RESEARCH ARTICLE Protein-induced mass increase of the gastrointestinal tract of locusts improves net nutrient uptake via

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1 329 The Journl of Experimentl Biology 216, Published by The Compny of Biologists Ltd doi:1.1242/jeb RESEARCH ARTICLE Protein-induced mss increse of the gstrointestinl trct of locusts improves net nutrient uptke vi lrger mels rther thn more efficient nutrient bsorption Fion J. Clissold 1, *, Zuben P. Brown 1, nd Stephen J. Simpson 1,2 1 School of Biologicl Sciences nd 2 The Chrles Perkins Centre, The University of Sydney, NSW 26, Austrli *Author for correspondence (fion.clissold@sydney.edu.u) Present ddress: Lbortory of Protein Synthesis nd Expression, Institute for Protein Reserch, Osk University, 3-2 Ymdok, Suit , Jpn SUMMARY Incresing the tissue biomss nd/or volume of the gstrointestinl trct (GIT) is commonly seen when nimls feed on poorqulity diets. This increse cn simply permit lrger mel sizes, but my lso reblnce nutritionlly imblnced ingest by llowing selective bsorption of limiting nutrients. In n insect herbivore, the migrtory locust, synthetic diet with high rtio of protein to crbohydrte ws found to induce mss enhncement of the GIT. When normlised for sex nd overll body size, increses to the mss of the foregut nd midgut cec resulted in higher bsorption (2 3%) of both protein nd crbohydrte when subsequently feeding on three chemiclly nd structurlly different grsses. Greter net bsorption of mcronutrients occurred becuse these locusts te lrger mels tht trnsited t the sme time nd with the sme digestive efficiency s locusts in which the GIT ws not enlrged. Thus, plsticity of the GIT did not improve nutritionl homeostsis, but incresed the rte of nutrient uptke. Supplementry mteril vilble online t Key words: phenotypic plsticity, nutrition, digestion, geometric frmework. Received 2 June 212; Accepted 15 September 212 INTRODUCTION It is becoming incresingly pprecited tht the gstrointestinl trct (GIT) is remrkbly dynmic orgn, exhibiting plsticity in size, structure nd function (reviewed by Krsov nd Hume, 1997; Strck, 25; Ny et l., 27; Krsov et l., 211). Such plsticity is thought to ccommodte fluctutions in the qulity nd quntity of food over time by sustining rtes of nutrient extrction t homeosttic norm, supporting both generl metbolism nd the mintennce costs of the intestine itself (Cnt et l., 1996; Hume, 25). Digestive function nd subsequent nutritionl outcomes hve been modelled using chemicl rectorbsed theory (Penry nd Jumrs, 1986; Penry nd Jumrs, 1987) nd considered in terms of optiml digestive strtegies (Sibly, 1981; Hume, 1989; Krsov, 1999). However, dt re lmost lwys inconsistent with predictions from such models (e.g. Dimond nd Hmmond, 1992; Yng nd Joern, 1994; Jumrs nd Mrtínez del Rio, 1999; Krsov, 1999; Levey nd Mrtínez del Rio, 1999), suggesting problems with the underlying ssumptions (reviewed by Krsov, 1999; McWhorter, 25). Typiclly, these models hve been tested using clorific estimtes or by single nutrient nlyses, nd it is now recognised tht nimls cross ll trophic levels blnce the intke of multiple nutrients (e.g. Myntz et l., 25; Rubenheimer nd Jones, 26; Behmer, 29; Rubenheimer et l., 29; Jensen et l., 212) rther thn mximize intke or provide the tissues with constnt flux of energy (Sibly, 1981; Slnsky nd Wheeler, 1989; Krsov nd Hume, 1997; Woods nd Kingsolver, 1999). Consistent with this ide, it ws recently shown tht when locusts were confined to diet contining n unblnced rtio of protein to crbohydrte, they exhibited lower digestive enzyme ctivity for the mcronutrient present in reltive excess in the diet (Clissold et l., 21). An enlrged GIT hs generlly been ssocited with nimls ingesting poor-qulity diets (reviewed by Yng nd Joern, 1994b; Krsov nd Hume, 1997; Jumrs, 2; Strck, 25; Ny et l., 27). By enlrging the GIT when feeding on foods in which nutrients re diluted within n indigestible mtrix, it is thought tht nutrient supply to the tissues cn be mintined by either incresing the efficiency with which ech mel is digested or incresing intke rtes, lbeit with