớch nghi vi hu ht cỏc loi mụi trng trờn trỏi t v cú cỏc hot tớnh sinh hc a dng tựy thuc vo mụi trng sng Cõu hi quan trng nghiờn cu vi sinh vt mụi trng t nhiờn ca chỳng l: cỏc cng ng vi sinh vt hot ng nh th no v s thay i v cht cng ng xy s thay i mụi trng nh th no Cú rt nhiu phng phỏp mụ t a dng vi sinh vt nh phng phỏp sinh húa, nuụi cy, phõn tớch acid bộo phospholipid , cỏc phng phỏp sinh hc phõn t nh lai acid nucleic, a hỡnh chiu di cỏc on gii hn (RFLP), a hỡnh chiu di cỏc on gii hn cui (T-RFLP), DNA microarrays, in di gradient bin tớnh (DGGE) hoc in di gradient nhit (TGGE), a hỡnh th cu to si n (SSCP) 26 Footer Page 26 of 126 Header Page 27 of 126 Vi s i ca gii trỡnh t th h mi (NGS), kh nng phỏt hin cỏc nhúm vi sinh vt mi cỏc h mụi trng phc m khụng cn nuụi cy c tớch ly v cỏc k thut gii trỡnh t mi ny ó lm sỏng t s phc ca cỏc qun th vi sinh vt S dng NGS, cú th xỏc nh thnh phn qun xó vi sinh vt cc k phc tp, cng nh liờn kt s a dng cng ng vi sinh vt vi chc nng mụi trng sng Bng 1.1: Phn mm, cỏc cụng c da trờn Web v cỏc c s d liu s dng phõn tớch metagenome Tờn phn mm/Web server ng dng Ti liu tham kho UniFrac Cụng c online so sỏnh a Lozupone http://bmf.colorado.edu/unifrac/ dng vi sinh vt JANE Lp bn ESTs v reads Liang http://jane.bioapps.biozentrum.uni- trỡnh t genome prokaryote 2009 wuerzburg.de chiu di khỏc trờn cỏc et al., 2006 et al., mu genomes h hng WebCARMA Phõn loi ti loi v chc Gerlach et al., http://webcarma.cebitec.unibilefeld.de nng ca reads metagenomic 2009 khụng c lp rỏp DraGnET Phõn loi, biờn v phõn Duncan et al., http://www.dragnet.cvm.iastae.edu tớch cỏc s liu trỡnh t 2010 genome c phỏc tho chỳ thớch Prodigal Nhn bit gen prokaryotes Hyatt http://compbio.ornl.gov/pordial v dch mó im bt u 2010 et al., et al., nhn dng MEGAN Cụng c phõn tớch reads Mitra 27 Footer Page 27 of 126 Header Page 28 of 126 www.ab.informatik.uni- metagenome c gii trỡnh 2010 tuebingen.de/software/megan t bng illumina MG-RAST Metagenome http://metagenomics.anl.gov/ server CAMERA Metagenomic http://camera.calit2.net server cha cỏc trỡnh t t 2009 annotation Aziz 2010 database Maumus et al., cỏc mu mụi trng thu thp c quỏ trỡnh ly mu i dng ton cu (GOS) FUNGIpath C s d liu v tool server Grossettete http://www.fungipath.upsud.fr cho orthology nm et v al., 2010 metagenomics envDB C s d liu v tool server Tamames http://metagenomics.uv.es/envDB/ cho s phõn b mụi al., 2010 trng cỏc taxa prokaryotes 28 Footer Page 28 of 126 et Header Page 29 of 126 TI LIU THAM KHO TING VIT Nguyn Xuõn Cng, Trn Anh Tun, Nguyn Thu Phng, V Vn Thnh, Phan Vn Kim, Chõu Vn Minh(2007), Nghiờn cu thnh phn húa hc ca loi hi miờn Xestospongia testudinaria thu ti Vit Nam, Tp khoa hc v cụng ngh, 45(3), Tr.43-50 Phm Vit Cng, Nguyn Mai Anh, V Th Quyờn, Nguyn Th Kim Cỳc(2014), Phõn lp, tuyn chn v nh danh mt s chng vi khun liờn kt sỏu loi hi miờn vựng bin Sn Ch, sinh hc, 36(3), tr 345-350 Mnh Ho, Phm Th Th(2010), Mt s kt qu nghiờn cu v vi sinh vt ti vựng ven bin Hi Phũng, Tp Khoa hc v Cụng ngh bin, 10(1), pp 51-65 Phan Vn Kim(2013-2015), Nghiờn cu khai thỏc dc liu t Hi miờn vựng bin ụng bc Vit Nam theo nh hng hot tớnh dit t bo ung th, Vin Húa sinh bin TING ANH Abdulaziz Anas, C Jasmin, , Shanta Nair(2015), Bacterial diversity associated with Cinachyra cavernosa and Haliclona pigmentifera, cohabiting sponges in the coral reef ecosystem of Gulf of Mannar, Southeast Coast of India, PLoS ONE, 10(5) Ahn, Y.B., Rhee, S.K., Fennell, D.E., Kerkhof, L.J., Hentschel, U., and Họggblom, M.M(2003), Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba, Appl Environ Microbiol 69, pp 4159-4166 29 Footer Page 29 of 126 Header Page 30 of 126 Azzini F., Calcinai B., Cerrano C., Bavestrello G., Pansini M(2007), Sponges of the marine karst lakes and of the coast of the islands of Ha Long Bay (North Vietnam), Porifera Research: Biodiversity, Innovation and Sustainability, pp 157- 164 Bayer, K., Schmitt, S., and Hentschel, U (2008), Physiology, phylogeny and in situ evidence for bacterial and archaeal nitrifiers in the marine sponge Aplysina aerophoba, Environ Microbiol 10, pp 2942-2955 Bell, J.J.(2008), The functional roles of marine sponges, Estuar Coast Shelf Sci, 79, pp 341-353 Brỹmmer, F., Pfannkuchen, M., Baltz, A., Hauser, T., and Thiel, V (2008), Light inside sponges, JExp Mar Biol Ecol 367, pp 61-64 Calcinai B, Azzini F, Bavestrello G, Cerrano C., Pansini M, Do Cong Thung(2006), Boring sponges from Ha Long Bay, 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141-149 62 Yahel, G., Whitney, F., Reiswig, H.M., Eerkes-Medrano, D.I., and Leys, S.P (2007), In situ feeding and metabolism of glass sponges (Hexactinellida, Porifera) studied in a deep temperate fjord with a remotely operated submersible, Limnol Oceanogr 52, pp 428-440 63 Zhi-Yong Li *, Li-Ming He, Jie Wu, Qun Jiang(2005), Bacterial community diversity associated with four marine sponges from the South China Sea based on 16S rDNA-DGGE fingerprinting, Marine Biotechnology Laboratory, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China 37 Footer Page 37 of 126 Header Page 38 of 126 64 https://www.boundless.com/biology/textbooks/boundless-biologytextbook/invertebrates-28/phylum-porifera-166/morphology-of-sponges-64211864/ https://sites.google.com/a/rsu35.org/seventh-grade-science/unit-6-aquaticecology 38 Footer Page 38 of 126 ... 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