And the Atlantic Puffin (Fratercula Arctica)
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ENERGY METABOLISM IN THE LOCOMOTOR MUSCLES OF THE COMMON MURRE (URIA AALGE) AND THE ATLANTIC PUFFIN (FRATERCULA ARCTICA) M. BENJAMIN DAVIS AND HELGA GUDERLEY D•partementde biologie,Universit• Laval, Quebec, Quebec GIK 7P4, Canada ABSTRACT.--Tocompare the metabolicsystems that supportthe combinationof flying and diving with thoseused to supportburst flying and sustainedflying, myoglobinconcentrations and maximum enzyme activitieswere determined for selectedenzymes of glycolysis,the Krebs cycle, and amino acid metabolism in the pectoral, supracoracoideus,and sartorius musclesof the Common Murre (Uria aalge),Atlantic Puffin (Fraterculaarctica), Rock Dove (Columbalivia; hereafter "pigeon"), and Ring-neckedPheasant (Phasianus colchicus). Glycolytic enzyme levels in the flight muscleswere lower in the murre and the puffin than in the pheasant,while both glycolyticand Krebs-cycleenzyme levels resembledthose in the pigeon. We believe puffinsand murresdo not rely extensivelyon anaerobicglycolysis during diving. In concordancewith a role in oxygenstorage for diving, the levels of myoglobin in the flight musclesof murres and puffins were higher than those in pigeons or pheasants.They were lower than publishedvalues for penguins,however. In contrastto the trends for pigeon and pheasant muscles,the alcid sartorius muscleshad a considerably lower aerobic orientation than the flight muscles.Received 21 November1986, accepted 9 June1987. IN birds a high anaerobiccapacity, as indi- dives (Scholander 1940), voluntary dives in catedby typesand levels of glycolyticenzymes, ponds are aerobic(Butler and Woakes 1984) and is correlated positively with locomotory pat- free-ranging dives are generally short (re- ternsdemanding burst flying, extensivediving, viewed by Baldwin et al. 1984).The studiesthat or quick maneuvering (Wilson et al. 1963,Kies- established the correlation between diving sling 1977,Mill and Baldwin 1983,Baldwin et depth and glycolyticcapacity for penguins(Mill al. 1984).During theseactivities oxygen tension and Baldwin 1983, Baldwin et al. 1984), how- in musclemay be reducedto the point where ever, did not directly compareenzyme activities ATP is produced from substrate-level phos- in penguinswith thosein terrestrial birds with phorylations coupled to lactateproduction. An- varying locomotorstrategies. The need for such aerobicglycolysis seems the primary sourceof an integrated approach is underlined by the ATP during flight for burst fliers like the Ring- data from mammals. Although early studies necked Pheasant (Phasianuscolchicus; Wilson et suggestedthat diving mammalshad exception- al. 1963, Kiessling 1977). Similarly, the take-off, al glycolytic capacities(Simon et al. 1974), an hovering,and quick directionalchanges of Rock extensive comparison of pyruvate kinase and Doves (Columbalivia; hereafter "pigeon") are lactate dehydrogenaselevels in terrestrial and thought to be anaerobically supported (Pen- marine mammals showed no differences (Cas- nycuick 1968, Parker and George 1975). The tellini et al. 1981).In addition, becauseenzyme functionalutility of the high glycolyticcapacity activities are markedly affected by assaycon- of diving birds is less clear. Anaerobic glycol- ditions,simultaneous determination of enzyme ysiscould fully supportdiving, in analogywith activities in the different speciesis a prerequi- pheasant flight, or it could permit rapid direc- site for rigorous comparison. tional changesduring diving, in analogy with Severalalcid speciesare the ecologicalcoun- pigeonflight. Deeper-divingbirds also may have terparts of penguins in the Northern Hemi- a greater glycolytic capacityrelative to shallow- sphere. Two of these, the Atlantic Puffin (Fra- diving birds as a backup system proportional tercula arctica) and the Common Murre (Uria to the depths to which they dive. While most aalge),dive to depths of 60 m and 180 m, re- penguin specieshave anaerobiccapacities that spectively (Piatt and Nettleship 1985), using are proportional to their diving depths (Bald- their wings and legs for propulsion and steer- win et al. 1984) and show marked bradycardia ing (Tuck 1961,Spring 1971).Besides being pro- and lactateproduction during forcedlaboratory ficient divers, the alcids migrate over long dis- 733 The Auk 104: 733-739. October 1987 734 DAVISAND GUDERLEY [Auk,Vol. 104 tancesfrom overwintering areas at sea to land shooting as they surfaced (Environment Canada/Ca- for the breeding season.Just as their morphol- nadian Wildlife Permit ASK 18-84 and with the per- ogy representsa compromise between the op- mission of the Newfoundland Wildlife Division). The posingrequirements of diving and flying, their carcasseswere placed in -2øC seawaterduring trans- port