Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus

Ni ic oxide syn hesis by ni a e educ ase is egula ed du ing de elopmen in Aspe gillus Ana T. Ma cos,1Ma ía S. Ramos,1Jose F. Ma cos,2 Lou des Ca mona,2† Joseph S auss3,4 and Da id Cáno as1,3* 1Depa amen o de Gené ica,Facul ad de Biología, Uni e sidad de Se illa,Se illa, Spain. 2Depa men o Food Science,Ins i u e o Ag ochemis y and Food Technology (IATA),Valencia, Spain. 3Fungal Gene ics and Genomics Uni ,Depa men o Applied Gene ics and Cell Biology,Uni e si y o Na u al Resou ces and Li e Sciences (BOKU) Vienna,Vienna, Aus ia. 4Depa men o Heal h and En i onmen ,Bio esou ces, Aus ian Ins i u e o Technology (AIT),Vienna, Aus ia. Summa y Ni ic oxide (NO) is a signalling molecule in ol ed in many biological p ocesses in bac e ia, plan s and mammals. Howe e , li le is known abou he ole and biosyn hesis o NO in ungi. He e we show ha NO p oduc ion is inc eased a he ea ly s ages o he ansi ion om ege a i e g ow h o de elopmen in Aspe gillus nidulans. Full NO p oduc ion equi es a unc ional ni a e educ ase (NR) gene (niaD) ha is up egula ed upon induc ion o conidia ion, e en unde N- ep essing condi ions in he p esence o ammo- nium. A his s age, NO homeos asis is achie ed by balancing biosyn hesis (NR) and ca abolism ( la o- haemoglobins). niaD and la ohaemoglobin hbA a e ansien ly up egula ed upon induc ion o conidia ion, and bo h egula o s A eA and Ni A a e necessa y o his ansc ip ional esponse. The second la ohae- moglobin gene hbB shows a di e en exp ession p o ile being mode a ely exp essed du ing he ea ly s ages o he ansi ion phase om ege a i e g ow h o conidia ion, bu i is s ongly induced 24 h la e . NO le els in luence he balance be ween conidia ion and sexual ep oduc ion because a i icial s ong ele a- ion o NO le els educed conidia ion and induced he o ma ion o cleis o hecia. The ni a e-independen and ni ogen me aboli e ep ession-insensi i e an- sc ip ional up egula ion o niaD du ing conidia ion sugges s a no el ole o NR in linking me abolism and de elopmen . In oduc ion Rep oduc ion is an essen ial pa o he biological cycle o ungi and a mode o dispe sion in he en i onmen . Among ungi, Aspe gillus nidulans has been used as a model o ganism o s udy ep oduc ion o decades. A. nidulans displays wo modes o ep oduc ion: sexual o asexual. The asexual de elopmen p og am (conidia ion) is ini ia ed when supe icial hyphae a e exposed o an ai in e phase. The asexual ep oduc i e s uc u e o A. nidu- lans is he conidiopho e, which o ms g een-pigmen ed conidiospo es app oxima ely 24 h a e induc ion o de el- opmen (Adams e al., 1998; E xebes e e al., 2010; Yu, 2010). A cen al egula o y pa hway encompassing B lA, AbaA and We A con ols conidia ion (see e iews by Adams e al., 1998; E xebes e e al., 2010; Yu, 2010, and e e ences he ein). The i s componen in he egula o y cascade, B lA, is essen ial o d i e conidia ion (Adams e al., 1988). b lA is no exp essed du ing ege a i e g ow h, bu i s induc ion du ing de elopmen is con olled by a numbe o genes, e.g. he lu y genes (Adams e al., 1998; Yu e al., 2006). These lu y genes a e exp essed in ege a i e mycelium and a e able o espond o s imuli o induce he co-o dina ed ac i a ion o he mas e egu- la o b lA (E xebes e e al., 2010). The sexual p og am is p omo ed by signals ha can be g ouped in ou ca ego ies: absence o ligh , nu ien a ail- abili y, empe a u e and a mosphe ic gases. Unde labo a- o y condi ions, pla e sealing limi s he exchange o gases and esul s in lowe ing he oxygen le els and inc easing he CO2le els, which p omo es sexual ep oduc ion (Dye and O’Go man, 2012). A. nidulans is homo hallic, i.e. each s ain ha bou s bo h ma ing ype genes, MAT1/ma B and MAT2/ma A (Paole i e al., 2007). Du ing his p og am, A. nidulans de elops sphe ical ui ing bodies called cleis o- hecia, which con ain meiospo es called ascospo es (Dye and O’Go man, 2012). Genes in ol ed in he egula ion o cleis o hecia o ma ion we e mainly iden i ied h ough A. nidulans mu an s de ec i e a dis inc s ages o sexual de elopmen : acleis o hecial s ains such as ΔnsdD and Accep ed 3 Sep embe , 2015. *Fo co espondence. E-mail da idc@ us.es; Tel. +34954554405; Fax +34954557104. †P esen add ess: Depa men o Plan Bio echnology, Fundo de De esa da Ci icul u a, Vila Melhado – A a aqua a, São Paulo, B azil. Molecula Mic obiology (2016) 99(1), 15–33 ■doi:10.1111/mmi.13211 Fi s published online 14 Oc obe 2015 © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Δs uA (Wu and Mille , 1997; Han e al., 2001), mu a ions ha s op he p opaga ion p og am a e p oduc ion o Hülle cells such as Δs eA and ΔmedA (Clu e buck, 1969; Vallim e al., 2000) and s ains ha a e blocked in he ma u a ion o cleis o hecia (Ecke e al., 2000; Ho mann e al., 2000; Busch e al., 2001). Ea ly wo k in H. Ninnemann’s labo a o y connec ed ni a e educ ase ac i i y o blue ligh -p omo ed conidia- ion in Neu ospo a c assa (Klemm and Ninnemann, 1979; Ninnemann, 1991). The pa hway o he assimila ion o ni a e has been ex ensi ely s udied in A. nidulans o se e al decades. Ni a e assimila ion equi es he ac ion o wo enzymes: ni a e educ ase (NR, encoded by niaD), media ing he educ ion o ni a e o ni i e; and he ni i e educ ase (NiR, encoded by niiA) in ol ed in he con e - sion o ni i e o ammonium. Ni a e assimila ion is gene - ally ep essed in ungi by he p esence o he p e e ed ni ogen sou ces ammonium o glu amine and, in A. nidu- lans, is ac i a ed by ni a e o ni i e h ough he syne gis- ic ac ion o he pa hway speci ic egula o Ni A and he gene al egula o o ni ogen me abolism A eA (A s and Co e, 1973; Caddick e al., 1986; Bu ge e al., 1991; Ma zlu , 1997; S auss e al., 1998; Mu o-Pas o e al., 1999; Todd e al., 2005; Be ge e al., 2006; 2008; Be n ei e e al., 2007; Schinko e al., 2010). Recen ly, he egula ion o ni a e assimila ion was con- nec ed o ni ic oxide (NO) me abolism in A. nidulans (Schinko e al., 2010). NO is widely ecognised o i s ole as signalling compound om bac e ia o mammals (Rosselli e al., 1998; He e al., 2004; Kwon e al., 2012; Se h e al., 2012). This dia omic molecule is a sho -li ed adical wi h a hal -li e o a ew seconds. I plays essen ial egula o y oles in a a ie y o biological p ocesses bu can also cause ni osa i e s ess o cells. Fla ohaemo- globins a e known o unc ion in NO de oxi ica ion, con- e ing he eac i e NO adical in o ni a e and consequen ly p o ec ing agains ni osa i e s ess de i ed om ei he exogenous o endogenous sou ces (Ga dne e al., 1998a; Fos e e al., 2009). Di e en ou es o cel- lula NO biosyn hesis ha e been desc ibed in na u e, including bo h oxida i e and educ i e pa hways. The oxi- da i e syn hesis has been epo ed in bac e ia, plan s and mammals in ol ing he con e sion o L-a ginine and O2 in o ci ulline and NO by he ni ic oxide syn hase (Alde on e al., 2001; Lamo e e al., 2005; Go en and Maye , 2007). The educ i e NO syn hesis was epo ed as i s in legumes (Dean and Ha pe , 1988; Yamasaki, 2000), bu la e was shown o be widesp ead in plan s, and i in ol es he ac ion o he ni a e educ ase a sa u- a ing ni i e concen a ions unde educ i e condi ions (excess o NAPDH) (Yamasaki, 2000; Yamasaki and Sakihama, 2000; Rockel e al., 2002; Lamo e e al., 2005). In mammals al e na i e educ i e pa hways o he con e sion o ni i e o NO ha e been iden i ied in ol ing di e en enzymes such as cy och ome c, P450 o deoxy- haemoglobin in he hea and wall essels o by non- enzyma ic mechanisms (Zweie e al., 2010). No ably, ungal genomes a e de oid o NO syn hase homologues (Go en and Maye , 2007; Samalo a e al., 2013) and he physiological and gene ic mechanisms ha de e mine changes in he le el o NO in ungi a e poo ly unde s ood in hese o ganisms. Addi ion o NO chemical dono s induces asexual spo e de elopmen in he asco- myce e N. c assa (Ninnemann and Maie , 1996), spo an- giopho e de elopmen in he zygomyce e Phycomyces blakesleeanus (Maie e al., 2001), conidia ion in Conio- hy ium mini ans (Gong e al., 2007) and sexual ui ing bodies in he ascomyce e A. nidulans (Baidya e al., 2011) and in he basidiomyce e Flammulina elu ipes (Song e al., 2000). Magnapo he o yzae syn hesised NO du ing ge mina ion and ea ly de elopmen ; howe e , he dele ion o candida e genes in bo h oxida i e and educ i e ou es did no impai NO biosyn hesis in his ungus (Samalo a e al., 2013). In A. nidulans he wo la ohaemoglobins FhbA and FhbB we e shown o be in ol ed in he me abo- lism o NO o ni a e (Ga dne e al., 1998b; Poole and Hughes, 2000). In addi ion, hey a e essen ial o p o ec ni a e educ ase and ni i e educ ase enzymes agains ni osa i e damage elici ed by g ow h on ni i e a low en i onmen al pH condi ions (Schinko e al., 2010; 2013). In his wo k, we p o ide da a indica ing he exis ence o a ni a e educ ase-dependen pa hway o he syn hesis o NO in A. nidulans and ound ha NO le els inc ease immedia ely a e swi ching om ege a i e g ow h o conidia ion. Bo h la ohaemoglobins a e in ol ed in he balance o NO du ing he de elopmen al swi ch. Resul s Aspe gillus p oduces NO Se e al echniques ha e been employed o de ec and quan i y NO p oduc ion (Yamasaki and Sakihama, 2000; Maie e al., 2001; Planche and Kaise , 2006; Nagano, 2009; Samalo a e al., 2013). We selec ed he NO-sensi i e luo escen p obes de eloped by Nagano and collabo a o s (Kojima e al., 1998a,b; 1999). This app oach was p e iously epo ed o be use ul in ungi (Ca mona e al., 2012; Samalo a e al., 2013). DAF-FM is e y sensi i e o NO, wi h a de ec ion limi o 3 nM, whe eas DAF-FM diace a e (DAF-FM DA) is cell- pe meable and passi ely di uses ac oss cellula mem- b anes. Once inside he cells i is deace yla ed o DAF-FM by es e ases (Kojima e al., 1999). To es whe he hey could also be employed in Aspe gillus, ungal cells we e g own in liquid ammonium minimal medium. Ei he DAF-FM o DAF-FM DA was added o he cul u es. A e cells we e loaded wi h he dyes o 20 min, he excess o 16 A. T. Ma cos e al. ■ © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 bo h dyes was washed ou in hal o he samples, and all o hem we e ans e ed o a 96-well pla e o moni o ing luo escence. As shown in Fig. 1A–B, he e was an inc ease in he luo escence o e ime when he excess o dye we e no washed o he cells. Howe e , when he cells we e washed, he luo escence eco ded was d as- ically lowe han in non-washed cells o in con ol medium. To analyse whe he he moni o ed luo escence was he esul o he eac ion wi h he NO-sensi i e p obe inside o ou side he cells, luo escence was imaged unde a luo escen mic oscope (Fig. 1C). When he cells