Digestive enzyme activities and gastrointestinal fermentation in wood-eating catwshes

Transcription

1 J Comp Physiol B (29) 179: DOI 1.17/s z ORIGINAL PAPER Digestive enzyme ctivities nd gstrointestinl fermenttion in wood-eting ctwshes Donovn P. Germn Roslie A. Bittong Received: 27 Mrch 29 / Revised: 11 June 29 / Accepted: 16 June 29 / Pulished online: 1 July 29 The Author(s) 29. This rticle is pulished with open ccess t Springerlink.com Astrct To determine wht cpilities wood-eting nd detritivorous ctwshes hve for the digestion of refrctory polyscchrides with the id of n endosymiotic microil community, the ph, redox potentils, concentrtions of short-chin ftty cids (SCFAs), nd the ctivity levels of 14 digestive enzymes were mesured long the gstrointestinl (GI) trcts of three wood-eting tx (Pnque cf. nigrolinetus Mrñon, Pnque nocturnus, nd Hypostomus pyrineusi) nd one detritivorous species (Pterygoplichthys disjunctivus) from the fmily Loricriide. Negtive redox potentils ( 6 mv) were oserved in the intestinl Xuids of the Wsh, suggesting tht fermenttive digestion ws possile. However, SCFA concentrtions were low (<3 mm in ny intestinl region), indicting tht little GI fermenttion occurs in the Wshes GI trcts. Cellulse nd xylnse ctivities were low (<.3 U g 1 ), nd generlly decresed distlly in the intestine, wheres mylolytic nd lminrinse ctivities were Wve nd two orders of mgnitude greter, respectively, thn cellulse nd xylnse ctivities, suggesting tht the Wsh more redily digest solule polyscchrides. Furthermore, the Michelis Menten constnts (K m ) of the Wshes Communicted y I. D. Hume. Electronic supplementry mteril The online version of this rticle (doi:1.17/s z) contins supplementry mteril, which is ville to uthorized users. D. P. Germn R. A. Bittong Deprtment of Zoology, University of Florid, Ginesville, FL, USA Present Address: D. P. Germn (&) Deprtment of Ecology nd Evolutionry Biology, University of Cliforni, Irvine, CA 92697, USA e-mil: dgermn@uci.edu β-glucosidse nd N-cetyl-β-D-glucosminidse enzymes were signiwcntly lower thn the K m vlues of microil enzymes ingested with their food, further suggesting tht the Wsh eyciently digest solule components of their detritl diet rther thn refrctory polyscchrides. Coupled with rpid gut trnsit nd poor cellulose digestiility, the wood-eting ctwshes pper to e detritivores relint on endogenous digestive mechnisms, s re other loricriid ctwshes. This stnds in contrst to truly xylivorous tx (e.g., evers, termites), which re relint on n endosymiotic community of microorgnisms to digest refrctory polyscchrides. Keywords Introduction Digestive enzymes Xylivory Fermenttion The consumption of wood for food is rre mong nimls. Unlike the greener portions of plnts, woody tissues re mde of cells tht re ded t functionl mturity nd, hence, lck the cell contents on which mny herivorous nimls thrive. Becuse wood is composed lmost entirely of structurl polyscchrides (e.g., lignocellulose), it is considered to e nutrient poor (Krsov nd Mrtínez del Rio 27). Thus, mny wood-eting, or xylivorous, nimls (e.g., lower termites, evers) require the id of symiotic microorgnisms in their gstrointestinl (GI) trcts to digest cellulose nd mke the energy in this compound ville to the host (Prins nd Kreulen 1991; Vispo nd Hume 1995). Indeed, xylivorous nimls possess n expnded hindgut or cecum in which microes reside nd produce cellulolytic enzymes to id in the digestion of woody mteril (Prins nd Kreulen 1991; Vispo nd Hume 1995; Mo et l. 24). Becuse the conditions in this expnded hindgut re typiclly neroic, microil endosymionts

2 126 J Comp Physiol B (29) 179: operte under fermenttive pthwys, reducing glucose (nd other monomers) to yproducts clled short-chin ftty cids (SCFAs; e.g., cette), which re then sored y the host niml nd used to generte ATP (Bergmn 199; Krsov nd Mrtínez del Rio 27). In 1993, Schefer nd Stewrt descried severl new species s prt of linege of neotropicl ctwshes, genus Pnque, which my possily e xylivorous. The enlrged teeth these nimls use to scrpe wood from the surfce of fllen trees in the river, nd the presence of wood s the only mcroscopic mteril in the Wshes GI trcts intrigued the uthors (Schefer nd Stewrt 1993). Furthermore, xylivory evolved twice in loricriid ctwshes, s clde in the genus Hypostomus (Armruster 23) is recognized s wood-eting in ddition to the Pnque (Fig. 1). Both xylivorous cldes re derived within the phylogeny, ut little is known of the digestive physiology of these Wshes, nd whether they cn digest cellulose from wood. The xylivorous ctwshes elong to the Loricriide, diverse ctwsh fmily (68 descried species in 8 gener) endemic to the neotropics (Armruster 24). The diets of reltively few species of loricriids re known (Delriv nd Agostinho 21; Pouilly et l. 23; de Melo et l. 24; Novkowski et l. 28; Germn 29) nd pper to include niml, plnt, nd detritl mteril from the enthos. Loricriids re known to consume morphic (e.g., wood) nd morphic (i.e., unidentiwle colloidl mteril) detritus (Germn 29). It is cler, however, tht these Wshes hve undergone evolutionry rerrngements of jw structure, llowing for diversity in feeding modes nd trophic speciliztion (Schefer nd Luder 1986; Lujn 29). Furthermore, loricriids hve long, thin-wlled intestines (Delriv nd Agostinho 21; Germn 29), which suggests tht they hve high levels of intke of lowqulity food (Sily nd Clow 1986; Horn nd Messer 1992; Krsov nd Mrtínez del Rio 27), such s detritus (Arujo-Lim et l. 1986). High intke equtes to rpid gut trnsit nd little endosymiotic fermenttion (Stevens nd Hume 1998; Crossmn et l. 25; Krsov nd Mrtínez del Rio 27; Germn 29). Nelson et l. (1999) exmined digestive enzyme ctivities nd cultured microes from the GI trcts of Pnque mccus, nd n undescried species of Pterygoplichthys (formerly Liposrcus; Armruster 24), oth of which they otined vi the qurium trde. Nelson et l. were le to isolte eroic microes with cellulolytic cpilities from the guts of the two species nd mesured cellulse ctivities in the Wshes GI trcts. From these results, Nelson et l. (1999) concluded tht loricriids possess n endosymiotic community in their guts cple of digesting cellulose under eroic conditions. Conversely, Germn (29) showed tht Pnque nigrolinetus nd Pterygoplichthys disjunctivus pssed wood through their guts in less thn 4 h, could not ssimilte signiwcnt mounts of cellulose from wood nd, hence, did not thrive on woody diet in the lortory. Wht is clerly needed is n nlysis of digestive trct function to etter understnd the digestive strtegy of the wood-eting ctwshes. Do these ctwsh GI trcts function more like those of other xylivorous nimls (Breznk nd Brune 1994; Vispo nd Hume 1995; Felicetti et l. 2), with some mechnism for slowing the Xow of digest nd llowing microes to ferment refrctory Fig. 1 Prtil phylogenetic hypothesis for three tries in the ctwsh fmily Loricriide (Armruster 24). Phylogeny sed on prsimony nlysis of 214 morphologicl chrcters. See Armruster (24) for sttisticl support. Gener in old include wood-eting species, nd the sterisks () indicte gener from which species were investigted in this study. Numers in prentheses indicte pproximte numer of tx not shown Hypostomus cochliodon group (7) Hypostomus (2) Hemincistrus (2) Pterygoplichthys (14) Pnque (smll; 5) Pnque (lrge; 3) Pekolti (2) Hypncistrus/Prncistrus (2) Six gener (7) Hemincistrus/Pekolti (3) Chetostom nd others (3) Hemincistrus sp. (1) Hypostomini Pterygoplichthini Ancistrini