back to the wharf (maximum 2 h). There tissues metabolicorganization must be compatiblewith were dissected,wrapped in aluminum foil, and fro- both. For alcids flying and diving are closely zen in 2-methylbutane (EastmanKodak, Rochester, associatedactivities. During the breeding sea- New York) previously chilled in a liquid nitrogen son, foraging dives are sandwiched between bath. Samples were stored at -25øC until assayed. flights between the nest and feeding areas(Har- Five pheasantsand 5 pigeonswere obtainedlive from ris and Hislop 1978). commercialsuppliers, decapitated,and dissectedim- We examined the metabolic organization of mediately. Tissueswere stored as above. the locomotormuscles in puffins and tourresto Enzymeassays.--Muscle samples were freed of con- elucidate the metabolic strategiesthat support nectivetissue and fat, weighed, mincedwith a scalpel, the combinationof diving and flying. To com- and homogenizedwith a Polytron (Brinkman Instru- ments) for 30 s in 10 ml/g ice-cold extraction buffer. pare the metaboliccapacities of thesealcids with For all enzymes except NADP+-linked isocitratede- those of birds whose metabolic strategies are hydrogenase,the extraction buffer was 50 mM im- reasonablywell defined, we studied the Ring- idazole-HC1, 50 mM KC1, 1 mM EDTA, 2 mM 2-mer- necked Pheasant, a burst flyer (Wilson et al. captoethanol(omitted for citrate synthaseassay), pH 1963, Kiessling 1977), and the pigeon, a bird 7.4. For NADP+-linked isocitratedehydrogenase, the capable of sustainedflight (Pennycuick 1968, extraction buffer was 50 mM Pipes [piperazine-NN'- Parker and George 1975).Our specificaims were bis(2-ethanesuphonicacid)], 10 mM MgCI2, 5 mM to compare the metabolic systemsthat support 2-mercaptoethanol,1 mM EDTA, 5 mM MnC12,2 mM extensiveflying and diving with those used to ADP, 5 M glycerol, 0.1 mM phenylmethylsulphonyl support burst or sustainedflying, to examine fluoride, pH 7.4. The homogenatewas centrifugedat whether glycolyticcapacity was proportional to 25,000 x g for 40 min at 4øC(600 x g for 3 min for NADP + isocitratedehydrogenase) in a Sorvall SS-34 diving depth in these alcids, and to compare rotor. The supernatantwas used immediately for the the metabolic organization of the major flight assays.All biochemicalswere obtained from Sigma muscles(pectoral and supracoracoideus)and an ChemicalCo. (St. Louis, Missouri).Enzymes were as- important leg muscle (sartorius) becauseboth sayed with a UV/Vis recording spectrophotometer wings and legs are used in underwater pro- (Varian Cary 210).The cuvettetemperature was main- pulsion. tained at 38 +_ 0.5øCthroughout the assaysusing a Puffinsand tourresare logisticallydifficult to circulatingwater bath.Determinations of activitywere obtain, making determination of in situ meta- done in triplicate. Enzyme activities are expressedas bolic flux impossible.Instead, we measuredthe •tmol of substrate transformed.min •.g • wet mass maximal activitiesof glycolyticand Krebs-cycle of tissue.Enzymes were assayedby following changes in absorbanceat 340 nm, exceptcitrate synthase, which enzymesto assessanaerobic and aerobicpoten- wasassayed at 412 nm. Preliminary experimentswere tial; this has been done in previous studies,in- run to determine the substrate concentrations re- cluding those on penguins (Crabtree and quired to give maximal activities. All pH values were Newsholme 1972,Kiessling 1977, Castellini and adjustedat 38øC.The compositionof the reactionmix- Somero 1981, Mill and Baldwin 1983, Baldwin tures is given below. et al. 1984). We measured myoglobin concen- Aldolase(ALD) (E.C. 4.1.2.13): 0.2 mM NADH, 1.5 trations as an indication of the oxygen stores mM fructose-l,6-bisphosphate, 50 mM triethanol- availableto the muscles(Weber et al. 1974,Pages amine/HC1, excess glycerol-3-phosphatedehydro- and Planas1983) and the levels of enzymesthat genase,triose phosphateisomerase, pH 7.4. Fructose- 1,6-bisphosphatewas omitted for the control.Pyruvate contributeintermediates to the Krebscycle. Be- kinase(PK) (E.C. 2.7.1.40):100 mM KC1,10 mM MgC12, cause we worked with frozen tissues, we mea- 0.2 mM NADH, 5 mM ADP, 5 mM phosphoenolpy- sured only enzymes that are stable to freezing ruvate, 50 mM triethanolamine/HC1, excess lactate (Farrar and Farrar 1983). dehydrogenase,pH 7.4. Phosphoenolpyruvatewas omitted for the control. Lactatedehydrogenase (LDH) MATERIALS AND METHODS (E.C. 1.1.1.27):0.2 mM NADH, 1.5 mM pyruvate for pheasantand pigeon muscleand 2 mM pyruvatefor Experimentalanimals.--Five Common Murres and 5 murre and puffin muscle, 50 mM triethanolamine/ Atlantic Puffins were collectedin early July 1984 at HC1, pH 7.4. Pyruvate was omitted for the control. Witless Bay, Newfoundland (47ø15'N, 52ø40'W) by Citratesynthase (CS) (E.C. 4.1.3.7): 50 mM triethanol- October1987] MetabolicAdaptations toDiving