we e no washed, luo escence was obse ed bo h ou side and inside he cells ega dless o he dye (DAF-FM o DAF-FM DA). A e he excess o dye was washed ou , luo escence was obse ed p e e en ially inside he cells. To con i m he biological o igin o he signal, Aspe gillus cells g own o 16 h in liquid ammo- nium minimal medium we e au ocla ed. DAF-FM DA was added o li e cells, dead cells and he minimal medium and luo escence was eco ded (Fig. 1D). While luo es- cence in li e cell samples inc eased con inuously wi h ime, he luo escence gene a ed in he samples con ain- 3 3.5 4 4.5 5 . x 1,000) Non-washed cells Washed cells DAF-FM Diace a e A 0.5 1 1.5 2 2.5 3 o escence (a.u Washed Con ol (medium) 0 0 10 20 30 40 50 60 70 80 90 100 110 120 Flu Time (min) 18 0) DAF-FM B 8 10 12 14 16 ce (a.u. x 1,000 Non-washed cells Washed cells 0 2 4 6 0 10 20 30 40 50 60 70 80 90 100 110 120 Fluo escen Washed Con ol (medium) shed DAF-FM Diace a e DAF-FM Time (min) C Non-was Washed D E 7000 8000 Li e ungus 4000 5000 6000 c ence (a.u.) Killed ungus 1000 2000 3000 Fluo es c 0 0 20 40 60 80 100 120 Time (min) -1000 4000 9000 14000 19000 24000 29000 0 25 50 75 100 125 Fluo escence (a.u.) Time (min) NH4 NH4 + dNO NH4 + PTIO 100 NH4 + PTIO 500 NH4 + PTIO 1000 Fig. 1. Quan i ica ion o NO p oduced by Aspe gillus.A. nidulans was g own in liquid ammonium minimal medium. DAF-FM DA (A) o DAF-FM (B) was added o he cul u es and incuba ed o 20 min in he da k. A e cell loading o he dyes, one sample con aining each dye was washed h ee imes, whe eas con ol samples we e no washed. F esh medium was used as an addi ional con ol o de ec he backg ound signal. Fluo escence was moni o ed by luo ome y (A and B) o cells we e imaged by luo escen mic oscopy (C). (D) A. nidulans was g own in liquid ammonium minimal medium o 16 h. One sample was kep as a li ing con ol, whe eas he es o he cells we e killed by au ocla ing. DAF-FM DA was added o he samples and he luo escence was eco ded by luo ome y. Backg ound signal ob ained wi h esh medium was sub ac ed om signals ob ained wi h bo h cell samples. (E) Aspe gillus cells we e g own in he p esence o in he absence o he NO- eleasing compound dNO (1.5 mM) o di e en concen a ions o he NO-sca enge PTIO (100–1000 μM). DAF-FM DA was added o he samples and luo escence was eco ded by luo ome y. Unde all hese condi ions, ungal g ow h was simila . In all cases, one ep esen a i e expe imen is shown. Ni ic oxide syn hesis in Aspe gillus 17 © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 ing hea -killed ungi emained cons an o e ime, sug- ges ing ha NO p oduc ion was media ed by li e cells. I has been p e iously epo ed ha he DAF amily o NO-sensi i e dyes can also eac wi h asco bic acid and dehyd oasco bic acid (Zhang e al., 2002a,b). Al hough his possibili y was al eady dis ega ded by Samalo a and collabo a o s in Magnapo he (Samalo a e al., 2013), we es ed his possibili y in Aspe gillus by employing he NO-sca enge 4,4,5,5- e ame hylimidazoline-l-oxyl3- oxide (PTIO). Addi ion o di e en concen a ions o PTIO esul ed in a dose-dependen dec ease o luo escence compa ed wi h he con ol sample in he absence o he NO sca enge (Fig. 1E). Addi ion o he NO- eleasing compound de aNONOATE (dNO) p oduced a high inc ease o luo escence, as expec ed. Collec i ely, i shows ha DAF-FM and DAF-FM DA we e able o de ec he NO p oduced by Aspe gillus, bo h in acellula and ex acellula ly. We selec ed DAF-FM DA o he es o he s udy o ha ing a be e signal a io be ween samples con aining cells and con ol media in ou 96-well pla e assays (3.3 wi h DAF-FM DA e sus 1.5- old wi h DAF-FM). NO syn hesis du ing g ow h in liquid media pa ially depends on niaD I has been known o a long ime ha ungi can p oduce NO (Ninnemann and Maie , 1996); howe e , he mecha- nisms o NO syn hesis ha e emained obscu e so a . In o de o s udy a possible ole o he ni a e assimila ion pa hway in he biosyn hesis o NO, ou expe imen al se up was as ollows: he A. nidulans wild ype s ain was g own in liquid media con aining ni a e, ni i e o ammo- nium as sole ni ogen sou ce o 16 h; hen NO le els we e ollowed o up o 200 min by addi ion o he NO-sensi i e luo escen dye DAF-FM DA di ec ly o he g ow h medium and by subsequen luo ome ic quan i i- ca ion (Fig. 2A). The le el o luo escence in ensi y inc eased in he samples con aining ungal cells o e ime, o a highe ex en han in con ol samples (which con ain only he cul u e media). Sub ac ion o he back- g ound signal de i ed om he g ow h media con i med ha , in e es ingly, NO le els we e highe in ammonium han in ni i e o ni a e-con aining media (Fig. 2B, wild ype s ain). We hen in es iga ed whe he a ni a e educ ase- dependen ou e o he syn hesis o NO is ope a ional in A. nidulans, simila o he si ua ion desc ibed in plan s (Dean and Ha pe , 1988; Yamasaki and Sakihama, 2000; Rockel e al., 2002). We employed a ΔniaD mu an lacking ni a e educ ase. Ob iously his mu an canno g ow on ni a e as sole ni ogen sou ce, bu i can g ow on ni i e, which is, in plan s, he subs a e o he ni a e educ ase o p oduce NO. No majo di e ences we e ound in he NO le els be ween he wild ype and he mu an s ain on ammonium (Fig. 2B). Howe e , when bo h s ains we e g own in ni i e, he ΔniaD mu an p oduced 30% less NO han he wild