Who ar e t he Myt i l us t r ossul us? • The M. t r os s ul us a r e c ommon ma r i ne mus s e l s • Found a l ong r oc ky s hor e l i ne s of Nor t h Ame r i c a i n t he i n t e r t i da l z one a nd s pa n f r om hi gh t i da l z one t o l owe s t t i da l poi n t . • Col oni z e i n c l umps on r oc ks • Ar e a f ounda t i on s pe c i e s - a domi na nt s pe c i e s i n a ha bi t a t t ha t a r e a bunda nt a nd ha ve ma j or i nf l ue nc e on t he e c os ys t e m • Ar e a l s o I ndi c a t or s pe c i e s – a s t he y r e pr e s e nt t he ove r a l l he a l t h of a n e c os ys t e m a nd a r e i nf l ue nc e d by e nvi r onme nt a l c ondi t i ons : d i s e a s e out br e a k, pol l u t i on, s pe c i e s c ompe t i t i on or c l i ma t e c ha nge . Who ar e t he Myt i l us t r ossul us Phyl um Mol l us c a Cl a s s Bi va l vi a Subc l a s s Pt e r i omor phi a Or de r Myt i l o i da Fa mi l y Myt i l i da e Ge nus Myt i l us Spe c i e s t r os s ul us Mussel s: An i deal model t o st udy oxygen met abol i sm • Re s i de i n i n t e r t i da l z one – s ubj e c t e d da i l y t o e xt r e me f l uc t ua t i ons i n oxyge n l e ve l s • Se s s i l e or ga ni s ms - e a s i e r t o mi ni mi z e s t r e s s on a ni ma l s r e s ul t i ng f r om c a pt i vi t y a nd t e s t i ng pr oc e dur e s . No move me nt = no i nt e r f e r e nc e wi t h e qui pme nt • Mus s e l s c a n l i ve f or e xt r e me l y l ong pe r i ods i n t he pr e s e nc e of a noxi a ( no oxyge n) . – 35 da ys a t 10 oC ! ! ! ! ! Mussel s: Over comi ng adver si t y • Mus s e l s a r e we l l a da pt e d t o t he pr obl e ms f a c e i n l ow a nd hi gh t i de s : – Fi l t e r f e e di ng or ga ni s ms t ha t us e de l i c a t e gi l l s t o t r a p nut r i e nt s i nc l udi ng oxyge n – I n t he s ubme r ge d s t a t e , oxyge n mus t be di s s ol ve d i n wa t e r i n or de r t o be t a ke n up by gi l l s – Low t i de mus s e l s wi l l c l a m up a nd e mpl oy s t r a t e gi e s t o s ur vi ve hypoxi a ( l ow oxyge n l e ve l s ) Over comi ng Chal l enges i n Oxygen Fl uct uat i ons • Low t i de pos e s s pe c i a l pr obl e ms f or or ga ni s ms . • Gi l l r e s pi r i ng a ni ma l s ha ve di f f i c ul t y e xt r a c t i ng oxyge n f r om a i r • For t hos e l uc ky e nough t o ha ve wa t e r , t i da l pool s a r e wa r me r – oxyge n s a t ur a t i on i n wa t e r i s muc h l owe r , c a r bon d i oxi de di s s ol ve s r e a di l y i n t o t he pool a nd oxyge n e s c a pe s i t . - c o- i nha bi t a nt s c ompe t e f or a va i l a bl e oxyge n How do Mussel go wi t hout ? How do t hey sur vi ve t he dr y spel l ? Wi t h l e s s oxyge n, t he r e c ome s a poi nt whe r e t he c os t of r e s pi r a t i on e xc e e ds t he be ne f i t of e xt r a c t i ng oxyge n. The t ur ni ng poi nt i s c a l l e d t he Pc r i t – t he c r i t i c a l oxyge n pr e s s ur e The Pc r i t r e pr e s e nt s t he poi nt whe r e e nvi r ome nt a l oxyge n i s t oo l ow f or a n or ga ni s m t o ma i nt a i n c ur r e nt oxyge n me t a bol i s m Whe n f a c e d wi t h oxyge n s hor t a ge , a n or ga ni s m ha s a c hoi c e : - To obt a i n e ne r gy f r om ot he r s our c e s - To de pr e s s me t a bol i s m - To do bot h of t he a bove I nvest i gat i ng Hypoxi a Tol er ance • The pur pos e of t h i s