3 J Comp Physiol B (29) 179: polyscchrides (Clements nd Ruenheimer 26; Krsov nd Mrtínez del Rio 27), or re their guts more similr to those of other detritivorous Wshes (Horn nd Messer 1992; Crossmn et l. 25; Germn 29), with rpid gut trnsit, relince on endogenous digestive mechnisms nd, hence, little digestion of refrctory polyscchrides? These divergent digestive strtegies not only feture diverences in digest trnsit rte nd gut morphology, ut lso involve completely diverent prowles of digestive enzyme ctivities nd SCFA concentrtions long the gut (Horn nd Messer 1992; Jumrs 2; Crossmn et l. 25; Ske et l. 25; Ske et l. 27; Germn 29). For exmple, n niml relint on hindgut fermenttion would e expected to hve high concentrtions (>2 mm; Chot nd Clements 1998) of SCFAs in its hindgut (Vispo nd Hume 1995; Mountfort et l. 22; Crossmn et l. 25; Pryor nd Bjorndl 25) nd high ctivities of microilly produced digestive enzymes in this gut region (e.g., cellulse, Potts nd Hewitt 1973; Nkshim et l. 22; Mo et l. 24). On the other hnd, detritivorous Wshes relint on endogenous digestion show decreses in digestive enzyme ctivities distlly in their intestines, low SCFA concentrtions, nd no pttern of SCFA concentrtions long their GI trcts (Smith et l. 1996; Crossmn et l. 25; Germn 29). In this study, we exmined digestive enzyme ctivities, luminl crohydrte prowles, nd gstrointestinl fermenttion in xylivorous nd detrivorous loricriid ctwshes to determine if these nimls were cple of digesting diet rich in refrctory polyscchrides, nd whether they were relint on n endosymiotic community to do so. Fishes were collected from their ntive hitt in the Río Mrñon in northern Perú, where xylivorous ctwshes re most diverse nd undnt (Schefer nd Stewrt 1993). In ll, we collected two species from the genus Pnque (P. nocturnus Schefer nd Stewrt 1993, nd n undescried species tht we re clling P. cf. nigrolinetus Mrñon ; J. Armruster, pers. comm.), representing the two cldes of this genus, nd one species of Hypostomus (H. pyrineusi Mirnd-Rieiro 192) representing the other clde of xylivorous ctwshes (Fig. 1). All of these tx re symptric in the Río Mrñon. Additionlly, we mde use of n introduced popultion of detritivorous loricriid, Pterygoplichthys disjunctivus (Weer 1991), which hs een living in Florid for nerly two decdes (Nico 25; Nico et l. 29). This study, therefore, included oth cldes of xylivorous ctwshes nd less-derived detritivore from the sme fmily (Fig. 1). Thus, we were le to exmine the digestive physiology of closely relted Wshes with diverent diets, nd those tht converged independently on woody diet. This study hd four min components. First, we mesured the ph nd redox conditions long the GI trcts of the Wsh to determine whether ny portion of the gut would e hospitle to n neroic popultion of endosymiotic microorgnisms, or whether the Wshes guts were eroic, s proposed y Nelson et l. (1999). Second, luminl crohydrte prowles nd SCFA concentrtions were mesured long the GI trct to determine where nutrients were eing hydrolyzed nd sored, nd where microes might e most concentrted in the GI trct. If the Wsh were relint on endosymiont fermenttion to gin energy from cellulose nd other refrctory polyscchrides, we would expect SCFA concentrtions to e highest in the hindgut region. Third, we mesured the iochemicl ctivity levels of 14 digestive enzymes cting in the gut lumen or long the rush order of the intestine tht rexect the ility of the Wsh to hydrolyze sustrtes commonly encountered in wood, lge, nd detritus (Tle 1). Following the methodology of Ske et l. (25), we mesured enzyme ctivities reltive to loction long the gut nd determined whether the sources of these enzyme ctivities were endogenous (host-produced) or exogenous (produced y microorgnisms). This ws done y collecting three frctions from the gut sections: gut wll tissue (endogenous), gut Xuid (enzymes secreted either y the Wsh or microorgnisms), nd microil extrct (exogenous). If, similr to other xylivorous nimls, the ctwshes were relying on endosymionts in their hindgut to digest cellulose, we would expect refrctory polyscchride-degrding enzyme ctivities (e.g., cellulse) to e highest in the microil extrct of the hindgut region of the GI trct (Tle 1). Digestive enzymes of endogenous origin (i.e., those produced y the Wsh, such s mylse, trypsin, nd lipse) would e expected to show pttern of decresing ctivity towrd the hindgut (Germn 29). And fourth, we mesured the Michelis Menten (K m ) constnts of discchridses (mltse, β-glucosidse, nd N-cetyl-β-D-glucosminidse) produced y the Wsh (i.e., in gut wll tissue) nd y microes (i.e., in microil extrct) to determine if the Wsh were more eycient in digesting nd ssimilting discchrides found in detritus thn were microes ingested with detritus. Mterils nd methods Fish collection Ten dult individuls ech of Pnque cf. nigrolinetus Mrñon nd P. nocturnus, nd Wve dult individuls of Hypostomus pyrineusi were cptured y seine nd ckpck electroshocker from the upper Río Mrñon in northern Peru ( S, W) in August 26. Fourteen individuls of Pterygoplichthys disjunctivus were cptured y hnd while snorkeling from the Wekiv Springs complex in north centrl Florid ( N, W) in Mrch 26. Upon cpture, Wshes were

4 128 J Comp Physiol B (29) 179: Tle 1 Digestive enzymes ssyed in this study of digestive physiology in loricriid ctwshes Enzyme Loction Sustrte Dietry source Frctions ssyed Expected pttern c >Frction d Amylolytic Lum., cont. Strch, α-glucns Alge, detritus Fluid, contents Decrese Fluid Lminrinse Lum., cont. Lminrin Ditoms Fluid, contents Decrese Fluid Cellulse Lum., cont. Cellulose Wood, lge, detritus Fluid, contents Increse Contents Xylnse Lum., cont. Xyln Wood, detritus Fluid, contents Increse Contents Mnnnse Lum., cont. Mnnn Wood, detritus Fluid, contents Increse Contents Chitinse Lum., cont. Chitin Fungi, insects, detritus Fluid, contents Decrese Fluid Trypsin Lum., cont. Protein Alge, detritus, nimls Fluid, contents Decrese Fluid Lipse Lum., cont. Lipid Alge, detritus, nimls Fluid, contents Decrese Fluid Mltse BB, cont. Mltose Alge, detritus Contents, gut wll Decrese Gut wll β-glucosidse BB, cont. β-glucosides Alge, wood, detritus Contents, gut wll Increse Contents β-xylosidse BB, cont. β-xylosides Wood, detritus Contents, gut wll Increse Contents β-mnnosidse BB, cont. β-mnnosides Wood, detritus Contents, gut wll Increse Contents N-cetyl-β-D-glucos e BB, cont. N-cetyl-β-D-glucom Fungi, insects, detritus Contents, gut wll Decrese Gut wll Aminopeptidse BB, cont. Dipeptides Alge, detritus, nimls Contents, gut wll Decrese Gut wll Lum lumen of the intestine, cont. contents (ingest) of the intestine, BB rush order of the intestine Indictes where the enzyme is ctive The portions of gut content or intestinl tissue in which the ctivity of the enzyme ws ssyed c This column shows the expected ptterns of ctivity long the GI trcts of the Wshes, if they re relint upon endosymiotic communities of microorgnisms in their hindguts to digest refrctory polyscchrides. For exmple, increse mens tht the ctivity of this enzyme should increse towrd the distl intestine of the Wsh d Predictions of which ssyed frctions will hve higher ctivity of prticulr enzyme. For exmple, Xuid mens tht the ctivity of tht enzyme is expected to e greter in the intestinl Xuid thn in the intestinl contents of given gut region e Complete nme of the enzyme is N-cetyl-β-D-glucosminidse, nd the sustrte is N-cetyl-β-D-glucominides plced in coolers of erted river wter nd held until euthnized (up to 2 h). Fishes were euthnized in uvered wter contining 1 g l 1 tricine methnesulfonte (MS-222, Argent Chemicls Lortory, Inc., Redmond, WA, USA), mesured [stndrd length (SL) 1 mm], nd dissected on chilled (»4 C) cutting ord. Guts were removed y cutting t the esophgus nd t the nus nd processed in mnner pproprite for speciwc nlyses. Gut ph nd redox mesurements Upon dissection, the complete digestive trcts of four individuls ech of P. cf. n. Mrñon, P. nocturnus, nd Pt. disjunctivus were plced on sterilized, stinless-steel dissection try t mient temperture (22 25 C) nd gently uncoiled without tering or stretching. The ph nd redox conditions of the digestive trcts were mesured following Clements et l. (1994) with clirted ph nd redox microelectrodes (models PHR-146S nd ORP-146, respectively; Lzr Lortories Inc., Los Angeles, CA, USA) connected to portle ph-redox meter (model 61A, Jenco Inc., Sn Diego, CA, USA). Incisions lrge enough to llow penetrtion of the microelectrode tip (».25 mm) into the gut Xuid were mde in the stomch nd intestinl wll, nd the ph nd redox conditions were mesured immeditely fter ech incision ws mde. Overll, ph nd redox conditions were mesured in Wve sections of the stomch nd ten sections ech of the proximl, mid-, nd distl intestine of ech individul Wsh. The men ph nd redox conditions were then determined for ech region of the digestive trct in n individul Wsh, nd men vlues determined for ech gut region for ech species. The ph nd redox conditions were not mesured in the intestines of H. pyrineusi ecuse this species ws not s undnt s the other tx nd, thus, we did not cpture enough individuls for ll of the nlyses. Tissue preprtion for digestive enzyme nlyses For Wshes designted for digestive enzyme nlyses, guts were dissected out, plced on sterilized, chilled (»4 C) cutting ord, nd uncoiled. The stomchs were excised, nd the intestines divided into three sections of equl length representing the proximl, mid-, nd distl intestine. The gut contents were gently squeezed from ech of the three intestinl regions with forceps nd the lunt side of rzor lde into sterile centrifuge vils. These vils (with their contents) were then centrifuged t 1, g for 5 min (Ske et l. 25) in n Eppendorf 5415R desktop centrifuge powered y 12 V cr ttery vi power inverter. Following centrifugtion, the superntnts (heretofore clled intestinl Xuid ) were gently pipetted into seprte sterile centrifuge vils, nd the pelleted gut

5 J Comp Physiol B (29) 179: contents nd intestinl Xuid were frozen in liquid nitrogen. Gut wll sections were collected from ech intestinl region of ech specimen y excising n pproximtely 3 mm piece ech of the proximl, mid-, nd distl intestine. These intestinl pieces were then cut longitudinlly, rinsed with ice-cold.5 M Tris HCl uver, ph 7.5, to remove ny trce of intestinl contents, plced in sterile centrifuge vils, nd frozen in liquid nitrogen. All of the smples were then trnsported on dry ice ck to the University of Florid where they were stored t 8 C until nlyzed. The intestinl Xuids nd pelleted gut contents were homogenized on ice following Ske et l. (25). Intestinl Xuids were defrosted, diluted 5 1 volumes in.5 M Tris HCl, ph 7.5, nd gently homogenized using Polytron homogenizer (Brinkmnn Instruments, Westury, NY) with 7-mm genertor t setting of 1,1 rpm for 3 s. The intestinl Xuid smples were then stored t 8 C in smll liquots (1 2 μl) until use. To ensure the rupture of microil cells nd the complete relese of enzymes from the gut contents, the pelleted gut contents were defrosted, diluted 3 5 volumes in.5 M Tris HCl, ph 7.5, sonicted t 5 W output for 3 2 s, with 4-s intervls etween pulses, nd homogenized with the Polytron homogenizer t 3, rpm for 3 3 s. The homogenized pelleted gut contents were then centrifuged t 12, g for 1 min t 4 C, nd the resulting superntnt designted microil extrct. Gut wll smples were homogenized ccording to Germn et l. (24). Gut wll sections were defrosted, diluted in 5 1 volumes of.3 M mnnitol in.1 M Hepes/ NOH (Mrtínez Del Rio et l. 1995; Levey et l. 1999), ph 7., homogenized with the Polytron homogenizer t 3, rpm for 3 3 s, nd centrifuged t 9,4 g for 2 min t 4 C. Following centrifugtion, the superntnts from the pelleted gut contents (microil extrct) nd the gut wll sections were collected nd stored in smll liquots (1 2 μl) t 8 C until just efore use in spectrophotometric ssys of ctivities of digestive enzymes. The protein content of the homogentes ws mesured using icinchoninic cid (Smith et l. 1985), s detiled y Germn (29). Liver nd heptopncres tissues were lso prepred for enzymtic nlyses s descried y Germn (28). All ssys of digestive enzyme ctivity were crried out t 25 C, consistent with the mesured tempertures (24 26 C) of the Río Mrñon, in triplicte using the BioRd Benchmrk Plus microplte spectrophotomer nd Flcon Xt-ottom 96-well micropltes (Fisher ScientiWc). All ph vlues listed for uvers were mesured t room temperture (22 C), nd ll regents were purchsed from Sigm-Aldrich Chemicl (St. Louis). All rections were run t sturting sustrte concentrtions s determined for ech enzyme with gut tissues from the four species. Ech enzyme ctivity (Tle 1) ws mesured in ech gut region of ech individul Wsh, nd lnks consisting of sustrte only nd homogente only (in uver) were conducted simultneously to ccount for endogenous sustrte nd/or product in the tissue homogentes nd sustrte solutions (Ske et l. 25; Germn et l. 29). Assys of polyscchride degrding enzymes Polyscchridse ctivities (i.e., ctivities ginst strch, lminrin, cellulose, mnnn, nd xyln) were mesured in the intestinl Xuid nd microil extrcts ccording to the Somogyi Nelson method (Nelson 1944; Somogyi 1952). Polyscchride sustrte ws dissolved [strch (2%), lminrin (.5%), croxymethyl cellulose (.5%), or mnnn (.5%)] or suspended (xyln,.5%) in.8 M sodium citrte uver, ph 7.5, contining.1% sodium zide. In microcentrifuge vil, 5 μl of polyscchride solution ws comined with 5 μl of mixture of sodium citrte uver nd intestinl Xuid, tissue, or microil extrct homogente. Homogente volumes rnged from 1 to 3 μl, depending on the enzyme concentrtion in the homogentes. The incution period vried with sustrte: the ssys were crried out for 1 min for strch, 2 h for lminrin, ech in wter th, nd 24 h for ech of croxymethyl cellulose, mnnn, nd xyln, under constnt shking on rotry shker in n incutor. The 24-h incutions lso included 1 μl of protese inhiitor (Sigm P834) to prevent the degrdtion of polyscchride degrding enzymes y proteses during the ssy period. The incutions were stopped y dding 2 μl of 1 M NOH nd 2 μl of Somogyi Nelson regent A. Somogyi Nelson regent B ws dded fter the ssy solution ws oiled for 1 min (see Germn et l. 24 for regent recipes). The resulting solution ws diluted in wter nd centrifuged t 6, g for 5 min. The reducing sugr content of the solution ws then determined spectrophotometriclly t 65 nm, nd polyscchridse ctivity ws determined from stndrd curve constructed with the respective monomer (i.e., glucose for strch, lminrin, nd croxymethyl cellulose; mnnose for mnnn; nd xylose for xyln). Enzyme ctivities re expressed in U (1 μmol reducing sugr lierted per minute) per grm wet weight of Xuid, tissue, or content. Chitinse ctivities were mesured following Germn et l. (29), ut no ctivity ws detected in the four species used in this study. In ll ssys, the ckground levels of N-cetyl-glucosmine detected in the lnks (>1 mm) mtched wht ws mesurle in the ssy mixtures, mking ctivity determintions impossile. However, the mesurle N-cetyl-glucosmine in the gut in ddition to mesurle N-cetyl-glucosminidse ctivities mkes it likely tht the Wsh cn utilize chitin s nutrient source.

6 13 J Comp Physiol B (29) 179: Assys of discchridses Mltse ctivity ws mesured in gut wll tissues nd pelleted gut contents following Dhlqvist (1968) s descried y Germn (29). In microcentrifuge tue, 1 μl of 56 mm mltose dissolved in 1 mm mlete uver, ph 7., ws comined with 1 μl of regionl gut wll or microil extrct homogente. After 1 min, the rection ws stopped y the ddition of 3 μl of ssy regent (Sigm GAGO2) dissolved in 1 M Tris HCl, ph 7.. The rection mixture ws incuted for 3 min t 37 C nd ws stopped y the ddition of 3 μl of 12 N H 2 SO 4. The mount of glucose in the solution ws then determined spectrophotometriclly t 54 nm. The mltse ctivity ws determined from glucose stndrd curve nd expressed in U (1 μmol glucose lierted per minute) per grm wet weight of gut tissue or pelleted contents. The Michelis Menten constnt (K m ) for mltse ws determined for gut wll nd microil extrct smples with sustrte concentrtions rnging from.56 to 112 mm. Tris is known to e n inhiitor of mltse ctivity (Dhlqvist 1968), ut in higher concentrtions (e.g., 1 M; Levey et l. 1999) thn those used in our homogente uver (.5 M). Nevertheless, to conwrm tht the diverent uvers used for the gut wll (Hepes mnnitol) nd microil extrct (Tris HCl) homogentes did not directly Vect the K m or ctivity for mltse, the gut wlls nd pelleted gut contents of the proximl intestine of Wve dditionl Pt. disjunctivus were homogenized in the opposite uvers: gut wlls in Tris HCl nd pelleted gut contents in Hepes mnnitol. For mltse, the diverent uvers did not produce diverent K m (Tris HCl: mm; Hepes mnnitol: mm; t =.1, P =.92, df = 1) or ctivity (Tris HCl: U g tissue 1 ; Hepes mnnitol: U g tissue 1 ; t =1.38, P =.2, df = 1) vlues in the microil extrct, or K m (Tris HCl: mm; Hepes mnnitol: mm; t =1.2, P =.26, df = 1) or ctivity (Tris HCl: U g tissue 1 ; Hepes mnnitol: U g tissue 1 ; t =.7, P =.5, df = 1) vlues in the gut wll homogentes. The low-concentrtion Tris HCl ws oserved to hve little evect on mltse ctivity in two previous investigtions (Germn et l. 24; Germn 29) in which the gut tissues were homogenized in.5 M Tris HCl uver. The diverent uvers lso did not Vect the K m nd ctivity levels of the other discchridses mesured in this study (see elow) nd, thus, we cn e conwdent tht ny diverences in K m nd enzyme ctivity mong the gut wll nd microil extrct homogentes re not due to the diverent uvers used in their homogeniztion. The ctivities of the discchridses β-glucosidse, β-mnnosidse, β-xylosidse, nd N-cetyl-β-D-glucosminidse (NAG) were mesured in gut wll tissues nd microil extrcts using p-nitrophenol conjugted sustrtes (Nelson et l. 1999; Xie et l. 27) dissolved in.1 M sodium citrte, ph 7.. In microplte well, 9 μl of 11.1 mm sustrte (1.33 mm for NAG) ws comined with 1 μl of gut wll or microil extrct homogente nd the rection ws red kineticlly t 45 nm for 15 min. The discchridse ctivities were determined from p-nitrophenol stndrd curve nd expressed in U (1 μmol p-nitrophenol lierted per minute) per grm wet weight of gut tissue or pelleted contents. The K m ws determined for gut wll nd microil extrct smples for β-glucosidse nd NAG. The sustrte concentrtions rnged from.1 to 12 mm for β-glucosidse nd mm for NAG. Assys of proteses nd lipse Trypsin ctivity ws ssyed in the intestinl Xuid nd microil extrct using modiwed version of the method designed y Erlnger et l. (1961), s descried y Gwlick et l. (2). The sustrte, 2 mm Nα-enzoyl-L-rginine-p-nitronilide hydrochloride (BAPNA), ws dissolved in 1 mm Tris HCl uver (ph 7.5) y heting to 95 C (Preiser et l. 1975; Germn et l. 24). In microplte, 95 μl of BAPNA ws comined with 5 μl of homogente, nd the increse in sornce ws red continuously t 41 nm for 15 min. Trypsin ws lso ssyed in the liver nd heptopncres, ut tissues homogentes from these orgns were Wrst incuted with enterokinse for 15 min to ctivte trypsinogen prior to comining the homogentes with sustrte (Germn et l. 24). Trypsin ctivity ws determined with p-nitroniline stndrd curve nd expressed in U (1 μmol p-nitroniline lierted per minute) per grm wet weight of tissue, gut Xuid, or microil extrct. Aminopeptidse ctivity ws mesured in gut wll tissues nd microil extrcts ccording to Roncri nd Zuer (1969), s descried y Germn et l. (24). In microplte, 9 μl of 2.4 mm L-lnine-p-nitronilide HCl dissolved in 2 mm sodium phosphte uver (ph 7.5) ws comined with 1 μl of homogente. The increse in sornce ws red continuously t 41 nm for 15 min nd ctivity determined with p-nitroniline stndrd curve. Aminopeptidse ctivity ws expressed in U (1 μmol p-nitroniline lierted per minute) per grm wet weight of gut tissue or pelleted gut contents. Lipse (nonspeciwc ile-slt ctivted E.C ) ctivities were ssyed in the intestinl Xuids nd microil extrcts using modiwed version of the method designed y Iijim et l. (1998). In microplte, 86 μl of 5.2 mm sodium cholte dissolved in 25 mm Tris HCl (ph 7.5) ws comined with 6 μl of homogente nd 2.5 μl of 1 mm 2-methoxyethnol nd incuted t room temperture for 15 min to llow for lipse ctivtion y ile slts.

