Ecology and Physiology of the Pathogenic Cyanobacterium Roseofilum.pptx

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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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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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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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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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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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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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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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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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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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

Ecology and Physiology of the Pathogenic Cyanobacterium Roseofilum.pptx

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