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2.*MATERIALS*AND*METHODS*

2.1!Experimental/organisms/and/growth/conditions/

Experiments! were! carried! out! using! thirty?five! strains! of! Microcystis)
aeruginosa) (Kützing)! Kützing! from! the! Algal! Culture! collection,! Genetics!
laboratory,! Facultad! de! Veterinaria,! Universidad! Complutense! de! Madrid,! Spain.!
Strains! were! collected! from! water! samples! of! four! M.) aeruginosa! blooms.! Three!
blooms!occurred!in!the!water!supply!reservoirs!of!El!Gergal,!La!Minilla!and!Cuerda!
del!Pozo!(Andalusia,!Spain)!and!the!other!bloom!occurred!in!Doñana!National!Park!
(Spain)!causing!mass!mortality!of!wildlife!(data!in!16,!30,!37).!These!water!supply!
reservoirs! provide! water! to! more! than! 1.5! millions! people! in! Spain.! Doñana!
National! Park! is! classified! as! a! Special! Protection! Area! by! the! European! Union!
under! Directive! 79/409,! because! of! its! ecological! value! and! international!
relevance.!!

Isolation! of! cells! and! colonies! was! made! using! a! Zeiss?Eppendorf!
micromanipulator?microinjector! and! each! strain! was! obtained! from! a! single!
vegetative! cell! separated! from! a! M.) aeruginosa! colony.! Cultures! were! grown!
axenically! in! tissue! culture! flasks! (Greiner,! Bio?One! Inc.! Longwood,! NJ,! USA)!
containing! 20! ml! of! cyanobacterial! BG?11! medium! (Sigma,! Aldrich! Chemie,!
Taufkierchen,! Germany),! at! 22ºC! and! 120! µmol! m?2! s?1! over! waveband! supplied!
by! daylight! fluorescent! tubes.! Clones! were! maintained! in! mid?log! exponential!
growth!by!serial!transfers!of!an!inoculum!to!fresh!medium!once!every!two!weeks.!
Under! these! conditions,! all! the! cultures! grew! asexually.! The! absence! of! bacteria!
was!tested!periodically!by!epifluorescence!observation!after!staining!with!acridine!
orange.!Only!cultures!lacking!bacteria!were!employed.!

2.2./Measurement/of/toxicity//

The! toxin! production! of! the! different! strains! was! measured! as! previously!
described! (29,! 34).! Two! quantitative! tests! for! the! detection! of! microcystins!
residues! in! water! were! employed.! A! microcystin?specific! enzyme?linked!
immunoabsorbent!(ELISA)!test!was!used!(EnviroGard!Microcystin!Quantitube!Test!
Kit;! Strategic! Diagnostic,! Newark,! N.J.,! USA)! as! well! as! a! phosphatase?inhibition!
based! test! for! the! detection! of! microcystins! (Microcystest,! ZEU?Immunotech,!
Zaragoza,! Spain),! according! to! the! manufacturer´s! recommendations.! The! cells! of!
each! aliquot! were! broken! by! freezing,! sonication,! and! intense! agitation.! The!
insoluble! cell! debris! was! removed! by! centrifugation! (2500! ×! g,! 10! min.).! Six!
measurements! of! each! strain! were! run! to! estimate! confidence! intervals.!
Additionally,!the!toxicity!of!the!water!samples!from!the!four!different!blooms!was!
also!measured!using!the!same!procedure.!

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