The Auk 109(4):758-770, 1992 CHITIN DIGESTION AND ASSIMILATION BY SEABIRDS SUE JACKSON?4 ALLEN R. PLACE?AND LINDSAYJ. SEIDERER3's •PercyFitzPatrick Institute of AfricanOrnithology, University of CapeTown, PrivateBag, Rondebosch 7700, South Africa; 2Universityof Maryland,Center of MarineBiotechnology, 600 East Lombard St., Baltimore,Maryland 21202,USA; and 3ZoologyDepartment, University of CapeTown, Private Bag, Rondebosch 7700, South Africa ABSTRACT.--ASa structural componentof crustaceanexoskeletons, chitin is the most im- portantcarbohydrate in the dietsof many marine carnivores.To investigatethe physiological and biochemicaladaptations that may enable seabirdsto break down this "prey defense," we estimatedchitin digestibilitiesfor Sooty Albatrosses(Phoebetria fusca), White-chinned Petrels (Procellariaaequinoctialis), Rockhopper Penguins (Eudypteschrysocome), Gentoo Pen- guins (Pygoscelispapua), King Penguins(Aptenodytes patagonicus) and Leach'sStorm-Petrels (Oceanodromaleucorhoa) fed Antarctickrill (Euphausiasuperba). These species retain a substantial proportion (46.5 + $D of 13.1%,39.1 + 4.9%, 52.8 + 37.6%,45.3 + 5.6%, 84.8 + 11.7%and 35 + 12.2%, respectively) of ingested chitin. We also obtained preliminary estimatesof chitinolytic activity in the gastricmucosae of the abovesix speciesby incubating extractsof tissuesamples with a chitin substrateand measuring the production of the end product of chitin hydrolysis,N-acetyl-D-glucosamine (NAG). Chitinolytic activity (up to 5,000•tg NAG h • g • expressedper gram tissue)was measuredfrom proventriculartissue and within the activity range (1,350 to 61,650•tg NAG h • g-l) reported for eight other avian species.In order to assessthe energeticand nutritional benefitsof chitinolytic activity in seabirds,we studied gastrointestinalabsorption of the end productsof chitinolysisin Leach'sStorm- Petrels.The overall absorptionefficiency of NAG and its deacetylatedprecursor, glucosamine (Gin), in this specieswas 44.0 + 3.0% and 11.0 + 1.9%,respectively. These absorptioneffi- ciencieswere significantlyless than for glucose,which was absorbedwith an efficiencyof 90.6 + 2.5%. No absorptionof NAG and Gin occurred in the proventriculus. Overall, we showed that seabirdshave a capacityto assimilatea considerableportion of the carbon and nitrogen present as chitin in the exoskeletonof their prey, but we have not demonstrated that assimilationactually occurs. The potentialcosts and benefitsof chitin hydrolysis,as well as the absorption of the breakdown products, need to be assessed.Received 24 May 1991, accepted18 December1991. THE MUCOPOLYSACCHARIDEpolymer chitin crustaceans.Although chitin digestion and as- ([ 1/•4-2]-acetamido-2-deoxy-/•-D-glucan) in similation in marine fish are well studied (e.g. crustaceanexoskeletons represents a substantial Fange et al. 1976, Lindsay et al. 1984, Lindsay sourceof potential energy and carbohydratefor and Gooday1985), little attentionhas been paid marine predators (Anderson et al. 1978, Reh- to utilization of dietary chitin by seabirdswhose bein et al. 1986). Estimates of krill biomass, for daily mass-specificenergy demandsfar exceed example,range from 80 to 500 million metric those of marine fish. tons (Sahrage and Steinberg 1975), the equiv- Seabirds such as those that feed on crusta- alent of approximately!.6 to 10 million tons of ceans in the Southern Ocean would benefit chitin, and 0.24 to 3 million tons of other car- greatly from an ability to penetratethe "struc- bohydrates(Clarke 1980). Hence, chitin is the tural defenses"of their prey, and to utilize the mostimportant carbohydratein the dietsof ma- energy residing in chitinous exoskeletons.By rine predatorsof krill, and probably of other studying specieswith a range of natural diets (Table 1), we testedthe predictionthat seabirds known to habitually eat crustaceansalso secrete 4 Departmentof Physiology,UCLA Schoolof Med- chitinolytic enzymes,which enhancetheir abil- icine, Center for the Health Sciences, 10833 Le Conte ity to break down and assimilate this carbo- Ave., Los Angeles, California 90024, USA. hydrate.Although a studyof microbialand ver- 5 Marine EcologicalSurveys Ltd., P.O. Box 6, Fav- tebrate chitin degradation by crabeater seals ersham,Kent, ME13 AAA, United Kingdom. (Lobodoncarcinophagus) and Ad•lie Penguins 758 October1992] ChitinDigestion by Seabirds 759 T^I•LE1. Predominantprey types of seabirdsstu•tied (less-frequently-eaten prey in parentheses). Species Predominantprey Reference Leach's Storm-Petrel (Oceanodroma Fish (crustacea) Linton (1978) leucorhoa) White-chinned Petrel (Procellariaae- Fish (squid, crustacea) Jackson(1988), A. Berruti (unpubl. quinoctialis) data) SootyAlbatross (Phoebetria fusca) Squid (fish) Cooper and Klages(unpubl. data) RockhopperPenguin (Eudypteschry- Crustacea(fish) Brown and Klages(1987) SO½ODle) Gentoo Penguin (Pygoscelispapua) Fish (crustacea) Adams and Wilson (1987) King Penguin (Aptenodytespatagoni- Fish (squid) Adamsand Klages(1987), Croxall and Prince (1980) (Pygoscelisadeliae) is currentlyunder way (Stal- radation route, involving the breakdown of chi- ey 1986), we are not aware of published data tin into glucosamineby specific deacetylases other than our own (for a preliminary report, (Reyeset al. 1986),has not been shown to occur see Place 1990, Place and Jackson 1991) on the in vertebrates.There are four recognizedroles levels of chitinolytic activity in seabirdguts. for chitinasesin nature, namely morphogenic, Most forms of chitin are insoluble in many nutritional, digestive and defensive (Gooday solvents, highly hydrophobic, and relatively 1990). In this paper, we use a variety of tech- inert to biodegradation(Pangburn et al. 1984, niques to assesswhich of these roles chitinase Gooday 1990). With only one exception,the plays in seabirddigestion. •-chitin of diatoms, chitin is always found We report the resultsof chitinolytic assayson crosslinked or associated with other structural stomachcontents and tissuesamples from five components. In insectsand other invertebrates, speciesof seabirdsoccurring in the Southern chitin is associatedwith specific proteins Ocean,and one speciesthat breedson the North through covalent and noncovalentbonding, Atlantic coast. We examine the nutritional im- producing an ordered structure(Blackwell and portanceof chitin using two approaches,the Weih 1984).Chitin often exhibitsvarying de- first of which is a balancestudy estimatingap- grees of mineralization, in particular, calcifi- parent digestibilitiesof chitin in three penguin cation or sclerotizationinvolving interactions speciesand three procellariiform speciesfound with phenolic and lipid molecules(Peter et al. to have gastric chitinase activity. Second, we 1986,Poulicek et al. 1986).In nature, varying demonstratethe capacityof a procellariiform to degreesof deacetylationof the polymer result absorb the hydrolytic products of chitin, in a structuralcontinuum between chitin (fully N-acetyl-D-glucosamineand glucosamine,and acetylated) and chitosan (fully deacetylated; comparethis capacityto that for absorptionof Aruchami et al. 1986, Datema et al. 1977, Davis glucose,a structurally similar monosaccharide and Bartnicki-Garcia 1984, Gowri et al. 1986). that is probablythe secondmost important car- Once consideredthe action of a nonspecific bohydrate in seabirddiets after chitin itself. In enzymefor the catabolismof chitin,chitinolysis addition, we investigated some in vitro condi- is now known to involve a complexgroup of tions (i.e. pH and Ca concentrations)that may enzymeswith discretephylogenies and subtle regulate chitinolytic activity and comparedthe differencesin specificity,role, and biochemical effects with in vivo conditions found in seabird properties.Chitin degradationrequires the ac- stomachs. tion of at least two enzymes, chitinase (E.C. 3.2.1.14; poly-•-l,4-[2-acetamido-2-deoxy]-D- METHODS glucoside glycanohydrolyase)and chitobiase (E.C. 3.2.1.30; •-N-acetyl-D-glucosaminidase; We purchasedthe following chemicalsfrom Sigma Chemical(St. Louis,Missouri): chitin; chitosan;D-(+ )- Jeuniaux1961). Chitinase hydrolyzes chitin to glucosamine hydrochloride; N-acetyl-D-glucos- yield trimers and dimers (chitotriose and chi- amine;N,N'-diacetylchitobiose; D-glucose; polyeth- tobiose)of N-acetyl-D-glucosamine(NAG), and ylene glycol 4000;Serratia marcescens chitinase; Helix chitobiasehydrolyzes these trimers and dimers pomatiafl-glucuronidase; and almond fl-glucosidase. to the monomerof NAG. An alternativedeg- Aceticanhydride was purchasedfrom Aldrich Chem- 760 JACKSON,PLACE, AND SEIDERER [Auk,VoL 109 ical (Milwaukee, Wisconsin). All other chemicals were weight) olive oil. Nightly mealsof 5 to 8 cc (approx- reagent grade unlessspecified otherwise. All solvents imately 88 to 141 kJ/meal) were warmed to 37øCand were either pesticideor FIPLC grade.Whatman 3-MM delivered to the stomachvia the esophaguswith a chromatographicfilter paper wasobtained from VWR disposable5-ml syringe attachedto a 10-cmlength of Scientific(Philadelphia, Pennsylvania). polyethylene tubing. All chicks took the feeding Radiolabelsand fiuors.--We used acetic anhydride without regurgitation, and their daily wing-chord [•FI] (50 mCi/mmol), D-['4C(U)] glucose(4.28 mCi/ growth indicated normal development.After inges- retool), acetyl-N-[glucosamine-l,6-3H(N)]-D-glucos- tion, each chick was placed on a polyethylene mesh amine (44.2 mCi/mmol), D-[1,6-3H(N)] glucosamine (6-ram mesh size) platform suspendedin a 9-L Baine hydrochloride(24 mCi / retool), and [1,2-•4C]polyeth- Marie polyethylenecontainer