7 J Comp Physiol B (29) 179: The sustrte p-nitrophenyl myristte (5.5 μl of 2 mm p-nitrophenyl myristte dissolved in 1% ethnol) ws then dded nd the increse in sornce ws red continuously t 45 nm for 15 min. Lipse ctivity ws determined with p-nitrophenol stndrd curve nd expressed in U (1 μmol p-nitrophenol lierted per minute) per grm wet weight of gut tissue. The ctivity of ech enzyme ws regressed ginst the protein content of the homogentes to conwrm tht there were no signiwcnt correltions etween the two vriles. Becuse no signiwcnt correltions were oserved, the dt re not reported s U per mg protein. Gut Xuid preprtion, gstrointestinl fermenttion, nd luminl crohydrte prowles Mesurements of symiotic fermenttion ctivity were sed on the methods of Pryor nd Bjorndl (25). Fermenttion ctivity ws indicted y reltive concentrtions of short-chin ftty cids (SCFA) in the Xuid contents of the guts of the Wshes t the time of deth. As homogentes were prepred from the intestinl Xuid smples (see Tissue preprtion for digestive enzyme nlyses ), 3 μl of undiluted intestinl Xuid ws pipetted into sterile centrifuge vil equipped with.22 μm cellulose cette Wlter (Costr Spin-X gmm sterilized centrifuge tue Wlters, Coming, NY) nd centrifuged under refrigertion t 13, g for 15 min to remove prticles from the Xuid (including cteril cells). The Wltrtes were collected nd frozen until they were nlyzed for SCFA nd nutrient concentrtions. Concentrtions of SCFA in the intestinl Xuid smples from ech gut region in ech species were mesured using gs chromtogrphy s descried y Pryor et l. (26) nd Germn et l. (29). Glucose concentrtions were nlyzed in 2 μl of gut Xuid using the sme glucose content ssy descried for the mltse ssy ove, the only deprture eing tht there ws no pre-incution with mltose. To exmine the presence of reducing sugrs of vrious sizes in the intestinl Xuids of the Wsh, 1 μl of Wltered intestinl Xuid ws spotted on to pre-coted silic gel pltes (Whtmn, PE SIL G) together with stndrds of glucose, mltose, nd tri- to pent-oligoscchrides of glucose. The thin lyer chromtogrm (TLC) ws developed with scending solvent (isopropnol/cetic cid/wter, 7:2:1 (v/v)) nd stined with thymol regent (Adchi 1965; Ske et l. 25). Sttisticl nlyses Prior to ll signiwcnce tests, Levene s test for equl vrince ws performed nd residul versus Wts plots were exmined to ensure the ppropriteness of the dt for prmetric nlyses. All tests were run using SPSS (version 11) nd Minit (version 12) sttisticl softwre pckges. Amylolytic, lminrinse, cellulse, nd xylnse ctivities were compred etween the intestinl Xuid nd microil extrct frctions of ech gut region in ech species with t test, using Bonferroni correction. IntrspeciWc comprisons of totl enzymtic ctivities (intestinl Xuid + microil extrct) nd totl SCFA concentrtions mong the gut regions of ech species were mde with ANOVA followed y Tukey s HSD with fmily error rte of P =.5. The numericl dt for the enzyme ctivities re presented seprtely (in Wgures) from the ctul sttisticl nd P vlues (in tles). The ctivities of mltse, β-glucosidse, N-cetyl-β-D-glucosminidse, β-mnnosidse, nd minopeptidse were compred etween the gut wll nd microil extrct frctions of ech gut region in ech species with t test, using Bonferroni correction. Similrly, the K m vlues of mltse, β-glucosidse, nd N-cetyl-β-D-glucosminidse from the proximl intestine of the Wsh were compred etween the gut wll nd microil extrct frctions of ech species with t test. Results Gut ph nd redox conditions The ph of the digestive trcts of P. cf. n. Mrñon, P. nocturnus, nd Pt. disjunctivus were ll neutrl, wheres the redox conditions of the stomch were positive (Pt. disjunctivus) or less negtive (P. cf. n. Mrñon nd P. nocturnus), nd the redox conditions of the intestines of ll three species were negtive (see Supplementl Tle S1 in online version). Thus, the guts of the three species were eroic or slightly neroic in the stomch region, nd dewnitively neroic long the intestine. Polyscchride degrding enzyme ctivities No diverences were oserved in mylolytic, lminrinse, or cellulse ctivities etween the intestinl Xuid nd the microil extrcts of ny species (See Supplementl Tle S2 in online version). However, xylnse ctivity ws signiwcntly greter in the microil extrcts of the proximl nd mid-intestine of P. nocturnus thn in the intestinl Xuids of these regions. Totl mylolytic ctivity ws signiwcntly greter in the proximl intestine thn in the distl intestine of ll four species (Tle 2; Fig. 2). Lminrinse ctivity ws signiwcntly higher in the proximl intestine of ll four species thn in their mid- or distl intestines (Tle 2; Fig. 2). No lminrinse ctivity ws detected in the distl intestines of P. nocturnus nd H. pyrineusi.

