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Assessing hypoxia tolerance in the Mytilus trossulus Mader, Heidi 2008-03-08

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Who ar e t he My t i l us t r os s ul us ? • Th e M. t r o s s u l u s a r e c o mmo n ma r i n e mu s s e l s • Fo u n d a l o n g r o c k y s h o r e l i n e s o f No r t h Ame r i c a i n t h e i n t e r t i d a l z o n e a n d s p a n f r o m h i g h t i d a l z o n e t o l o we s t t i da l poi nt . • Co l o n i z e i n c l u mp s o n r o c k s • Ar e a f o u n d a t i o n s p e c i e s - a d o mi n a n t 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 o r i n f l u e n c e o n t h e e c o s y s t e m  • Ar e a l s o I n d i c a t o r s p e 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 n f l u e n c e d b y e n v i r o n me n t a l c o n d i t i o n s :  Who ar e t he My t i l us t r os s ul us Ph y l u m Mo l l u s c a Cl a s s Bi v a l v i a Su b c l a s s Pt e r i o mo r p h i a Or d e r My t i l o i d a Fa mi l y My t i l i d a e Ge n u s My t i l u s Sp e c i e s t r o s s u l u s  Mus s el s : An i deal model t o s t udy oxygen met abol i sm • Re s i d e i n i n t e r t i d a l z o n e – 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 o r g a n i s ms -  eas i er 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  • Mu s s e l s c a n l i v e f o r e x t 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) . –  o  Mussel s: Over comi ng adver si t y • Mu s s e l s a r e we l l a d a p t e d t o t h e p r o b l e ms f a c e i n l o w a n d h i g h t i de s : – Fi l t e r f e e d i n g o r g a n i s ms t ha t us e de l i c a t e g i l l s t o t r a p n ut r i e nt s i nc l udi ng oxyge n – I n t h e s u b me r g e d s t a t e , oxyge n mu s t b e d i s s o l v e d i n wa t e r i n or d e r t o b e t a k e n u p b y gi l l s – Lo w t i d e mu s s e l s wi l l c l a m up a nd e mp l o y s t r a t e g i e s t o s u r vi ve  Ov er c omi ng Chal l enges i n Oxygen Fl uct uat i ons • Lo w t i d e p o s e s s p e c i a l p r o b l e ms f o r o r g a n i s ms . • Gi l l r e s p i r i n g a n i ma l s h a v e di f f i c ul t y e xt r a c t i ng oxyge n f r om ai r • Fo r t h o s e l u c k y e n o u g h t o h a v e wa t e r , t i d a l p o o l s a r e wa r me r – oxyge n s a t ur a t i on i n l o we r , c a r b o n d i o x i d e r e a di l y i nt o t he pool e s c a pe s i t . - c o - i n h a b i t a n t s c o mp e t  wa t e r i s mu c h di s s ol ve s a nd oxyge n e f or a va i l a bl e  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 o x y g e n, t he r e c ome s a poi nt whe r e t h e c o s t o f r e s pi r a t i on e xc e e ds t he b e n e f i t o f e x t r a c t i ng oxyge n. Th e t ur n i n g p o i 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 o x y ge n pr e s s ur e Th e Pc r i t r e p r e s e nt s t he poi nt whe r e e n v i r o me n t a l oxyge n i s t oo l ow f or a n o r g a n i s m t o ma i nt a i n c ur r e nt oxyge n me t a b o l i s m Wh e 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 o b t a i n e ne r gy f r om ot he r s our c e s - To d e p r e s s me t a bol i s m - To d o b o t h of t he a bove  I nvest i gat i ng Hypoxi a Tol er ance • Th e p u r p o s e o f t h i s e x p e r i me n t i s t o f u r t h e r k n o wl e d g e a b o u t t h e me c h a n i s ms mu s s e l s u s e t o s ur vi ve hypoxi c