poorer digestibility (e.g. Gross et l., 1985; Hume, 1989; Hmmond nd Wunder, 1991; Yng nd Joern, 1994b; Yng nd Joern, 1994; Krsov nd Hume, 1997; Strck, 25). However, recent reserch on both locusts nd mice hs shown the GIT lso increses in size when feeding on foods tht re energy dense but where the blnce of mcronutrients is not supplied in the required rtio (Rubenheimer nd Bssil, 27; Sørensen et l., 21). Interpreting the nutritionl outcome in this circumstnce is problemtic (Rubenheimer nd Bssil, 27; Sørensen et l., 21). Does upregultion of the physicl dimensions of the GIT represent compenstory response, whereby nutrient homeostsis is mintined by fcilitting n increse in the uptke of the deficient mcronutrient, or is the response counter-compenstory, nd my ctully reduce fitness by mximizing the uptke of ll nutrients nd supplying the tissues with even more of mcronutrient surplus (Rubenheimer et l., 25; Boersm nd Elser, 26)? Explining how djustments to the physicl dimensions of the GIT ffect nutritionl outcomes from existing studies is chllenging given the numerous co-vrying fctors typiclly occurring simultneously with structurl remodelling of the GIT. In the present

2 33 The Journl of Experimentl Biology 216 (2) study, the reltionship between diet, the structurl plsticity of the GIT nd overll nutrient extrction ws investigted in locusts. Experiments were estblished to determine whether: (1) the increse in GIT mss ws uniform or confined to specific regions; (2) enlrging the GIT incresed overll mcronutrient uptke (ingested minus voided); nd (3) the uptke of ll mcronutrients incresed eqully, or ws confined to limiting mcronutrient only. The overll protocol ws (1) to mnipulte the plsticity of the GIT using synthetic diets in the fourth nd fifth nymphl stdi, nd (2) to follow this with short period of feeding on grsses, during which GIT msses nd nutrient extrctions were determined. We used grsses to ssess the functionl implictions of chnges in GIT mss becuse Locust migrtori bsorbs close to ll the vilble protein nd crbohydrte from synthetic diets (Miller et l., 29). This is most likely due to the lck of structure, in the sense of pckging of nutrients with synthetic diets, rther thn the chemicl differences with the type of proteins or crbohydrtes between nturl nd synthetic foods. Hence, grsshoppers ingesting dried nd ground grsses, where the effects of biomechnicl properties hve been removed, behve nutritionlly identiclly to grsshoppers feeding on synthetic diets with the sme percentge of protein nd crbohydrte (Clissold et l., 26). MATERIALS AND METHODS Locusts nd diets Locust migrtori (L.) cme from long-term culture t The University of Sydney (originlly collected from the Centrl Highlnds of Queenslnd, Austrli). Stock locusts were rered in lrge plstic bins ( cm) with 5 1 locusts per bin in room kept t 3 C under 14 h:1 h light:drk photoperiod, with ech bin hving n dditionl het source (25 W het lmp mounted on the mesh roof of the bin) during the lights on phse. Locusts were provided with d libitum seedling whet nd whet germ. Four dry, grnulr synthetic diets differing in the percentge of protein (P) nd crbohydrte (C) (35P:7C, 28P:14C, 14P:28C nd 7P:35C) were mde s described in Simpson nd Abisgold (Simpson nd Abisgold, 1985), with protein being 3:1:1 mixture of csein, bcteriologicl peptone nd egg lbumen, nd crbohydrte 1:1 mixture of sucrose nd dextrin. All diets contined 4% micronutrients (slts, vitmins nd sterols) nd 54% indigestible α- cellulose (C82, Sigm-Aldrich, St Louis, MO, USA) nd were ground to fine powder. Bldes of two grss species, Cynodon dctylon (L.) Pers. (Poles: Pocee) nd Themed ustrlis (R. Br.) Stpf (Poles: Pocee), were hrvested loclly s previously described (Clissold et l., 21). Seedling whet, Triticum estivum L. em Thell. (Poles: Pocee), ws grown from seed in glsshouse for ~2 dys. Grss bldes were detched t the ligule nd the bses were plced in florist vil with wter (Clissold et l., 24). Experimentl design We first provide n overview of the experimentl design nd then describe detiled methodologies for ech. In Experiment 1, we determined