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Assessing hypoxia tolerance in the Mytilus trossulus Mader, Heidi 2008

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Who are the Mytilus trossulus?The M. trossulus are common marine musselsFound along rocky shorelines of North America in the intertidal zone and span from high tidal zone to lowest tidal point.Colonize in clumps on rocksAre a foundation species  - a dominant species in a habitat that are abundant and have major influence on the ecosystemAre also Indicator species – as they represent the overall health of an ecosystem and are influenced by environmental conditions:  disease outbreak, pollution, species competition or climate change.Who are the Mytilus trossulusPhylum Mollusca Class Bivalvia Subclass Pteriomorphia Order Mytiloida Family Mytilidae Genus Mytilus Species trossulus Mussels: An ideal model to study oxygen metabolismReside in intertidal zone – subjected daily to extreme fluctuations in oxygen levelsSessile organisms -  easier to minimize stress on animals resulting from captivity and testing procedures.  No movement = no interference with equipmentMussels can live for extremely long periods in the presence of anoxia ( no oxygen). 35 days at 10oC !!!!!Mussels: Overcoming adversityMussels are well adapted to the problems face in low and high tides:Filter feeding organisms that use delicate gills to trap nutrients including oxygenIn the submerged state, oxygen must be dissolved in water in order to be taken up by gillsLow tide mussels will clam up and employ strategies to survive hypoxia ( low oxygen levels)Overcoming Challenges in Oxygen FluctuationsLow tide poses special problems for organisms.  Gill respiring animals have difficulty extracting oxygen from airFor those lucky enough to have water, tidal pools are warmer – oxygen saturation in water is much lower, carbon dioxide dissolves readily into the pool and oxygen escapes it.- co-inhabitants compete for available oxygenHow do Mussel go without?How do they survive the dry spell?With less oxygen, there comes a point where the cost of respiration exceeds the benefit of extracting oxygen.The turning point is called the Pcrit – the critical oxygen pressureThe Pcrit represents the point where enviromental oxygen is too low for an organism to maintain current oxygen metabolismWhen faced with oxygen shortage, an organism has a choice:	- To obtain energy from other sources	- To depress metabolism	- To do both of the aboveInvestigating Hypoxia ToleranceThe purpose of this experiment is to further knowledge about the mechanisms mussels use to survive hypoxic conditions NORMOXIA= normal oxygen levelsHYPOXIA = lower than normal oxygen levelsANOXIA = no oxygen presentMethods35 mussels were harvested in October 15th, 2007 to assess the Pcrit from mid-tidal zones at Locarno Beach 40 were collected Feb 1st, 2008 for the second phase of the experimentMussel identified based on previous molecular genetic study of p53 gene (Muttray and Cox, 2005)  Methods continuedMussels were transported back to the Richards Lab at UBC:Barnacles removed and shells cleaned from debrisPlaced in a large aquarium and allowed to acclimate for a month at 12oC and 30ppt salinity.15 mussels from the first batch used to test setup and 16 were used to assess the Pcrit.Methods continuedMussels were transported back to the Richards Lab at UBC:Barnacles removed and shells cleaned from debrisPlaced in a large aquarium and allowed to acclimate for a month at 12oC and 30ppt salinity.15 mussels from the first batch used to test setup and 16 were used to assess the Pcrit.Measuring Oxygen Consumption Mussels were placed in respirometer and allowed a 24h recovery to de-stress prior to collecting dataRespirometer was connected to a Foxy Probe – transmits blue LED light at ~470nm and quenching of signal is read by a spectrometer, USB2000-FL Fluorescence Spectrometer, Using OOISensors Software oxygen consumption was assessed using the Stern-Volmer equation.Mussels were allowed a 24h recovery to de-stress prior to collecting dataApparatus SetupResults on Oxygen Consumption16 mussels were tested in a closed respirometer and allowed to consume the limited available oxygen until they ceased to respireThe above graph shows the consumption of oxygen when a mussel is placed in a sealed respirometer.For reference, 125 torr ~ 16% O2 , which is the amount of oxygen in air.Converting Oxygen Consumption Rate to calculate PcritThe above graph shows the molecular oxygen consumption rate  calculated from the previous slide .Consumption of Molecular Oxygen Consumption Rate relative to Oxygen Pressure yields the Pcrit using regression lines.Converting Oxygen Consumption Rate to calculate PcritUsing regression lines to determine the consumption of Molecular Oxygen Consumption Rate relative to oxygen pressure.Results of Pcrit AssessmentsPcrit is the threshold pressure of oxygen in which the mussel cannot sustain usual metabolic consumption rate of oxygenFrom data results, it was assessed that the mussels had a mean critical oxygen tension of 20.94± 2.09 torr ~ 2.7% atmospheric oxygenWhat does this mean? What happens after PcritPhase II of Mussel Hypoxia StudyNow that we have data on the transition point in the oxygen metabolism and we know what their threshold is.Very little is known about the emergency mechanisms that mussels employ when faced with an oxygen crisesStudies have focused on long term effect which show that Mussels will reduce metabolic activity – requiring fewer resources and producing less toxic byproducts The Emergency ResponseThe second portion is to assess metabolic markers measure ATP, Phosphoarginine, Octopine ( the invertebrate equivalent of lactate).This will show whether the mussel’s primary survival mechanism is to immediately lower metabolism or switch to alternate pathways to get their energyFor example, if ATP levels are the same before and after Pcrit, the muscle has not lowered metabolic activity. The Emergency ResponseAt this point 40 mussels have been acclimating:10 will be used at normoxia - control10 will be subjected at 20 torr10 will be subjected to 12 torr (2 standard deviations below mean)10 subjected to complete anoxiaStudy is ongoing to acquire data to determine ATP, Phosphoarginine, Octopine in Mussel TissuesData will allow further or understanding into how the Mytilus trossulus’s uses emergency metabolic coping mechanisms to hypoxia stressesAcknowledgmentI would like to thank the audience for their attentionThe Richards Labs for use of their facilitiesDr. Jeff Richards PhD for his patience and guidance.Images of mussels are altered pictures.  Originals were obtained from  the index found on http://www.wallawalla.edu/academics/departments/biology/rosario/inverts. D. Cowles (2002)Image for OOSensor Foxy probe can be found at:	http://www.instrumentregister.com/2006/09/oximeter-blood-infrared.html . (2006)  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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