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7/23/2019 Ecology and Physiology of the Pathogenic Cyanobacterium Roseofilum.pptx
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Ecology and Physiology
the PathogenicCyanobacterium
Roseoflum reptotaeniuRICHARDSON, SANI!, "A#, $RO%NE&&, 'ANARCA"PA'NA
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() Introduction
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Roseoflum reptotaeniumogliding, *lamentous, +hycoerythrinrich cyonabacteriumofound only in the hori-ontally migrating, +athogenic, mmat, blac. band disease /$$D0
odominates the $$D matobiomass and motilityo*laments form mat fabric
omicrocystin "C&R
otolerate high le1els of sul*de by sulfate reducing bacte
o+lay ma2or role in $$D
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()( Cyanobacteria and $lac. $and Disof Corals
Produce cyanoto3in Degrade 4ater 5uality 1ia e3tracellul
accumulation of to3ins one s+ecies in1ol1ed in +olymicrobial
disease Predominant cyanobacterium in $$D
Cyanobacteria as harmfulmicroorganisms6
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()( Cyanobacteria and $lac. $and Disof Corals
7irst obser1ed in scleractinan /stony0 corals 'lobally distributed on tro+ical and subtro+ical reefs Dense blac. band Se1eral mm to cm 4ide and (mm thic. &yse tissue Initiates from a central +oint on to+ or side and migrates as
e3+anding ring Seasonal disease Highly infecti1e and easily transmitted 7ilamentous sul*de o3idi-ers
$lac. $and Disease /$$D0
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()( Cyanobacteria and $lac. $and Disof Corals
7ormerly classi*ed into the genera Oscillatoria andPhormidium
$lac. $and Disease /$$D0
Geitlerinema and Leptolyngbya
Carribean $lac. $and Disease /$$D0
de1elo+s from cyanobacterial 8+atches9 Blennothrix
$$D on the 'reat $arrier Reef
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()( Cyanobacteria and $lac. $and Disof Corals
Caribbean BBD Narro4, has smooth trichomes One rounded and one +ointed
ti+ on each *lament +hycoerythrin Produces cyanoto3in
microcystin
Palau reef B Rounded ti+s
ends Ob1ious cons
bet) cells
Red Sea BBD $lunt ends Phycocyanin as the ma2or
light har1esting +igment
BBD on the GBarrier Reef
Phycoerythrin Do not +rodu
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():) he Ecology of Caribbean $lac$and Disease
$$D cyanobacteria olerate sul*de Sul*deinsensiti1e o3ygenic +hotosynthesis Play im+ortant role in $$D microbial community +roduce bioacti1e com+ounds that su++ress the gro4th of co
associated bacteriaR. reptotaenium Produces cyanoto3in microcystin "C&R ; most to3ic 1ariant
+romote or inhibit gro4th of isolates of $$D heterotro+hic bac Structuring the $$D microbial community
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():) he Ecology of Caribbean $lac$and Disease
E3+osure of $$D inoculum to sodium molybdate s+eci*c inhibitor of sulfate reduction
+rior to inoculation of healthy coral fragments +re1ented infection
e3+osure after coral fragments 4ere infected did noa
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:) "ethods
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:)( Isolation of Roseoflumreptotaenium Strains
strains (>(( and (>>( t4o coral s+ecies, Diploria strigosa a
Siderastrea siderea ASNIII and marine $'(( at room
tem+erature under natural light One a3enic strain 4as attained
lost 1iability after t4o 4ee.s t4o isolates that maintained 1iability
small heterotro+hic bacteria e3tracellular +olysaccharide sheath
Roseoflumreptotaeni
um
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Fig 2.Photomicrogra+h of Roseo/@>> magni*cation0) Note the +ta+ering and rounded end cells)
Fig. 3.Photomicrogra+h of Rose/$0 /@>> magni*cation0) Note thta+ering and rounded end cells)
Fig. 4.Scanning electron microg
of Roseoflum reptotaeniumstrai
Fig. 5.Scanning electron microgof Roseoflum reptotaeniumstrai
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:):) Infection of Coral 7ragm
R. reptotaenium strain (>((
used to inoculate coral fragments of the s+ecies Diploria strigos fragments 4ere +laced in (> & a5uaria 4ith arti*cial sea4ater /A
second a5uarium ser1ed as a control
no inoculation of fragments
A5uaria 4ere +laced under a (:6(: h light6dar. cycle 4i4hite Buorescent light : C
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Inoculation Procedure
small clum+of R.