c ondi t i ons NORMOXI A= n o r ma l o x y g e n l e ve l s HYPOXI A = l o we r t h a n n o r ma l oxyge n l e ve l s ANOXI A = n o o x y g e n p r e s e n t  Met hods • 3 5 mu s s e l s we r e h a r v e s t e d i n Oc t o b e r 1 5 t h , 2 0 0 7 t o a s s e s s t h e Pc r i t f r o m mi d - t i d a l z o n e s a t Lo c a r n o Be a c h •  4 0 we r e c o l l e c t e d Fe b 1 s t , 2 0 0 8 f o r t h e s e c o n d p h a s e o f t h e e x p e r i me n t  • Mu s s e l i d e n t i f i e d b a s e d o n p r e v i o u s mo l e c u l a r g e n e t i c s t u d y o f p 5 3 g e n e ( Mu t t r a y a n d Co x , 2 0 0 5 )  Met hods c ont i nued • Mu s s e l s we r e t r a n s p o r t e d b a c k t o t h e Ri c h a r d s La b a t UBC: – Ba r n a c l e s r e mo v e d a n d s h e 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 g e a q u a r i um a nd a l l o we d t o a c c l i ma t e f o r a mont h a t 1 2 o C a n d 3 0 p p t s a l i n i t y. – 15 mu s s e l s f r o m t h e f i r s t ba t c h us e d t o t e s t s e t u p a n d 1 6 we r e us e d t o a s s e s s t h e Pc r i t .  Met hods c ont i nued • Mu s s e l s we r e t r a n s p o r t e d b a c k t o t h e Ri c h a r d s La b a t UBC: – Ba r n a c l e s r e mo v e d a n d s h e 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 g e a q u a r i um a nd a l l o we d t o a c c l i ma t e f o r a mont h a t 1 2 o C a n d 3 0 p p t s a l i n i t y. – 15 mu s s e l s f r o m t h e f i r s t ba t c h us e d t o t e s t s e t u p a n d 1 6 we r e us e d t o a s s e s s t h e Pc r i t .  Meas ur i ng Ox y gen Cons umpt i on • Mu s s e l s we r e p l a c e d i n r e s p i r o me t e r a n d a l l o we d a 2 4 h r e c o v e r y t o d e s t r e s s pr i or t o c ol l e c t i ng da t a • Re s p i r o me t e r wa s c o n n e c t e d t o a Fo x y Pr o b e – t r a n s mi t s b l u e LED l i g h t a t ~4 7 0 n m a n d q u e n c h i n g o f s i g n a l i s r e a d b y a s p e c t r o me t e r , USB2 0 0 0 - FL Fl u o r e s c e n c e Sp e c t r o me t e r , • Us i n g OOI Se n s o r s So f t wa r e o x y g e n c o n s u mp t i o n wa s a s s e s s e d u s i n g t h e St e r n - Vo l me r e q u a t i o n . • Mu s s e l s we r e a l l o we d a 2 4 h r e c o v e 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  Water PO2 (torr)  140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00 0.00  50.00  100.00  150.00  200.00  250.00  300.00  Time (min)  • 16 mu s s e l s we r e t e s t e d a l l o we d t o c o n s u me t h e l t he y c e a s e d t o r e s p i r e • The a b o v e g r a p h s h o ws t a mus s e l i s p l a c e d i n a •For r e f e r e n c e , 1 2 5 t o r r  i n a c l o s e d r e s pi r ome t e r a nd i mi t e d a v a i l a b l e oxyge n unt i l h e c o n s u mp t i o n of oxyge n whe n s e a l e d r e s p i r o me t e r . ~ 1 6 % O2 , wh i c h i s t he a mount  Conv er t i ng Oxygen Consumpt i on Rat e t o cal cul at e Pcr i t Time Chart 0.02 0.01  umol/g/hr  MO2  0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00  20.00  40.00  60.00  80.00  100.00  120.00  Water PO2 (torr)  • The a bove gr a p h s h o ws t h e mo l e c u l a r o x y g e n c ons umpt i on r a t e c a l c u l a t e d f r o m t h e p r e v i o u s s l i de . • Cons umpt i on of Mo l e c u l a r Ox y g e n Co n s u mp t i o n Ra t e r e l a t i ve t o Ox y g e n Pr e s s u r e y i e l d s t h e Pc r i t us i ng r e g r e s s i o n l i n e s .  