the reltionship between diet nd growth cross the finl nymphl stdi, the mss of ech region of the GIT, nd subsequent nutrient cquisition from T. ustrlis. From the strt of the penultimte nymphl stdium to midwy through the finl nymphl stdium, locusts were provided d libitum ccess to one of the five tretments nd then fed T. ustrlis for 24 h. The tretments consisted of four synthetic diets vrying in the rtio of protein to crbohydrte (P:C) s described bove, nd fifth, where the locusts were llowed to self-select their protein nd crbohydrte intke from either 35P:7C versus 7P:35C or 28P:14C versus 14P:28C. Previous work indicted tht n optiml P:C rtio for L. migrtori is close to 21:21 (Miller et l., 29). In Experiment 2, we seprted the effect of tretment diet, GIT mss nd subsequent nutrient bsorption by generting differences in the GIT without concurrent chnges occurring in food intke, development rte or the size or composition of the reminder of the body, nd then determined the response to one of three grsses tht differed in their protein nd crbohydrte composition (P:C): Cynodon dctylon [which hs P:C rtio tht is closest to optiml for L. migrtori (P=C)], Triticum estivum (P>C) or T. ustrlis (P<C). At the end of the experiment, the dry mss of ech region of the GIT ws determined nd correlted with feeding behviours nd the rte nd efficiency of protein nd crbohydrte bsorption from ech grss. Absorption ws defined s the difference in protein or crbohydrte mesured in the ingest nd feces. Experiment 1: effects of diet on lloction to the GIT nd response to T. ustrlis Equl numbers of ech sex of newly moulted (within 4 h of ecdysis) fourth-instr L. migrtori nymphs whose mss ws within stndrd devition of previously weighed popultion were rndomly llocted to ech tretment diet. Locusts were plced lone in cler plstic boxes ( cm, length width height) contining wter, metl perch nd the experimentl diet (~1 11 per diet tretment, totl N=53). All experiments were crried out t C under 14 h:1 h light drk photoperiod. Nymphs were provided with wter nd known mount of fresh synthetic diet (~2 mg) dily until Dy 3 of the fifth stdium (Dy is dy of moulting), when the synthetic tretment diet ws exchnged for T. ustrlis. A known mss of grss (~1.6 g) ws provided nd the locusts were llowed to feed for 24 h. At the end of the 24 h, ll remining grss ws removed nd nymphs were llowed to feed on the initil synthetic tretment diet, nd were provided with wter until the mels of grss hd pssed through the GIT (~5 h). At this point, nymphs were killed, nd the gut ws removed by severing the bdomen between the lst two segments, pulling the hed until the cervicl membrne ruptured nd then gently removing the entire gut. The foregut (F), midgut cec (C), midgut ventriculus (M) nd hindgut (H) were seprted. Once detched, ech GIT section ws slit open nd wshed in insect sline (125 mmol l 1 NCl, 4 mmol l 1 KCl, 5 mmol l 1 CCl 2, 2 mmol l 1 KH 2 PO 4, 2 mmol l 1 Hepes, ph 7.5) to remove ny contents before being blotted dry. Following lyophiliztion, the mss of ech section ws weighed to n ccurcy of.1 µg (Mettler, Melbourne, Austrli). All feces were removed from the continer, nd the feces derived from grss were seprted. Three response vribles were determined for the period (24 h) during which the locusts were confined to the grss diet: (1) intke (mesured directly s described below), (2) bsorption of nutrients from the GIT (intke minus feces) nd (3) efficiency of bsorption (with intke minus feces s the dependent vrible nd intke s the covrite), following the logic of Rubenheimer nd Simpson (Rubenheimer nd Simpson, 1992). Intke of totl dry mtter ws derived by subtrcting the dry mss of the remining uneten grss from n estimte of the initil dry mss provided. The ltter ws clculted by fresh-weighing the grss nd using regression bsed on liquots of fresh grss tht were weighed, lyophilized nd reweighed. These liquots cme from subsets of bldes of grss tht were collected dily. Absorption of protein nd crbohydrte ws determined from the percentge of protein or crbohydrte in the ingest less tht remining in the feces (see below).