ype, indica ing ha his enzyme pa icipa es in he p oduc ion o NO and is esponsible o a ound 30% o his me aboli e syn hesis when ni i e se ed as N-sou ce. On ni a e medium, he wild ype s ain showed he smalles amoun o NO compa ed wi h he o he wo ypes o ni ogen (Fig. 2B). In o de o es also NO p o- duc ion o ΔniaD on ni a e, he s ains we e p e-g own on ammonium o 12 h and hen ans e ed o ni a e- con aining ammonium liquid medium o back o sole ammonium medium as con ol. A e 6 h o g ow h unde hese condi ions, DAF-FM DA was added o he cul u es, and luo escence was quan i ied (Fig. 2C). The wild- ype s ain showed highe NO le els han he mu an when he medium was supplemen ed wi h ni a e indica ing again ha NR is in ol ed in he p oduc ion o NO h ough he ni a e assimila ion ou e. Ou da a sugges ed a pu a i e ole o niaD in medi- a ing he syn hesis o NO. P e ious da a easoned ha he la ohaemoglobin genes could be in ol ed in he ca abolism and hus de oxi ica ion o NO (Schinko e al., 2010). The dele ion o bo h la ohaemoglobin genes, hbA and hbB, did no a ec NO le els in ammonium medium (Fig. 2C–D, compa e NO le els wi h he wild ype s ain), bu he e was a d as ic inc ease in NO le els much highe han in he wild ype (up o 3.5- old inc ease) when ni a e was p esen in he medium (Fig. 2D). Impo an ly, e en in he p esence o he ep essing ni ogen sou ce ammonium, ni a e was an ob ious sou ce o NO p oduc ion. This is in ag eemen wi h indings by Schinko e al. (2010), who showed ha he ansc ip ion o hbA is independen om A eA unc- ion and ha ull induc ion o his gene also occu s unde induced- ep essed condi ions (i.e. ammonium plus ni a e). When he ni a e educ ase niaD was dele ed in he la ohaemoglobin dele ion mu an (ΔniaD Δ hbA Δ hbB), he inc ease o NO in he p esence o ni a e was no obse ed, con i ming ha a ni a e pa hway ope a es o syn hesise NO in A. nidulans and ha his pa hway equi es a unc ional niaD gene. Dele ion o niaD esul s in d as ic educ ion o NO p oduc ion du ing g ow h on solid media P e ious ansc ip omic da a ob ained in ou labo a o y sugges ed ha he ni a e u ilisa ion pa hway was exp essed du ing he induc ion o conidia ion (Cano as e al., 2014), which allowed us o hypo hesise ha , in addi ion o ege a i e g ow h, NO could also be p oduced h ough he ni a e pa hway du ing de elopmen . Fo his eason, we we e in e es ed in in es iga ing whe he NR was also ele an o NO p oduc ion on solid medium. We 18 A. T. Ma cos e al. ■ © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 compa ed he NO le els in he wild ype and he ΔniaD s ain. Bo h s ains we e g own on solid medium con ain- ing di e en ni ogen sou ces (ammonium, p oline o p oline supplemen ed wi h ni a e). P oline allows he g ow h o he wild ype and also he ΔniaD s ain, and i is a neu al (non- ep essing, non-inducing) ni ogen sou ce o he ni a e assimila ion sys em. Su p isingly, we ound ha NO le els o he ΔniaD mu an on ammonium solid medium was only 41% o he wild ype (Fig. 3A), whe eas in liquid ammonium medium bo h exhibi ed simila le els (Fig. 2). On p oline solid medium, he ΔniaD mu an p o- duced less NO compa ed wi h he wild ype. When ni a e was added o he p oline solid media, he wild ype inc eased NO p oduc ion (P<0.05; S uden ’s - es ), whe eas in he ΔniaD s ain educed le els we e ound: 30% less ela i e o p oline media (P<0.05; S uden ’s - es ), and 55% less NO han he wild ype in p oline plus ni a e (P<0.01; S uden ’s - es ). In his case, we obse ed ha he NO le els in he ni i e educ ase de ec- i e mu an (niiA4) we e simila o he wild- ype le els (Fig. 3A). The e o e, all hese da a sugges ha he e is a niaD-dependen pa hway o he syn hesis o NO bo h in liquid and on solid media and ha addi ional pa hway(s) o he syn hesis o NO mus ope a e in A. nidulans. 0 20 40 60 80 100 120 NH4 NO2 NO3 Pe cen age ( % luo escence / OD595) A B 0 2000 4000 6000 8000 10000 12000 14000 0 50 100 150 200 Fluo escence (a.u.) Time (min) Con ol_NH4 A. nidulans_NH4 Con ol_NO2 A. nidulans_NO2 Con ol_NO3 A. nidulans_NO3 Wild ype ΔniaD n.d. NH4+NO3- NO2- Con ol NH4+ Con ol NO3- Con ol NO2- A. nidulans NH4+ A. nidulans NO3- A. nidulans NO2- 0 10 20 30 40 50 60 70 80 90 100 Fluo escence (a.u.) / mg d y weigh C 0 50 100 150 200 250 300 350 Fluo escence (a.u.) / mg d y weigh D WT ΔniaD Δ hbAB ΔniaD Δ hbAB NH4+NH4+ + 10 mM NO3- NH4+NH4+ + 10 mM NO3- NH4+NH4++ 10 mM NO3- NH4++ 100 mM NO3- NH4+NH4++ 10 mM NO3- NH4++ 100 mM NO3- Fig. 2. NO quan i ica ion in mycelium in liquid medium. A. A. nidulans wild ype was inocula ed in media con aining he indica ed compounds as sole ni ogen sou ces. DAF-FM DA was added o he cul u es and non-inocula ed media (con ol samples). Media, which was no inocula ed, was employed as con ol o de e mine he biological o igin o he NO p oduced. Fluo escence was eco ded o e ime. One ep esen a i e expe imen is shown. B. A. nidulans wild ype (ligh g ey ba s) and ΔniaD (da k g ey ba s) s ains we e g own in liquid media wi h he indica ed ni ogen sou ces. The ΔniaD s ain could no g ow in ni a e, and consequen ly, NO p oduc ion was no es ed in his sample (n.d.). Da a shown co espond o one ime poin a e addi ion o DAF-FM DA (100 min) and a e e e ed o he wild- ype s ain g own in ammonium media (100%). Da a shown a e he mean and he s anda d e o o he mean