reptotaeniumbiomass /ca)
F mm indiameter0
+lace onsurface of
indi1idual coralfragments 4ith
sterile force+s
inoculum4as held in
+lace
ste
reto
in
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Inoculation Procedure
*laments 4ereobser1ed to
adhere to the
coral /(;: day0
G e3+erimentaland G controlfragments
e3+ere+ea
tim
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:)G) Scanning Electron"icrosco+y
Infected coralfragments,and also
cultured R.reptotaenium
/clum+s0,4ere *3ed in
:)Fglutaraldehyd
e andmaintained at
C until+rocessing
fragments4ere
dehydratedthrough a
gradedseries ofalcohol
+lacing themfor (> min in
ethanol atconcentrations of :>, >,>, J>, K>
/one 4asheach0 and(>> /three
4ashes0
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:)G) Scanning Electron"icrosco+y
aL3ed to analuminum stubusing carbon
adhesi1e ta+e
coated 4ith+alladiumMgoldusing a s+utter
coater
7rag1ie4
OE& Scann
"i
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:) "otility Patterns
R) re+totaenium chemota3is to microcystin and sul*de e
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Chemotactic res+onse to sul*:F mg of sodium sul*de nonahydrate /Na:SKH:O0 crystals4ere +laced four cm from the s+ot 4here cyanobacterial*laments 4ere inoculated
7or inoculation, biomass from stoc. cultures of both R.reptotaenium strains 4ere +laced on the agar surface usingsterile force+s
In each of three /re+eated0 e3+eriments, test +lates /thr
re+licate +lates 4ith sul*de +lus t4o re+licate control +la4ere incubated on a (:6(: h light6dar. cycle 4ith cool4Buorescent light in a : C incubator
Plates 4ere oriented so that the side of the +late cosul*de 4as closest to the light source)
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Chemotactic res+onse to sul*
QControl +lates contained only cyanobacterial *laments andno sul*de
documented in terms of their +osition on the +late asobser1ed using a dissecting microsco+e
"igration of the *laments 4as mar.ed, measured 4ith ametric ruler, and +hotogra+hed
otal migration distance bet4een the +oint 4heresul*de crystals 4ere +lated and the closest cyanob*lament
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"icrocystin chemota3ise3+eriments
+uri*ed microcystin&R /"C&R0
a hole /challenge 4ell0 4as +unched into agar +lates/one +er +late0
*lled 4ith F> & of ( gM&, F> gM&, or (>> gM& "Cfrom stoc. solutions made 4ith arti*cial sea4ater/AS%0
QControl +lates ; 4ell 4as created but not *lled
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microcystin chemota3ise3+eriments
:F & of safranin dye 4as mi3ed 4ith :F & of (>> gM&"C stoc. solution and +i+etted into a challenge 4ell
Assay +lates 4ere incubated
Plates 4ere oriented so that the challenge 4ell 4asclosest to the light source
actic res+onses of *laments 4ere obser1ed
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:) "otility Patterns
During these e3+eriments6 all control +lates of R. reptotaenium *laments mo1ed to the lighdocumenting +ositi1e +hotota3is
Ensure res+onses to "C di sec Homogeni-ed biomass /F>> T&0 of each strain 4as dis+ensed in
4ell +lates /in tri+licate for each +late0 one set of +lates e3+osed to light /G> TEmU(sU(0 other .e+t in the dar. by 4ra++ing the +late in aluminum foil
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:) "otility Patterns
a1erage biomass /dry 4eight0 used +er 4ell forthis e3+eriment 4as ) mg
e3tent of *lament clum+ing 4as recorded byta.ing digital +ictures after F, (>, (F and G> min
rate of clum+ing measuring the diameter ofbiomass in each 4ell using image analysissoft4are and 4as e3+ressed as +ercentage ofarea decrease relati1e to the freshly dis+ersedcultures
: F ' th d
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:)F 'ro4th andClum+ing as a 7actor of+H R.