e xpe r i me nt i s t o f ur t he r knowl e dge a bout t he me c ha ni s ms mus s e l s us e t o s ur vi ve hypoxi c c ondi t i ons NORMOXI A= nor ma l oxyge n l e ve l s HYPOXI A = l owe r t ha n nor ma l oxyge n l e ve l s ANOXI A = no oxyge n pr e s e nt Met hods • 35 mus s e l s we r e ha r ve s t e d i n Oc t obe r 15 t h, 2007 t o a s s e s s t he Pc r i t f r om mi d- t i da l z one s a t Loc a r no Be a c h • 40 we r e c ol l e c t e d Fe b 1 s t , 2008 f or t he s e c ond pha s e of t he e xpe r i me nt • Mus s e l i de nt i f i e d ba s e d on pr e vi ous mol e c ul a r ge ne t i c s t udy of p53 ge ne ( Mut t r a y a nd Cox, 2005) Met hods cont i nued • Mus s e l s we r e t r a ns por t e d ba c k t o t he Ri c ha r ds La b a t UBC: – Ba r na c l e s r e move d a nd s he l l s c l e a ne d f r om de br i s – Pl a c e d i n a l a r ge a qua r i um a nd a l l owe d t o a c c l i ma t e f or a mont h a t 12 oC a nd 30ppt s a l i ni t y . – 15 mus s e l s f r om t he f i r s t ba t c h us e d t o t e s t s e t up a nd 16 we r e us e d t o a s s e s s t he Pc r i t . Met hods cont i nued • Mus s e l s we r e t r a ns por t e d ba c k t o t he Ri c ha r ds La b a t UBC: – Ba r na c l e s r e move d a nd s he l l s c l e a ne d f r om de br i s – Pl a c e d i n a l a r ge a qua r i um a nd a l l owe d t o a c c l i ma t e f or a mont h a t 12 oC a nd 30ppt s a l i ni t y . – 15 mus s e l s f r om t he f i r s t ba t c h us e d t o t e s t s e t up a nd 16 we r e us e d t o a s s e s s t he Pc r i t . Measur i ng Oxygen Consumpt i on • Mus s e l s we r e p l a c e d i n r e s pi r ome t e r a nd a l l owe d a 24h r e c ove r y t o de - s t r e s s pr i or t o c ol l e c t i ng da t a • Re s pi r ome t e r wa s c onne c t e d t o a Foxy Pr obe – t r a ns mi t s b l ue LED l i ght a t ~470nm a nd que nc hi ng of s i gna l i s r e a d by a s pe c t r ome t e r , USB2000- FL Fl uor e s c e nc e Spe c t r ome t e r , • Us i ng OOI Se ns or s Sof t wa r e oxyge n c ons umpt i on wa s a s s e s s e d us i ng t he St e r n- Vol me r e qua t i on. • Mus s e l s we r e a l l owe d a 24h r e c ove r y t o de - s t r e s s pr i or t o c ol l e c t i ng da t a Appar at us Set up Resul t s on Oxygen Consumpt i on Time Chart 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 0.00 50.00 100.00 150.00 200.00 250.00 300.00 Time (min) W at er P O 2 (t or r) • 16 mus s e l s we r e t e s t e d i n a c l os e d r e s pi r ome t e r a nd a l l owe d t o c ons ume t he l i mi t e d a va i l a bl e oxyge n unt i l t he y c e a s e d t o r e s pi r e • The a bove gr a ph s hows t he c ons umpt i on of oxyge n whe n a mus s e l i s pl a c e d i n a s e a l e d r e s pi r ome t e r . •For r e f e r e nc e , 125 t or r ~ 16% O2 , whi c h i s t he a mount of oxyge n i n a i r . Conver t i ng Oxygen Consumpt i on Rat e t o cal cul at e Pcr i t Time Chart 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.00 20.00 40.00 60.00 80.00 100.00 120.00 Water PO2 (torr) M O 2 um ol /g /h r • The a bove gr a ph s hows t he mol e c ul a r oxyge n c ons umpt i on r a t e c a l c ul a t e d f r om t he pr e vi ous s l i de . • Cons umpt i on of Mol e c ul a r Oxyge n Cons umpt i on Ra t e r e l a t i ve t o Oxyge n Pr e s s ur e yi e l ds t he Pc r i t us i ng r e gr e s s i on l i ne s . Conver