8 132 J Comp Physiol B (29) 179: Tle 2 Summry of ANOVA nd t test sttistics for intrspeciwc comprisons of digestive enzyme ctivities mong diverent regions of the intestine in four species of loricriid ctwshes Enzyme P. cf. n. Mrñon (6) P. nocturnus (6) Pt. disjunctivus (1) H. pyrineusi (5) Amylolytic F 2,17 = 28.3 F 2,17 = F 2,29 =8.56 F 2,14 = P <.1 P <.1 P =.1 P <.1 Lminrinse F 2,17 = t =2.68 F 2,29 = 13.2 t =1.96 P <.1 P =.23 P <.1 P =.86 Cellulse F 2,17 =.2 F 2,17 =.74 F 2,29 = t =3.86 P =.818 P =.492 P <.1 P =.18 Xylnse F 2,17 =6.56 F 2,17 =.81 F 2,29 =3.4 t =3.18 P =.9 P =.463 P =.65 P =.13 Trypsin F 2,17 = F 2,17 = F 2,29 =9.3 F 2,14 = P =.9 P <.1 P =.1 P <.1 Lipse F 2,17 = 34.8 F 2,17 = F 2,29 =.74 F 2,14 = 22.6 P <.1 P <.1 P =.485 P <.1 If only two vlues were compred, t test ws used insted of ANOVA. For exmple, comprisons of lminrinse ctivities in P. nocturnus nd H. pyrineusi were only mde mong the PI nd MI with t test ecuse these species lcked lminrinse ctivity in their distl intestines. Smple sizes in prentheses following species nmes. Actul enzyme ctivity dt re presented in Fig. 2 for mylse, lminrinse, cellulse, nd xylnse, nd in Supplementl Fig. 1 (see online version) for trypsin nd lipse Pterygoplichthys disjunctivus nd H. pyrineusi exhiited signiwcntly higher cellulse ctivity in their proximl intestines thn in their mid- or distl intestines (H. pyrineusi lcked detectle cellulse ctivity in its distl intestine), wheres the two species of Pnque showed no diverence in cellulse ctivity long the gut (Tle 2; Fig. 2). Individuls of P. cf. n. Mrñon, Pt. disjunctivus, nd H. pyrineusi possessed signiwcntly greter xylnse ctivity in their proximl intestines thn in their mid- or distl intestines (like cellulse, H. pyrineusi lcked detectle xylnse ctivity in its distl intestine). Pnque nocturnus, on the other hnd, showed slight, ut insigniwcnt increse in xylnse ctivity moving distlly long its intestine (Tle 2; Fig. 2). No mnnnse ctivity ws detected in ny gut region of ny species. Discchridse ctivities The mltse ctivity in the microil extrct ws signiwcntly higher thn the ctivity of this enzyme in the gut wll of the proximl intestines of ll four species (Figs. 3, 4). No signiwcnt diverences were oserved in the mid-intestine. The mltse ctivity in the gut wlls of the distl intestines of the wood-eting tx ws higher thn the mltse ctivity of the microil extrct, wheres the opposite ws true for the detritivorous Pt. disjunctivus (Figs. 3, 4). All four species showed decresing mltse ctivities in the microil extrct distlly in the intestine, wheres ll four tx showed slight increses in gut wll mltse ctivity in the mid-intestine in comprison to the proximl intestine (Figs. 3, 4). The β-glucosidse ctivities in the microil extrcts of the proximl intestines of P. cf. n. Mrñon, P. nocturnus, nd Pt. disjunctivus were ll signiwcntly higher thn the ctivities of this enzyme in the gut wll frctions; however, the opposite ws true for H. pyrineusi (Figs. 3, 4). Only Pt. disjunctivus showed signiwcnt diverences in β-glucosidse ctivity in their mid- nd distl intestines, with the gut wll ctivity eing signiwcntly higher in the mid-intestine, nd the ctivity in the microil extrct eing higher in the distl intestine. All four species showed decresing β-glucosidse ctivity in the microil extrcts of their distl intestines (Figs. 3, 4). However, there were severl diverent ptterns for gut wll β-glucosidse ctivity: P. nocturnus nd H. pyrineusi showed decresing ctivity in their distl intestine, P. cf. n. Mrñon showed incresing ctivity towrd their distl intestine, nd Pt. disjunctivus showed spike in ctivity in the mid-intestine, followed y decrese in the distl intestine. Pnque nocturnus exhiited signiwcntly greter N-cetyl-β-D-glucosminidse (NAG) ctivity in the gut wll of its proximl intestine thn in the microil extrct, wheres none of the other species showed diverences in NAG ctivity etween these two frctions in their proximl intestines (Figs. 3, 4). The wood-eting tx ll exhiited signiwcntly higher NAG ctivity in the gut wlls of their mid-intestines thn in the microil extrcts from this gut region, wheres Pt. disjunctivus showed no diverences etween the two frctions. However, P. nocturnus nd Pt. disjunctivus hd signiwcntly greter NAG ctivity in the gut wlls of their distl intestine thn in their microil extrcts, wheres the other species showed no diverences etween the two frctions (Figs. 3, 4). Pnque cf. n.

9 J Comp Physiol B (29) 179: Amylse (U. g -1 ) Lminrinse (U. g -1 ) Cellulse (U. g -1 ) Xylnse (U. g -1 ) Proximl Intestine Mid Intestine Distl Intestine Fig. 2 Totl (intestinl Xuid + microil extrct) mylolytic, lminrinse, cellulse, nd xylnse ctivities in three regions of the intestine of Pnque cf. nigrolinetus Mrñon (Pm), P. nocturnus (Pn), Pterygoplichthys disjunctivus (Ptd), nd Hypostomus pyrineusi (Hp). Vlues re mens nd error rs represent SEM. IntrspeciWc comprisons of ech enzyme mong gut regions were mde with ANOVA followed y Tukey s HSD with fmily error rte of P =.5. Regionl ctivity levels for speciwc enzyme nd species tht shre letter re not signiwcntly diverent. No letters ove the gut regions for prticulr enzyme indicte tht there re no diverences in ctivity mong the intestinl regions for tht species. InterspeciWc comprisons mong species were not mde n.d. Pm Pn Ptd Hp c c n.d. n.d. n.d. Mrñon, P. nocturnus, nd Pt. disjunctivus showed increses in their gut wll NAG ctivities distlly in the intestine, wheres H. pyrineusi showed decrese. The NAG ctivities of the microil extrcts were vrile nd did not follow one pttern (increse or decrese) long the guts of ny of the four species (Figs. 3, 4). The mltse Michelis Menten constnts (K m ) from the wll of the proximl intestines of the Wsh were generlly lower, lthough not signiwcntly so, thn the K m vlues of the microil extrcts from the proximl intestines (Tle 3). However, the K m vlues of β-glucosidse were ll signiwcntly lower in the Wsh gut wlls thn in the microil extrcts, nd the sme ws generlly true for NAG, except for P. nocturnus (Tle 3). All four species generlly possessed signiwcntly greter β-mnnosidse ctivities in their gut wlls thn in the microil extrcts (See Supplementl Tle S3 in online version). β-mnnosidse ctivity incresed in the distl intestine of P. cf. n. Mrñon, decresed in the distl intestines of P. nocturnus nd Pt. disjunctivus, nd spiked in the mid-intestine of H. pyrineusi. β-xylosidse ctivity ws only oserved in the microil extrcts of the four tx nd ws sent in the distl intestines of P. nocturnus, Pt. disjunctivus, nd H. pyrineusi (Supplementl Tle S3). Pnque cf. n. Mrñon showed signiwcnt decreses in β-xylosidse ctivity distlly in its intestine (ANOVA F 2,17 = 1.24, P =.2). Similrly, Pterygoplichthys disjunctivus (t =2.57, P =.19, df = 18) nd H. pyrineusi (t = 2.25, P =.5, df = 8) showed signiwcnt decreses in β-xylosidse ctivity in their mid-intestines compred to their proximl intestines, wheres P. nocturnus (t =.84, P =.421, df = 1) did not. Protese nd lipse ctivities Trypsin ctivities signiwcntly decresed distlly in the intestines of ll four species (Tle 2, Supplementl Fig. S1 in online version). All four species generlly possessed signiwcntly greter minopeptidse ctivities in their gut wlls thn in the microil extrcts (Tle 4). Aminopeptidse ctivities incresed distlly in the intestine in the wood-eting tx, nd spiked in the mid-intestine of Pt. disjunctivus (Tle 4). Lipse ctivities signiwcntly decresed distlly in the intestines of ll of the wood-eting tx, ut slightly incresed in the distl intestines of Pt. disjunctivus (Tle 2, Supplementl Fig. S1). Enzymtic ctivities of the heptopncres nd liver (not shown) vried y enzyme. No cellulse or xylnse ctivities were detected in the heptpncres or liver of ny species, wheres mylolytic ctivity, lminrinse, trypsin, nd

