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….$CLONES$OF$MICROCYSTIS$AERUGINOSA$USING$LECTINS$

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software! package! GraphPad! Instat! 3.0! (Graph?Pad! Software! Inc,! USA).! Differences!
were!considered!to!be!significant!P!<!0.01).!!

Lastly,! a! dendrogram! was! realised! using! a! binary! hierarchical! cluster!
analysis! (method! average! linkage! using! SPSS! software;! 0! for! negative! and! 1! for!
positive!lectin!binding!respectively).!

3.*RESULTS**

The! results! confirmed! that! optical! examination! under! an! epi?fluorescence!
microscope!is!a!rapid!and!precise!procedure!to!evaluate!the!lectin?binding!activity.!
The! green! fluorescence! observed! was! very! intense! in! the! colonies! positively!
labeled! with! lectins,! both! at! the! cell! surface! as! well! as! on! inter?cellular! matrix.! In!
additon,!a!similar!staining!pattern!is!shown!by!all!the!M.)aeruginosa!colonies!from!
a!same!strain!(as!well!as!all!the!cells!of!the!same!colony).!!

Experiments!assessing!presented!that!several!lectins!(Con?A,!WGA)!are!able!
to!bind!most!of!M.)aeruginosa!strains.!In!contrast,!other!lectins!(PNA,!SBA)!bind!to!
only!a!limited!number!of!strains.!Moreover,!the!results!also!confirmed!that!lectin?
binding! pattern! could! allow! differentiation! among! the! different! strains! isolated!
from!a!same!bloom!(Table!2).!!

A! very! interesting! result! was! that! the! lectin?binding! pattern! and! toxin!
production! was! related.! Lectins! are! found! to! be! useful! tools! for! differentiating!
clones! of! M.) aeruginosa! between! toxic! and! non?toxic! strains! within! a! pool! of! 35!
different! clones! isolated! from! four! different! blooms! (with! a! great! variety! in! toxin!
production).! In! this! sense,! lectin! from! Ulex) europaeus! (UEA?1)! showed! the! most!
important! result:! all! non?toxic! M.) aeruginosa! strains! were! positively! binding! with!
UEA?1! lectin.! Contrarily,! lectin! from! Canavalia) ensiformis! (ConA)! was! able! to! bind!
to! all! clones! except! the! three! most! toxic! (MaD7,! MaG8! and! MaD1).! The! other! six!
lectins! used! in! this! study! demonstrated! non?significant! affinities! to! discriminate!
between!toxic!or!non?toxic!clones.!

The!results!reported!here!indicated!that!the!most!toxic!strains!were!labelled!
by!very!few!lectins.!In!contrast,!non?toxic!strains!were!tagged!by!numerous!strains!
of! lectins.! Statistically! significant! regression! was! found! between! toxin! production!
(in! fg! microcystin! LR?equivalent! per! cell)! and! the! number! of! lectin! that! positively!
label!the!strain!(slope!±!se!=!?0.0434!±!0.0044;!correlation!coefficient!r!=!?0.!8677;!
P<0.001).!

A! hierarchical! cluster! analysis! showed! three! differentiated! alignments!
(Figure! 2).! These! alignments! were! directly! related! with! the! toxin! production! of!
each!strain.!In!short,!all!the!non?toxic!strains!were!included!in!the!first!alignment.!
The! strains! with! a! moderate! microcystin! production! (from! 0.02! to! 0.17! µg!

129$

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