t i ng Oxygen Consumpt i on Rat e t o cal cul at e Pcr i t • Us i ng r e gr e s s i on l i ne s t o de t e r mi ne t he c ons umpt i on of Mol e c ul a r Oxyge n Cons umpt i on Ra t e r e l a t i ve t o oxyge n pr e s s ur e . Resul t s of Pcr i t Assessment s • Pc r i t i s t he t hr e s hol d pr e s s ur e of oxyge n i n whi c h t he mus s e l c a nnot s us t a i n us ua l me t a bol i c c ons umpt i on r a t e of oxyge n • Fr om da t a r e s ul t s , i t wa s a s s e s s e d t ha t t he mus s e l s ha d a me a n c r i t i c a l oxyge n t e ns i on of 20. 94± 2. 09 t or r ~ 2 . 7% a t mos phe r i c oxyge n What does t hi s mean? What happens af t er Pcr i t Phase I I of Mussel Hypoxi a St udy • Now t ha t we ha ve da t a on t he t r a ns i t i on poi n t i n t he oxyge n me t a bol i s m a nd we know wha t t he i r t hr e s hol d i s . • Ve r y l i t t l e i s known a bout t he e me r ge nc y me c ha ni s ms t ha t mus s e l s e mpl oy whe n f a c e d wi t h a n oxyge n c r i s e s • St udi e s ha ve f oc us e d on l ong t e r m e f f e c t whi c h s how t ha t Mus s e l s wi l l r e duc e me t a bol i c a c t i v i t y – r e qui r i ng f e we r r e s our c e s a nd pr oduc i ng l e s s t oxi c bypr oduc t s The Emer gency Response • The s e c ond por t i on i s t o a s s e s s me t a bol i c ma r ke r s me a s ur e ATP, Phos phoa r gi ni ne , Oc t opi ne ( t he i nve r t e br a t e e qui va l e nt of l a c t a t e ) . • Thi s wi l l s how whe t he r t he mus s e l ’ s pr i ma r y s ur vi va l me c ha ni s m i s t o i mme di a t e l y l owe r me t a bol i s m or s wi t c h t o a l t e r na t e pa t hwa ys t o ge t t he i r e ne r gy • For e xa mpl e , i f ATP l e ve l s a r e t he s a me be f or e a nd a f t e r Pc r i t , t he mus c l e ha s not l owe r e d me t a bol i c a c t i v i t y . The Emer gency Response • At t h i s poi nt 40 mus s e l s ha ve be e n a c c l i ma t i ng: – 10 wi l l be us e d a t nor moxi a - c ont r ol – 10 wi l l be s ubj e c t e d a t 20 t or r – 10 wi l l be s ubj e c t e d t o 12 t or r ( 2 s t a nda r d de vi a t i ons be l ow me a n) – 10 s ubj e c t e d t o c ompl e t e a noxi a • St udy i s ongoi ng t o a c qui r e da t a t o de t e r mi ne ATP, Phos phoa r gi ni ne , Oc t opi ne i n Mus s e l Ti s s ue s • Da t a wi l l a l l ow f ur t he r or unde r s t a ndi ng i n t o how t he Myt i l us t r os s ul us ’ s us e s e me r ge nc y me t a bol i c c opi ng me c ha ni s ms t o hypoxi a s t r e s s e s Acknowl edgment • I woul d l i ke t o t ha nk t he a udi e nc e f or t he i r a t t e nt i on • The Ri c ha r ds La bs f or us e of t he i r f a c i l i t i e s • Dr . J e f f Ri c ha r ds PhD f or hi s pa t i e nc e a nd gui da nc e . I ma ge s of mus s e l s a r e a l t e r e d pi c t ur e s . Or i gi na l s we r e obt a i ne d f r om t he i nde x f ound on ht t p: / / www. wa l l a wa l l a . e du/ a c a de mi c s / de pa r t me nt s / bi ol ogy/ r os a r i o/ i nve r t s . D. Cowl e s ( 2002) I ma ge f or OOSe ns or Foxy pr obe c a n be f ound a t : ht t p: / / www. i ns t r ume nt r e gi s t e r . c om/ 2006/ 09/ oxi me t e r - bl ood- i nf r a r e d. ht ml . ( 2006)
UBC Undergraduate Research
Assessing hypoxia tolerance in the Mytilus trossulus Mader, Heidi 2008-03-08
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