Conv er t i ng Oxygen Consumpt i on Rat e t o cal cul at e Pcr i t  • Us i n g r e g r e s s i o n l i n e s t o d e t e r mi n e t h e c o n s u mp t i o n o f Mo l e c u l a r Ox y g e n Co n s u mp t i o n Ra t e r e l a t i v e t o o x y g e n pr e s s ur e .  Res ul t s of Pc r i t As s es s ment s • Pc r i t i s t h e t h r e s h o l d p r e s s u r e o f o x y g e n i n wh i c h t h e mu s s e l c a n n o t s u s t a i n u s u a l me t a b o l i c c o n s u mp t i o n r a t e o f o x y g e n • Fr o m d a t a r e s u l t s , i t wa s a s s e s s e d t h a t t h e mu s s e l s h a d a me a n c r i t i c a l o x y g e n t e n s i o n o f 20. 94± 2. 09 t or r ~ 2. 7% a t mo s p h e r i c o x y g e n  What does t hi s mean? What happens af t er Pcr i t  Phase I I of Mussel Hypoxi a St udy • No w t h a t we h a v e d a t a o n t h e t r a ns i t i on poi nt i n t he oxyge n me t a b o l i s m a n d we k n o w wh a t t h e i r t hr e s hol d i s . • Ve r y l i t t l e i s k n o wn a b o u t t h e e me r g e n c y me c h a n i s ms t h a t mu s s e l s e mp l o y wh e n f a c e d wi t h a n o x y g e n cr i s es • St u d i e s h a v e f o c u s e d o n l o n g t e r m e f f e c t wh i c h s h o w t h a t Mu s s e l s wi l l r e d u c e me t a b o l i c a c t i v i t y – r e q u i r i n g f e we r r e s o u r c e s a n d pr oduc i ng l e s s t oxi c bypr oduc t s  The Emer genc y Res pons e • Th e s e c o n d p o r t i o n i s t o a s s e s s me t a b o l i c ma r k e r s me a s u r e ATP, Ph o s p h o a r g i n i n e , Oc t o p i n e ( t h e i nve r t e br a t e e qui va l e nt of l a c t a t e ) . • Th i s wi l l s h o w wh e t h e r t h e mu s s e l ’ s p r i ma r y s u r v i v a l me c h a n i s m i s t o i mme d i a t e l y l o we r me t a b o l i s m o r s wi t c h t o a l t e r n a t e p a t h wa y s t o g e t t h e i r e ne r gy • Fo r e x a mp l e , i f ATP l e v e l s a r e t h e s a me b e f o r e a n d a f t e r Pc r i t , t h e mu s c l e h a s n o t l o we r e d me t a b o l i c  The Emer genc y Res pons e • At t h i s p o i n t 4 0 mu s s e l s h a v e b e e n a c c l i ma t i n g : – 1 0 wi l l be us e d a t nor moxi a - c o n t r o l – 1 0 wi l l be s ubj e c t e d a t 20 t or r – 1 0 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 v i a t i o n s b e l o w me a n )  – 1 0 s ubj e c t e d t o c ompl e t e a noxi a  • St u d y i s o n g o i n g t o a c q u i r e d a t a t o d e t e r mi n e ATP, Ph o s p h o a r g i n i n e , Oc t o p i n e i n Mu s s e l Ti s s u e s • Da t a wi l l a l l o w f u r t h e r o r u n d e r s t a n d i n g i n t o h o w t h e My t i l u s t r o s s u l u s ’ s u s e s e me r g e n c y me t a b o l i c c o p i n g me c h a n i s ms t o h y p o x i a s t r e s s e s  Ac k nowl edgment • I wo u l d l i k e t o t h a n k a udi e nc e f or t he i r a t t • Th e Ri c h a r d s La b s f o r t he i r f a c i l i t i e s • Dr . J e f f Ri c h a r d s Ph D pa t i e nc e a nd gui da nc e .  t he e nt i on us e of f or hi s  I ma ge s of mu s s e l s a r e a l t e r e d p i c t u r e s . Or i g i n a l s we r e o b t 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 d e mi c s / d e p a r t me n t s / b i o l o g y / r os a r i o/ i nve r t s . D. Cowl e s ( 2 0 0 2 )  I ma ge f or OOSe n s o r Fo x y p r o b e c a n b e f o u n d a t :  

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