o a leas h ee independen biological eplica es. N.d.: no de e mined. C. A. nidulans wild ype and ΔniaD s ains we e g own in ammonium liquid medium o 12 h. The mycelia we e il e ed, washed and ans e ed o ammonium (con ol) and ammonium +10 mM ni a e. The cul u es we e g own o 6 addi ional hou s. DAF-FM DA was added o quan i y NO p oduc ion, and luo escence was eco ded o 60 min. Da a shown a e he mean and he s anda d e o o he mean o wo independen biological eplica es. D. A. nidulans Δ hbA Δ hbB and Δ hbA Δ hbB ΔniaD s ains we e g own in ammonium liquid medium o 12 h. The mycelia we e il e ed, washed and ans e ed o ammonium (con ol), ammonium +10 mM ni a e, and ammonium +100 mM ni a e. The cul u es we e g own o 6 addi ional hou s. DAF-FM DA was added o quan i y NO p oduc ion, and luo escence was eco ded o 60 min. Da a shown a e he mean and he s anda d e o o he mean o wo independen biological eplica es. Ni ic oxide syn hesis in Aspe gillus 19 © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 Wi h he aim o con i m he sou ce o NO, he la ohae- moglobin mu an s we e used again o educe NO ca abo- lism and hus o inc ease he o e all le els o his me aboli e. The h ee s ains we e g own on solid media con aining p oline as a sole ni ogen sou ce; he luo es- cence eco ding was s a ed by addi ion o DAF-FM, and hen di e en ni ogen sou ces we e indi idually added o he assay as he eco ding con inued. As shown in Fig. 3B, addi ion o ni a e did no ha e majo e ec s on Δ hbA Δ hbB. Howe e , addi ion o ni i e elici ed an inc ease in he luo escence. The ni i e educ ase mu an (Δ hbA Δ hbB niiA4), on he o he hand, exhibi ed an inc ease in he NO le els a e he addi ion o ei he ni a e o ni i e, bu no ammonium, demons a ing ha he ni i e educ ase (NiR) was no esponsible o he syn hesis o NO. Addi ion o ei he ni a e o ni i e o he Δ hbA Δ hbB ΔniaD mu an did no inc ease he NO le els o e he con ol sample; u he mo e, his s ain showed educed NO le els in he con ol condi ions (p oline) compa ed wi h he wild ype (Δ hbA Δ hbB) and he Δ hbA Δ hbB niiA4 s ains (Fig. 3B). Addi ion o ammonium o any o he s ains did no change he NO le els signi ican ly o e he con ol condi ion (p oline), as expec ed. The e o e, aken oge he , ou da a demons a e ha he ni a e educ ase is pa ially esponsible o he syn hesis o NO employing ni i e as subs a e. NO le els a e highe on solid medium han in liquid medium Du ing he p e ious expe imen s, we obse ed a highe p oduc ion o NO du ing g ow h on solid media han in liquid media. In o de o compa e he NO le els in bo h ype o media, he A. nidulans wild- ype s ain was g own in liquid o solid media con aining ammonium (con ol) o ammonium supplemen ed wi h 10 o 100 mM ni a e o 18 h. DAF-FM was added o he samples, and luo es- cence was eco ded o 200 min. In e es ingly, p oduc ion o NO was much highe when A. nidulans was g own on solid media han in subme ged cul u e unde hese expe imen al condi ions (Fig. 4A). Addi ionally, he inc e- men in he p oduc ion o NO in he p esence o ni a e was also signi ican ly highe on solid media. In o de o ind ou whe he NO was p oduced o accu- mula ed in hyphae o conidiopho es, Aspe gillus was g own on solid medium. DAF-FM DA was added and allowed o pe mea e he cells o 20 min be o e washing ou he excess o he dye. Visualisa ion unde he luo es- cen mic oscope e ealed ha bo h hyphae and conidi- opho es we e s ained; howe e , he signal was s onge in he conidiopho es han in he hyphae (Fig. 4B). Unde his low magni ica ion and using a luo escen mic oscope, he signal appea ed o be g ea e in s e igma a cells (me ulae and phialides) han in he s alk cells. Con ocal mic oscopy con i med ha he luo escen signal could be de ec ed in he s e igma a cells (Fig. 4C–E), bu also in he esicle and s alk cells, and in hyphae (Fig. 4C). Fungal g ow h media es ni i e and ni a e syn hesis in ammonium media The high le els o NO obse ed on ammonium we e in iguing, bu he ac ha he ΔniaD s ain p oduced less NO han he wild ype on ammonium solid medium (Fig. 3A) posed wo in e es ing ques ions. The i s one is: can NO be syn hesised om ni a e in a medium con ain- 0 20 40 60 80 100 120 % Fluo escence (a.u.) / OD595 Δ hbAB ΔniaD Δ hbAB Δ hbAB niiA4 P ol P ol + NO3- P ol + NO2- P ol + NH4+ ΔniaD WT niiA4 P ol P ol + NO3- NH4+ A B 0 25 50 75 100 125 150 175 % Fluo escence (a.u.) / OD595 Fig. 3. NO quan i ica ion on solid medium. A. A. nidulans wild- ype, ΔniaD and niiA4 s ains we e g own on solid media con aining he indica ed ni ogen sou ces a 10 mM (ammonium o ni a e) o 3 mM (p oline) a 37°C o 18 h. DAF-FM DA was added o quan i y NO p oduc ion, and luo escence was eco ded o 100 min. Da a shown a e e e ed o he wild- ype s ain g own on ammonium media (100%). Da a shown a e he mean and he s anda d e o o he mean o a leas h ee independen biological eplica es. B. A. nidulans Δ hbA Δ hbB, Δ hbA Δ hbB ΔniaD and Δ hbA Δ hbB niiA4 s ains we e g own on p oline solid media o 18 h. DAF-FM DA was added o quan i y NO p oduc ion. Then, he indica ed ni ogen sou ces we e added, and luo escence was eco ded o 60 min. Da a shown a e e e ed o he con ol s ain (Δ hbA Δ hbB) g own on p oline wi hou he addi ion o any o he ni ogen sou ce. E o ba s ep esen he s anda d e o o he mean. 20 A. T. Ma cos e al. ■ © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 ing ammonium as sole ni ogen sou ce? This sugges s ha ni a e could be p esen in his medium. To es his hypo hesis, we employed he A. nidulans ΔniaD s ain, which does no me abolise ni a e allowing i s accumula- ion, and quan i ied bo h ni a e and ni i e du ing g ow h in he media. The ΔniaD s ain was g own on he su ace o a pe i dish con aining liquid media supplemen ed wi h ammonium as sole ni ogen sou ce, as p e iously desc ibed (Ruge -He e os e al., 2011). Unde hese con- di ions, A. nidulans conidia ion p oceeded as on solid media. Nei he ni a e no ni i e could be de ec ed in he cul u e media be o e he inocula ion wi h he ungal spo es. Bu su p isingly, bo h ni a e and ni i e we e ound in he cul u e media a e 19, 24 and 48 h o ungal g ow h (Fig. 5). When he expe imen was pe o med wi h a wild- ype s ain, ni a e and ni i e we e also ound in he media bu a lowe le els, due o he me abolism o ni a e by an ac i e ni a e educ ase (Supplemen a y Fig. S1). niaD is induced du ing conidia ion The second ques ion is: i acco ding o he heo y o ni ogen egula ion, he ni a e educ ase gene niaD mus be ep essed in a media con aining he ep essing ni o- gen sou ce ammonium (A s and Co e, 1973), how is he ni a e educ ase ac i e in hese condi ions? T ansc ip- omics da a ob ained p e iously sugges ed ha he ni a e u ilisa ion pa hway was exp essed du ing induc ion o de elopmen on ammonium ni a e medium (Cano as e al., 2014). To u he con i m his, we i s ob ained 0 2000 4000 6000 8000 10000 12000 14000 16000 0 50 100 150 200 Fluo escence (a.u.) / OD595 Time (min) SOL AMM SOL AMM + 10 mM NIT SOL AMM + 100 mM NIT LIQ AMM LIQ AMM + 10 mM NIT LIQ AMM + 100 mM NIT SOL NH4+ SOL NH4+ + 10 mM NO3- SOL NH4+ + 100 mM NO3- LIQ NH4+ LIQ NH4+ + 10 mM NO3- LIQ NH4+ + 100 mM NO3- D E C DIC DAF-FM MERGE A B Fig. 4. NO p oduc ion is highe on solid medium han in liquid medium. A. A. nidulans wild- ype s ain was g own in liquid o solid media o 18 h. In liquid medium, A. nidulans was g own in ammonium liquid medium o 12 h. The mycelia we e il e ed, washed and ans e ed o ammonium (LIQ NH4+), ammonium +10 mM ni a e (LIQ NH4++10 mM NO3−) and ammonium + 100 mM ni a e (LIQ NH4++100 mM NO3−). The cul u es we e g own o 6 addi ional hou s ( o al g ow h ime was 18 h). On solid media spo es we e inocula ed on solid media con aining ammonium (SOL NH4+), ammonium +10 mM ni a e (SOL NH4++10 mM NO3−) and ammonium +100 mM ni a e (SOL NH4+ +100 mM NO3−), and he pla es we e incuba ed a 37°C o 18 h. DAF-FM DA was added o all cul u es o quan i y NO p oduc ion, and luo escence was eco ded o 200 min. The plo shown is he a e age o wo eplica e samples o a ep esen a i e expe imen . B–E. Aspe gillus wild- ype s ain was g own on solid ammonium medium o 24 h. Aga cubes con aining ungal biomass we e subme ged in a DAF-FM DA solu ion o 20 min and washed h ee imes. Conidiopho es we e imaged by luo escence mic oscopy (B) o con ocal mic oscopy (maximal p ojec ion in C, and single planes in D–E). Ni ic oxide syn hesis in Aspe gillus 21 © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 wild- ype ege a i e mycelia g own in ammonium liquid medium o 18 h, and hen, conidia ion was induced by ans e ing he mycelia o solid media con aining ammo- nium o ni a e as a sole ni ogen sou ce (Fig. 6). Two hou s a e he induc ion o conidia ion, niaD mRNA le els we e oughly 70- old highe on ni a e solid media han in ege a i e mycelia and, s ikingly, niaD was also an- sc ibed on ammonium solid media. Howe e , he ni a e- induced le el was 8.6- old highe compa ed wi h ammonium. Longe induc ion o conidia ion on ni a e solid media did no esul in much u he inc ease o niaD exp ession ( om 70- o 92- old induc ion compa ed wi h ege a i e mycelia). Howe e , longe induc ion o conidi- a ion on ammonium media inc eased he induc ion o niaD om 8.5- o 23- old ela i e o ege a i e mycelia. We also quan i ied he le els o he la ohaemoglobin mRNAs a e he induc ion o conidia ion. As p e iously epo ed, hbA was induced by ni a e (Schinko e al., 2010), and he e was no signi ican a ia ion in he exp ession le els in ammonium du ing he i s hou s o conidia ion (Fig. 6). On he o he hand, he hbB gene was sligh ly ep essed immedia ely a e induc ion o asexual de elopmen ega dless o he ni ogen sou ce and d opped h ee old a e 2 h (Fig. 6). Down egula ion o hbB was simila in bo h ni ogen sou ces ammonium and ni a e, sugges ing ha hbB is no cons i u i e, bu a he de elopmen ally egula ed. NO le els a e inc eased du ing he i s hou s o conidia ion The highe le els o NO p oduced du ing g ow h on solid media compa ed wi h liquid media sugges s ha he e migh be a connec ion be ween NO and de elopmen . Indeed he exp ession pa e n o hbB poin s o an ea ly egula ion o he NO le els du ing he ansi ion om ege a i e g ow h o conidia ion. In o de o s udy his possible connec ion be ween NO and asexual de elop- men , we i s quan i ied he NO le els a he ea ly s ages o he ansi ion om ege a i e g ow h o conidia ion. The wild- ype s ain and he mu an lacking bo h la ohaemo- globins (Δ hbA Δ hbB), which canno de oxi y NO, we e 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0h 19h 24h 48h NO3- o NO2- (ppm) NO3- NO2- Fig. 5. Quan i ica ion o ni a e and ni