10 134 J Comp Physiol B (29) 179: Fig. 3 Mltse, β-glucosidse, nd N-cetyl-β-D-glucosminidse (NAG) ctivities in the gut wlls nd microil extrcts of the proximl intestine (PI), mid-intestine (MI), nd distl intestine (DI) of Pnque cf. nigrolinetus Mrñon (left column) nd P. nocturnus (right column). Comprisons were mde of the ctivities of ech enzyme etween the gut wlls nd microil extrcts of ech gut region with t test. Following Bonferroni correction for ech enzyme nd species, diverences re considered signiwcnt t P =.13 [indicted with n sterisk ()] Mltse (U. g tissue -1 ) β-glucosidse (U. g tissue -1 ) Pnque cf. nigrolinetus Mrñon Gut Wll Microil Extrct Pnque nocturnus NAG (U. g tissue -1 ) PI MI DI PI MI DI lipse were ll detected in the heptopncres of the Wsh. Only mylolytic nd lipse ctivities were detectle in the liver. Gstrointestinl fermenttion nd luminl crohydrte prowles Hypostomus pyrineusi ws the only ctwsh species to show ny signiwcnt chnge in SCFA concentrtion long the gut, with signiwcntly higher SCFA concentrtions in the mid-intestine thn in the proximl intestine (Tle 5). The trends of SCFA concentrtions vried mong species, with Pt. disjunctivus showing n incresing concentrtion of SCFAs long the gut, nd P. cf. n. Mrñon showing decrese, leit no signiwcnt chnge (Tle 5). The TLC pltes (not shown) reveled tht ll four species hd solule oligo-, di-, nd monoscchrides in the proximl intestine, nd tht these concentrtions decresed until there were no solule sugrs remining in the distl intestine. Similrly, mesurle glucose ws oserved in the Xuid of the proximl intestine of P. cf. n. Mrñon ( mm) nd P. nocturnus ( mm), ut these concentrtions disppered in the mid- nd distl intestine. Only H. pyrineusi showed mesurle glucose in ll regions of the intestine nd these concentrtions decresed, signiwcntly so (ANOVA: F 2,14 =84.75, P <.1), from the proximl ( mm) to the mid- (.93.9 mm) to the distl (.73.3 mm) intestine. No glucose ws detected in the Xuid of ny gut region of Pt. disjunctivus. Discussion The results of this study support the null hypothesis tht wood-eting ctwshes re not relint upon endosymionts to digest refrctory polyscchrides in their GI trcts. First, even though negtive redox conditions were oserved in the intestinl Xuids of the Wshes, the SCFA concentrtions were low nd were not signiwcntly greter in the distl intestine thn in the other gut regions. Second, the prowles of solule oligoscchrides in the intestinl Xuids of the Wsh indicte tht most sorption of nutrients tkes plce in their proximl nd mid-intestines, nd not in their distl intestines. Third, the ptterns of digestive enzyme ctivities indicte tht the Wsh trget more solule components of their detritl diet rther thn refrctory polyscchrides:

11 J Comp Physiol B (29) 179: Fig. 4 Mltse, β-glucosidse nd N-cetyl-β-D-glucosminidse (NAG) ctivities in the gut wlls nd microil extrcts of the proximl intestine (PI), mid-intestine (MI), nd distl intestine (DI) of Pterygoplichthys disjunctivus (left column) nd Hypostomus pyrineusi (right column). Comprisons were mde of the ctivities of ech enzyme etween the gut wlls nd microil extrcts of ech gut region in ech species with t test. Following Bonferroni correction for ech enzyme nd species, diverences re considered signiwcnt t P =.13 [indicted with n sterisk ()] β-glucosidse (U. g tissue -1 ) Mltse (U. g tissue -1 ) Pterygoplichthys disjunctivus Hypostomus pyrineusi 6 Gut Wll Microil Extrct NAG (U. g tissue -1 ) PI MI DI PI MI DI Tle 3 Michelis Menten constnts (K m ) of discchridses in the gut wlls nd microil extrcts of the proximl intestines of Pnque cf. nigrolinetus Mrñon (Pm), P. nocturnus (Pn), Pterygoplichthys disjunctivus (Ptd), nd Hypostomus pyrineusi (Hp) Species Mltse β-glucosidse N-cetyl-β-D-glucosminidse Gut Wll Microil extrct Gut Wll Microil extrct Gut Wll Microil extrct Pm t = t = t =3.86 P =.97 P <.1 P =.3 Pn t = t = t =1.76 P =.53 P <.1 P =.18 Ptd t = t = t =3.4 P =.587 P <.1 P =.8 Hp t = t = t =3.34 P =.923 P <.1 P =.1 Vlues re men ( SEM), nd concentrtions re in mm. Gut wll nd microil extrct constnts were compred with t test for ech species nd enzyme, nd fter Bonferroni correction, re considered signiwcntly diverent t P =.13. Smples sizes were Pm: n =6; Pn: n =6; Ptd: n =6 (gut wll), n = 1 (microil extrct); Hp: n =5 cellulse nd xylnse ctivities were low, vrile, nd generlly decresed in the distl intestines of the Wsh; cellulse nd xylnse ctivities were not higher in the microil extrcts, s would e expected in nimls relint on n endosymiotic community; cellulse nd xylnse ctivities were severl orders of mgnitude lower thn mylolytic nd lminrinse ctivities; nd the K m vlues of mltse, β-glucosidse, nd N-cetyl-β-D-glucosminidse (NAG)

12 136 J Comp Physiol B (29) 179: Tle 4 Aminopeptidse ctivities (U g 1 ) in the gut wlls nd microil extrcts of the proximl (PI), mid- (MI), nd distl intestines (DI) of Pnque cf. nigrolinetus Mrñon (Pm), P. nocturnus (Pn), Pterygoplichthys disjunctivus (Ptd), nd Hypostomus pyrineusi (Hp) Vlues re men ( SEM). Comprisons of minopeptidse ctivity mong the gut wlls nd microil extrcts of ech gut region in ech species were mde with t test. Following Bonferroni correction, vlues re considered signiwcntly diverent t P =.17 PI MI DI Pm (6) Gut wll Microil extrct t P < Pn (6) Gut wll Microil extrct t P Ptd Gut wll (6) Microil extrct (1) t P. <.1.85 Hp (5) Gut wll Microil extrct t P Tle 5 Totl short-chin ftty cid concentrtions (mm) in the three gut regions of Pnque cf. nigrolinetus Mrñon, P. nocturnus, Pterygoplichthys disjunctivus, nd Hypostomus pyrineusi Gut Region P. cf. n. Mrñon P. nocturnus Pt. disjunctivus H. pyrineusi Proximl Mid Distl F 2,17 =.26 F 2,17 =.48 F 2,17 =1.28 F 2,14 =4.55 P =.77 P =.63 P =.31 P =.3 Vlues re men ( SEM). Comprisons of SCFA concentrtions mong gut regions within species were mde with ANOVA, with diverences considered signiwcnt t P =.5. If signiwcnt diverences were detected with ANOVA, this ws followed y Tukey s HSD multiple comprison test with fmily error rte of P =.5. Those vlues shring superscript letter re not signiwcntly diverent. Smple sizes were s follows: P. cf. n. Mrñon, n =6; P. nocturnus, n =6; Pt. disjunctivus, n = 1; H. pyrineusi, n = 5. Acette:propionte:utyrte rtios for totl SCFAs were s follows: P. cf. n. Mrñon = 62:23:15; P. nocturnus = 44:31:25; Pt. disjunctivus = 7:16:14; H. pyrineusi = 52:28:2 were generlly lower in the wlls of the proximl intestines of the Wsh thn in the microil extrcts, suggesting tht the Wsh more eyciently digest solule discchrides thn do microil enzymes ingested with their food. Thus, consistent with our oservtions of rpid gut trnsit nd poor cellulose digestiility in these Wshes (Germn 29), the wood-eting loricriid ctwshes pper to e more similr, from digestive physiology stndpoint, to their closely relted detritivorous reltives in the genus Pterygoplichthys thn to other xylivores (e.g., evers, termites). The ph levels nd redox potentils in the ctwsh intestines indicted the possiility of supporting popultion of neroic microes, ut only in the intestine. Mny loricriids rethe ir nd hve modiwed stomchs tht re considered to e ir rething orgns (Grhm nd Bird 1982; Armruster 1998), which explins why the redox potentils of the stomchs of the Wsh in this study were positive (Pt. disjunctivus) or only slightly negtive (P. cf. n. Mrñon nd P. nocturnus; Tle S1). However, the loricriid stomch is not involved in digestion. For exmple, the stomch of Pt. disjunctivus is usully Wlled with ir, is lkline, nd ingest re not held in the stomch for ny length of time; even individuls of this species killed minutes fter consuming food hd lredy pssed the ingest into the proximl intestine, ypssing the stomch vi smll groove t its se (DPG, pers. os.). Redox potentils mesured 1 mm eyond the pyloric sphincter in this study were lredy 6 mv, which indicted tht even the most

13 J Comp Physiol B (29) 179: proximl region of the intestine ws not oxygented y the Wsh s rething ctivity. Furthermore, similr to some detritivorous termites (Kppler nd Brune 22), detritivorous Wshes (including wood-eting species) proly consume humic cids, which, in ddition to other components of the intestinl Xuid (e.g., ile slts nd ingested metls; Kppler nd Brune 22), cn increse the reductive potentil, thus producing negtive redox conditions. Either wy, the redox potentils mesured in wild-cught Wsh in this study suggest tht the intestinl environment is highly reductive. The concentrtions of SCFAs oserved in the Wshes intestines further chllenge the hypothesis tht wood-eting ctwshes use n endosymiotic community to ferment reclcitrnt polyscchrides. Fishes tht rely on GI fermenttion to meet some proportion of their dily energy needs tend to hve >2 mm totl SCFAs in their hindguts (Chot nd Clements 1998). The highest concentrtions oserved in this study were in the mid-intestine of H. pyrineusi ( mm) nd were fr elow concentrtions oserved in Wshes with ctive endosymiotic communities in their GI trcts (Chot nd Clements 1998; Mountfort et l. 22). Furthermore, H. pyrineusi ws the only species to show ny signiwcnt diverence in SCFA concentrtions long its gut. Coupled with rpid gut trnsit, it ppers tht some detritivorous/microlgivorous Wsh species trget more solule components of their diet, especilly protein, nd do not redily digest refrctory polyscchrides (Crossmn et l. 25; Germn 29). Perhps the most informtive iochemicl dt gthered in this study re the ptterns of digestive enzyme ctivities long the Wshes intestines. A common pttern in lower termites, which digest cellulose in their hindgut vi n endosymiotic microil community, is incresing cellulse ctivities in the hindgut region (Nkshim et l. 22; Mo et l. 24). Similrly, mrine herivorous Wshes with ctive hindgut microil popultions hve incresing exogenously produced enzyme ctivities (e.g., crrgeense) in the microil extrcts of their hindguts (Ske et l. 25). However, none of the ctwsh species showed incresing cellulse ctivity in the distl intestine nd, insted, showed no pttern (no increse or decrese; P. cf. n. Mrñon nd P. nocturnus) or decresing ctivity (Pt. disjunctivus nd H. pyrineusi) towrd the distl intestine. Moreover, the cellulse ctivities in the ctwsh guts were Wve orders of mgnitude lower thn mylolytic ctivities, nd one to two orders lower thn lminrinse ctivities. Thus, the Wsh clerly digest solule polyscchrides, like strch nd lminrin, more rpidly thn refrctory polyscchrides, especilly given the rpid trnsit time of food through the gut (Germn 29). Decying wood in n qutic environment will likely hve more nutritious dietry items collecting on the surfce of the wood, nd in spces mong Wers, thn the wood itself. The epilithic lgl complex (EAC), which is loose ssemlge of cteri, cynocteri, Wlmentous green lge, ditoms, nd detritus tht grows on hrd sustrtes in qutic systems (Hoglnd et l. 1982; vn Dm et l. 22; Wilson et l. 23; Klock et l. 27; Germn et l. 29) contins solule polyscchrides in the lge (including ditoms; Pinter 1983) nd in exopolymeric sustnces produced y microes (Lepprd 1995; Wotton 24; Klock et l. 27). These solule polyscchrides re likely n importnt energy source, not only to grzing species like Pt. disjunctivus, ut lso to the xylivorous species digging into the decying wood. Other EAC-consuming Wshes (e.g., species in the genus Cmpostom) hve very similr ptterns nd mgnitudes of mylolytic nd lminrinse ctivities to the ctwshes (Germn 29; Germn et l. 29), suggesting tht they trget similr suites of nutrients from their foods. The xylnse ctivities in P. nocturnus were the only luminl enzyme ctivities to e diverent etween the intestinl Xuid nd the microil extrct, nd the ctivities of this enzyme slightly incresed, leit not signiwcntly so, towrd the distl intestine of this species. Xyln is component of hemicellulose (Petterson 1984; Breznk nd Brune 1994), ut mmmls (nd proly vertertes in generl) re not known to possess n endogenous xylnse or e le to metolize the monomer of xyln, xylose, without the id of intestinl microorgnisms (Johnson et l. 26, ). Additionlly, the ctwsh species exmined in this study lcked β-xylosidse ctivity in their gut wlls nd hd low β-xylosidse ctivities in their microil extrcts, which decresed distlly in the digestive trct. Given the low nd vrile cellulse nd xylnse ctivities oserved in the ctwsh, nd the lck of ny consistent pttern of ctivity long the guts of the Wsh, these enzymes re likely ingested (nd produced y microes ingested) with detritus rther thn produced y resident endosymiotic community. This stnds in contrst to the conclusions of Nelson et l. (1999), who isolted microes with cellulolytic cpilities from the guts of loricriid ctwshes, suggesting tht there ws resident microxor in the Wshes GI trcts. However, this does not men tht those microorgnisms re endosymionts digesting wood (Prejs nd Blszczyk 1977; Lindsy nd Hrris 198). For exmple, grss crp, which ets qutic mcrophytes rich in cellulose nd hve cellulse ctivities in their GI trcts (Lesel et l. 1986; Ds nd Tripthy 1991), poorly digests the cellulose component of their plnt diet (Vn Dyke nd Sutton 1977). This poor cellulose digestiility is likely due to rpid gut trnsit nd low levels of microil fermenttion in the grss crp guts (Stevens nd Hume 1998). Additionlly, Prejs nd Blszczyk (1977) oserved elevted cellulse ctivities in grss crp consuming detritus (i.e., degrded plnt mteril),