Reptotaeniumstrain (>((
R.Reptotaeniumstrain (>>(
ASN III mediumV "ES bu)F gM&0
ASN III mediumV "ES bu)F gM&0
NaOH or HCl to obtain H le!el" of 4.2# 5.5#$.%# &.%# '.%# (.%# )%.%G>ml of the
mediuminoculated 4ith
e5ual 1ol ofculture /:JC0
G>ml of themedium
inoculated 4ithe5ual 1ol of
culture /:JC0
*R+P,+C-*S
7ilter through%hatman '7M$
*lters
7ilter through%hatman '7M$
*lters
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E3tract biomass4ith conc)
"ethanol Cfor :h
E3tract biomass4ith conc)
"ethanol Cfor :h
Chl a concentration6measure absorbanceat FG nm and
nm using amicro+late reader
est signi*cancebet) gro4th atdi
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:) "icrocystinE3+osure and"etabolite Production
7ield study6 collection ofblac. band from G $$D
infected Diploria strigosa
%ater tem+ :J)FC at ade+th of
meters
After clum+formation,
sam+les 4erecombined
Homogeni-e $$D matby re+eated +um+ing of
combined sam+lesusing needleless
syringe
Sterile sea4ater4as added toincrease the
1olume
/G sets0 F ml of thedis+ersed sam+le+laced in (F ml
falcon tubes 4eree3+osed to "C&R/F>TgM&0 for : h
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ubes incubatedin shaded
natural light at areef 4ater tem+
of :C
Placed in ana5uarium 4MBo4through
sea4ater system
After : h, $$Dcommunities
4ere collectedusing 1acuum
*ltration tonylon *lters
7ilter folded andimmediately
stored incryogenic 1ialsand Bash fro-en
JC
7ro-en sam+lesstored
>C untile3tracted formetabolomic
analysis
: J "etabolite E3traction and
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:)J "etabolite E3traction and"etabolite Analysis Wsing WP&CHR"S
7ro-en *lters4ere +laced in
an em+ty Petridish on a bed ofice
Addition of ()G mlali5uot e3traction
sol1ent /:>C0
com+osition is />6>6:>acetonitrile6 methanol6 4ater
4ith >)(" formic acid0 to tha4and e3tract metabolome
7ilter unfolded,sam+le +laced inthe ES to allo4e3traction
:>C for :> mins
"etabolome measuredusing WP&CHR"S on
an E3acti1e PlusOrbitra+ *tted 4ith anWlti"ate G>>> WP&C
: "etabolomic Data Processing and
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:) "etabolomic Data Processing andAnalysis
he o+ensource soft4are,"a1en 4as used to
manually +ic. metabolite
+ea.s based on .no4nretention times and mM-1alues
based on6 e3actmass /X:>++m0,
retention time
Xmin
Pea.s 4ereanaly-ed using"etaboAnalystand *ltered by
the inter5uantile
range
7iltered datacom+ared to
reference data fromthe a1erage of the
control sam+les
7old changes and+ 1alues 4ere
calculated
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RESW&S
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G)() &aboratory Infection Wsing Rreptotaenium
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G):) "icrosco+ic Analysis
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G):) "icrosco+ic Analysis
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G)G) Chemotactic Res+onses to Sul*de"icrocystin
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RESW&S and DISCWSSION
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"otility Patterns of R.reptotaen
he ability to adhere to and gro4 as a mat
surface V continual and directional gliding com+act discrete band
R.reptotaenium e3hibits a ste+do4n +hotores+onse *laments mo1e to the band s
Negati1e ta3is to sul*de .ee+ing the *lamea4ay from the Srich layers of the band
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G) Clum+ing $eha1ior
G Cl i h i
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G) Clum+ing $eha1ior
Cons+icuous clum+ing
obser1ed in Roseoflumstrain(>>( and (>(( in li5uidculture
In the light, both strainse3hibited clum+ing 4ith areduced area of F /(>>(0
and JG /(>((0 In the dar., both strains
contracted to an area of J/(>>(0 and K /(>((0
7or each strain clrate 4as faster inthan in the dar.