i e in he cul u e media. A. nidulans ΔniaD s ain was g own on he su ace o a pe i dish con aining liquid media o he indica ed ime poin s. Ni a e (ligh g ey ba s) and ni i e (da k g ey ba s) we e quan i ied wi h he G iess eagen . Nei he ni a e no ni i e was de ec ed in he media be o e ungal g ow h. NO3- NH4+ 0.000 0.005 0.010 0.015 0.020 0.025 Rela i e exp ession hbB 0 h 2 h 4 h 6 h Time ( h ) 0 2 4 6 8 10 12 14 16 Rela i e exp ession hbA 0.0 0.5 1.0 1.5 2.0 Rela i e exp ession niaD Fig. 6. Exp ession o he la ohaemoglobins and ni a e educ ase genes in media con aining ni a e o ammonium as sole ni ogen sou ce ea ly du ing conidia ion. A. nidulans wild- ype s ain was g own ege a i ely in ammonium liquid minimal medium o 18 h and hen ans e ed o ni a e (da k g ey ba s) o ammonium (ligh g ey ba s) solid medium o induce conidia ion. Samples we e aken a he indica ed ime poin s o RNA isola ion. hbA, hbB and niaD exp ession was quan i ied by eal ime RT-PCR. Da a we e no malised agains he exp ession o he ß- ubuline gene (benA). The plo s show he a e age and s anda d e o o he mean o he ela i e exp ession alues in a leas h ee independen expe imen s. 22 A. T. Ma cos e al. ■ © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 g own in ammonium liquid medium, and hen ans e ed o solid media o induce conidia ion as abo e. Samples we e aken a 1 h in e als, and NO was quan i ied wi h DAF-FM. As can be seen in Fig. 7A–B, NO inc eased a e 1 h o induc ion compa ed wi h ege a i e g ow h in all he condi ions. In ac , we obse ed his inc ease a e induc ion o conidia ion o 1 h, and i emained ele a ed a leas o 7 h (da a no shown). The e we e no majo di e ences in he NO le els be ween ammonium and ni a e g ow h condi ions du ing he ea ly induc ion o conidia ion. We we e in e es ed in c ea ing a highe ange o NO le els o he ollowing expe imen s. The e o e, we added he NO- eleasing chemical compound de aNONOa e (dNO), which was p e iously es ed o inc eased le els o NO (Fig. 1E), and checked o an addi ional inc ease o NO le els (Fig. 7C). In his case, we ound a d as ic inc ease o NO in bo h s ains, as expec ed, and NO le els we e h ee old highe in he mu an han in he wild ype. Conidia ion (asexual de elopmen ) is ep essed by ele a ed NO le els In o de o ge insigh in o he e ec s o NO du ing ep o- duc ion, we i s es ed he e ec s o di e en NO le els on conidia ion using he wild- ype s ain and he mu an lacking bo h la ohaemoglobins (Δ hbA Δ hbB). Conidia- ion was induced as desc ibed abo e by ans e ing eg- e a i e mycelia o bo h s ains o solid media wi h o wi hou dNO. Based on he esul s shown abo e, we expec ed ha hese condi ions and s ains gene a ed a g adien o NO. As NO le els quickly inc eased a e induc ion o conidia ion, we quan i ied he le els unde hese condi ions and obse ed he expec ed g adien o inc easing NO le els (Fig. 8A). A e 3 days o incuba ion, he numbe o conidia was coun ed. Conidia ion was sligh ly educed in he double mu an compa ed wi h he wild ype in he con ol ni a e media (Fig. 8B). Addi ion o he NO-dono dNO o he media esul ed in a d as ic inc ease o NO le els, especially in he mu an s ain, and a u he educ ion o conidia ion (ca. 60%) bo h in he wild ype (P<0.1; S uden ’s - es ) and in he mu an s ain (P<0.05; S uden ’s - es ). Obse a ion o he ungal g ow h on he solid media wi h a s e eoscopic mic oscope e ealed a educ ion in he densi y o conidiopho es (Fig. S2). These esul s sugges ha an inc ease o NO co e- la es wi h a dec ease in conidia ion. Sexual de elopmen is induced by NO Conidia ion and sexual de elopmen a e no mally bal- anced in A. nidulans, meaning ha when one o hem inc eases, he o he one dec eases (Adams e al., 1998; Rod iguez-Rome o e al., 2010; Dye and O’Go man, 2012). To s udy he e ec s o NO in sexual de elopmen o A. nidulans, we ollowed a simila s a egy as desc ibed abo e bu g owing s ains unde condi ions o induce sexual de elopmen (i.e., sealed pla es in he da k). Again, his s a egy also allowed c ea ing he NO g adien unde he condi ions o induce sexual de elopmen (Fig. 8C). Opposi e o he e ec s obse ed in conidia ion, he e was an inc ease in he numbe o cleis o hecia ( he sexual ep oduc i e s uc u es o A. nidulans)in he mu an s ain compa ed wi h he wild ype in he con ol medium (Fig. 8D). When he NO- eleasing compound dNO was added o he media, he numbe o cleis o hecia e en inc eased in he pa en al s ain compa ed wi h he con ol media (Fig. 8D). Simila esul s we e ob ained by Baidya e al. (2011). This inc ease in he numbe o cleis- o hecia co ela ed wi h he inc ease o NO. Howe e , 0 200 400 600 800 1000 1200 1400 Fluo escence (a.u.) 0 200 400 600 800 1000 1200 1400 Fluo escence (a.u.) 0 2000 4000 6000 8000 10000 VEG 1 h 2 h 3 h 4 h Fluo escence (a.u.) Ammonium Ni a e Ni a e + dNO Wild ype Δ hbA Δ hbB A B C Time (h) Fig. 7. NO quan i ica ion a e induc ion o de elopmen . A. nidulans wild- ype s ain (ligh g ey ba s) and a Δ hbA Δ hbB mu an (da k g ey ba s) we e g own ege a i ely in ammonium liquid medium o 18 h (VEG) and hen ans e ed o solid media con aining di e en ni ogen sou ces o induce de elopmen : ammonium a a e as con ol ni ogen sou ce (A), sodium ni a e (B) and sodium ni a e supplemen ed wi h NO- eleasing chemical compound dNO (C). Samples we e aken a di e