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Assessing hypoxia tolerance in the Mytilus trossulus Mader, Heidi Mar 8, 2008

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 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) 

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