Strain (>>( clumfaster than (>((
G Cl i $ h i
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G) Clum+ing $eha1ior
SE" analysis of the Roseoflumclum+formation sho4s that there is aunidirectional orientation of *laments
similar to e3+erimentally induced $$D
G Cl i $ h i
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G) Clum+ing $eha1ior
Cultures gro4n undisturbed in ASN IIImedia formed +atterns of rings and circlesthat are of uniform si-e /Gmm diameter0
Cl i $ h i f R t t
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Clum+ing $eha1ior of R.reptotae
Clum+ing beha1ior in light and dar. enhances th
maintenance of the band Clum+ing res+onse to coral mucus /slightly acidic $$D mat formation6 circular motility that +roduce
gliding motility 4here the ta+ered end ser1es as leading end
At the leading edge of the $$D mat, R.reptotaenitunnels through coral tissue and its *laments borcoral s.eleton
G F E< t f H ' th d Cl i
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G)F E((, abiomass yield in all +H1alues sho4ed
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G)F E
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yProduction in the $$D community
G) E
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yProduction in the $$D community he most signi*cant e
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yProduction in the $$D community
"C&R e
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metabolism Production of microcystin may be inBuenci
$$D heterotro+hic bacteria community E3+osure to "C&R directly alters /increase
+ool of some im+ortant metabolites
"C &R e
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"C&R e
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DISCWSSION
Roles of R)re+otaenium as a member of
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+athogenic +olymicrobial $$D of corals
Initial adherence to the host coral infection
Creation of an ano3ic microen1ironment Sulfatreducing bacteria o3in +roduction Synergistic e
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R.Reptotaenium initiates $$
Wnder controlled conditions in the
lab, healthy coral fragments can beinfected 4ith $$D his cyanobacterium is in1ol1ed in
the *rst ste+6 adhesion to coral
surface +o+ulation gro4thanaerobic -ones form enrichinggro4th of SR$
Sul*de accumulates6 acti1e sulfurbiogeochemical cycle
Synergistic e
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Synergistic e
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Synergistic e
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CONC&WSION
Roseoflum reptotaenium
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Roseoflum reptotaenium
7irst .no4n cyanobacterium that is an acti1
member of a +athogenic +olymicrobial conin the Caribbean Plays se1eral roles in a 4orld4ide coral dis
that is contributing to reef destruction
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ac.$andDisease7inalPa+e)html htt+s6MM444)Bmnh)uB)eduM*shMsouthBoridaMcoralMdiseases)html htt+6MM444)atcc)orgM]MmediaMDGFJDG7J:GDA7$GK(AF$F htt+6MM444)statsdirect)comMhel+Mdefault)htm^basicsM+_1alues)htm htt+6MMblog)minitab)comMblogMad1enturesinstatisticsMho4tocorrect
1alues
htt/00111.re"earhgate.net0ubliation05)%&3(&)Fine"truturalanal"i"ofbla6banddi"ea"e7BBD8re!eal"boateriaandno!elbateria
http://www.sciencedirect.com/science/article/pii/S0165993615002575http://jrscience.wcp.muohio.edu/FieldCourses00/PapersMarineEcologyArticles/BlackBandDiseaseFinalPape.htmlhttp://jrscience.wcp.muohio.edu/FieldCourses00/PapersMarineEcologyArticles/BlackBandDiseaseFinalPape.htmlhttps://www.flmnh.ufl.edu/fish/southflorida/coral/diseases.htmlhttp://www.atcc.org/~/media/D3547D63F887423DAFB39841A5B588B9.ashxhttp://www.statsdirect.com/help/default.htmhttp://blog.minitab.com/blog/adventures-in-statistics/how-to-correctly-interpret-p-valueshttp://blog.minitab.com/blog/adventures-in-statistics/how-to-correctly-interpret-p-valueshttp://blog.minitab.com/blog/adventures-in-statistics/how-to-correctly-interpret-p-valueshttp://blog.minitab.com/blog/adventures-in-statistics/how-to-correctly-interpret-p-valueshttp://www.statsdirect.com/help/default.htmhttp://www.atcc.org/~/media/D3547D63F887423DAFB39841A5B588B9.ashxhttps://www.flmnh.ufl.edu/fish/southflorida/coral/diseases.htmlhttp://jrscience.wcp.muohio.edu/FieldCourses00/PapersMarineEcologyArticles/BlackBandDiseaseFinalPape.htmlhttp://jrscience.wcp.muohio.edu/FieldCourses00/PapersMarineEcologyArticles/BlackBandDiseaseFinalPape.htmlhttp://www.sciencedirect.com/science/article/pii/S0165993615002575