en imes, and he luo escence p oduced by he eac ion o NO and DAF-FM DA was quan i ied. Please no e he di e en g aph scales in A–C. Ni ic oxide syn hesis in Aspe gillus 23 © 2015 The Au ho s. Molecula Mic obiology published by John Wiley & Sons L d., Molecula Mic obiology,99, 15–33 ligh o conidia ion condi ions, o al e na i ely hey we e sealed and incuba ed in he da k o sexual de elopmen al condi ions. Plugs we e cu ou om he pla e 72 h a e induc- ion o de elopmen . Conidia in he plugs we e esuspended in Tween 0.1% bu e and coun ed. Fo coun ing cleis o he- cia, pla es incuba ed o 15 days we e i s sp ayed wi h 70% e hanol o acili a e he isualisa ion o he sexual s uc u es and hen pho og aphed. Da a shown a e he a e age o a leas ou independen expe imen s. NO quan i ica ion Ni ic oxide was quan i ied in samples by using he NO-sensi i e luo escen dye DAF-FM DA (In i ogen) ollow- ing he manu ac u e ’s ins uc ions excep in Fig. 1, in which DAF-FM (Sigma) was also employed. In Fig. 1, ungal cells we e g own in liquid ammonium minimal medium o 16 h, and 50 μlo 5μM DAF-FM DA o DAF-FM we e added o 100 μlo each sample. Cells we e loaded wi h he dye o 20 min and washed h ee imes wi h a 0.05% ween solu ion (when indi- ca ed). Samples we e ans e ed o 96-well pla es, and luo escence was eco ded in a Syne gy HT Mul i-mode Mic opla e Reade (Bio ek) equipped wi h FITC il e se s (λEm 488 nm, λEx 520 nm). In Fig. 2Aand B, conidia we e inocula ed in o 100 μl o minimal media a a inal concen a ion o 105conidia ml−1in 96-well pla es, and he pla es we e incu- ba ed wi h shaking a 37°C o 16 h o allow ae a ion o he cul u e. A e incuba ion, DAF-FM DA was added o he cul- u es o a inal concen a ion o 2.5 μM, and luo escence was eco ded in POLARs a Omega luo ome e (MG Lab ech) equipped wi h FITC il e se s (λEm 488 nm, λEx 520 nm) o up o 200 min. Da a we e analysed wi h Omega Con ol ( e sion 1.10) so wa e. Fo he es o he expe imen s, he s ains we e g own in ammonium liquid medium o 12 h, hen ans- e ed o liquid media con aining he app op ia e ni ogen sou ces and incuba ion con inued o 6 addi ional hou s. Cells we e loaded wi h he dye o 20 min and ans e ed o 96-well pla es. Al e na i ely, he s ains we e g own in ammonium liquid medium o 18 h and hen ans e ed o solid media o he indica ed ime poin s. Pla es we e incuba ed unde he condi ions desc ibed abo e o conidia ion o sexual de elop- men . Samples we e aken om he pla es a he indica ed ime poin s, subme ged in a solu ion o 5 μM DAF-FM DA o 1 h. Samples we e cen i uged, and he supe na an was ans e ed o a 96-well pla e o luo escence quan i ica ion. Fo he o he expe imen s pe o med on solid media in Figs 3 and 4, 105conidia we e inocula ed on 96-well pla es con ain- ing solid media con aining he indica ed ni ogen sou ces and incuba ed o 18 h be o e quan i ica ion o NO. In Fig. 3B, ungal cells we e g own in p oline solid media o 18 h, luo- escence quan i ica ion s a ed a e addi ion o DAF-FM DA and hen he indica ed ni ogen sou ces we e added and luo escence quan i ica ion con inued o 2 addi ional hou s. In all cases 2.5 μM DAF-FM DA was added o he samples. Fluo escence was de ec ed and quan i ied in a Syne gy HT Mul i-mode Mic opla e Reade (Bio ek) equipped wi h GFP il e se s. Da a we e analysed wi h Gen5TM Da a Analysis So wa e. In all expe imen s, da a we e no malised o d y weigh o OD595 as indica ed. Expe imen s we e epea ed a leas wo imes and pe o med in duplica es o iplica es, depending on he expe imen . Mic oscopy Aspe gillus was g own ei he on liquid ammonium medium o 14–16 h o solid ammonium medium o 24 h. A solu ion o 5μM DAF-FM DA(solid and liquid samples) o DAF-FM (liquid samples) was added o he mycelium. A e cell loading o he dye o 20 min, he excess o dye was washed ou wi h a 0.05% Tween 80 solu ion. Images we e eco ded in an Olympus IX2-UCB in e ed luo escence mic oscope equipped wi h a FITC il e se (λEm 488 nm; λEx 520 nm). Images in Fig. 1C we e aken using iden ical se ings o compa a i e pu poses. Images in Fig. 4B we e p ocessed and me ged using Adobe ImageReady CS2 (Adobe Sys ems Inco po a ed, CA, USA). Con ocal images we e ob ained in a ZEISS LSM 7 DUO con ocal mic oscope wi h an exci a ion a 488 nm using an OPSS lase beam. Con ocal images we e p ocessed using ZEN Li e 2012 (Zeiss, Ge many). Ni a e and ni i e measu emen s in he cul u e supe na an Ni a e and ni i e we e quan i ied in he cul u e media using he G iess eagen as p e iously desc ibed (Schinko e al., 2010). Acknowledgemen s We would like o hank Michael Hynes o he niiA4 s ain, Tho s en Schinko o help ul discussions, Modes o Ca ballo o help wi h he luo ome ic assays (Se icio de Biología, Cen o de In es igación Tecnología e Inno ación, Uni e sidad de Se illa) and Juan Luis Ribas o help wi h he con ocal mic oscopy (Cen o de In es igación Tecnología e Inno ación, Uni e sidad de Se illa). Wo k in Valencia was suppo ed by g an numbe BIO2012-34381 om he MINECO o JFM, and in Vienna was suppo ed by g an numbe LS12-009 om he n( +b) Lowe Aus ia Science Fund and M01693-B22 om he FWF. We would like o hank he anonymous e e ees o help ul sugges ions o imp o e his manusc ip . Re e ences Adams, T.H., Boylan, M.T., and Timbe lake, W.E. 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