3 Lrger mels not improved digestibility 331 Experiment 2: effect of GIT size on intke nd feeding behviour on different grsses Locusts were rndomly llocted within n experimentl design blnced by tretment diet (28P:14C or 14P:28C), sex nd grss species (C. dctylon, T. estivum nd T. ustrlis) (~11 12 per tretment, N=132) nd treted s described bove. Mel durtion nd intermel intervl were determined by mnul inspection for ll locusts when feeding on the grsses. Feeding behviour ws recorded t 1 min intervls for 3 h, commencing t lest 4 h fter the nymphs hd been provided with grss bldes. A mel ws considered complete if the locust fed for minimum of 2 min nd did not feed gin within 4 min (Simpson, 1982). Intermel durtion is highly correlted with the men time food is retined within the GIT. Determintion of diet composition nd nutrient bsorption from the grsses Totl protein, non-structurl crbohydrtes nd cell wll mteril (neutrl detergent fibre) were determined from finely ground (Retsch Mixer Mill MM 4, Hn, Germny) lyophilized smples of the grsses nd feces. Protein ws extrcted from replicte 1 mg smples of plnt nd fecl mteril with.1 mol l 1 NOH nd determined using the Bio-Rd micro ssy (Bio-Rd, Hercules, CA, USA) bsed on the Brdford ssy. Totl non-structurl crbohydrte ws determined colourimetriclly from 1 mg plnt nd fecl smples following extrction with.1 mol l 1 H 2 SO 4 (Smith et l., 1964), using the phenol-sulphuric ssy (DuBois et l., 1956). Neutrl detergent fibre ws determined grvimetriclly from replicte 5 mg plnt smples using the Vn Soest method, omitting sodium sulphite (Vn Soest et l., 1991; Clissold et l., 24). The mount of protein nd crbohydrte digested nd bsorbed ws clculted from the mount of ech nutrient ingested minus tht remining in the feces. Fibre is not digested nd bsorbed by L. migrtori (Hochuli et l., 1993) nd thus only the mount ingested ws clculted. For the grsses used in Experiment 2, lef mss re ws determined s surrogte mesure of toughness (Red nd Stokes, 26; Onod et l., 211). Ten leves from ech grss were scnned (Kyocer 45i, Sydney, Austrli) nd then the leves were lyophilized to constnt mss nd weighed. Sttisticl nlysis For ech dietry tretment, the mss of ech section of the gstrointestinl trct ws compred using ANCOVA, with body mss minus the mss of the entire GIT s the covrite. Although we were interested in bsolute differences in size, we used ANCOVA s this removed the effect of sex from ll nlyses. All differences due to sex were explined entirely by body mss nd there were no interctions between sex nd ny of the other fctors. ANCOVA ws used to test whether the totl mss of the GIT vried between tretments while djusting the covrites of body mss minus GIT. Responses (intke nd bsorption of protein nd crbohydrte) of the synthetic diets nd the grsses of insects pretreted s described for both experiments were compred using ANCOVA (Rubenheimer, 1995), with body mss t the end of the experiment s the covrite. The efficiency of bsorption ws nlysed using ANCOVA with feces s the min effect nd intke s the covrite (Rubenheimer, 1995). In Experiment 2, to ensure the effects observed were due to the size of the GIT rther thn ny effects of the dietry tretment per se, the reltionship within ech dietry tretment ws compred by removing the effect of body size on GIT mss, intke nd mcronutrient bsorption. The effect of body size ws removed by compring the residuls of the combined msses of the foregut nd cec (M R,FC ) with the residuls for nutrient intke nd bsorption generted by ANCOVA, with dietry tretment nd grss s fctors. The msses of the foregut nd cec were highly correlted nd these were combined rther thn used seprtely. Feeding behviours (men mel nd intermel durtions) were compred using ANCOVA, with body size s the covrite. Agin, we investigted the trends within dietry tretment by exmining the reltionship between the reltive size of the foregut nd/or cec nd feeding behviour. All nlyses were undertken using SYSTAT 12 (Systt Softwre, Chicgo, IL, USA), with ANOVA nd ANCOVA performed following the techniques outlined in Quinn nd Keough (Quinn nd Keough, 22). Prior to ll nlyses, box plots were used to check for normlity nd homogeneity of vrinces cross the tretments. For ANCOVA, the interction term between the covrite nd the dietry tretment fctor ws included in the initil model to test the equlity of slopes. RESULTS Experiment 1 Msses of regions of the gstrointestinl trct The entire GIT ws hevier in locusts consuming diets where protein ws supplied in excess reltive to crbohydrte (P:C 35:7 nd 28:14) thn for the other tretments (F 4,52 =5.29, P<.1). This difference ws due to increses in the foregut nd cec (Fig. 1; supplementry mteril Tble S1). Compred with locusts llowed to self-select their protein nd crbohydrte intkes, the foregut nd cec of nymphs fed 35P:7C were 8 nd 3% hevier, respectively, nd 4 nd 2% hevier for nymphs confined to 28P:14C. Across tretments, mss of the GIT (M GIT ) ws highly correlted with protein intke (both corrected for body size) nd to lesser degree with totl food intke, but showed no reltionship to the mount of crbohydrte eten [totl food ingested (I F ), M GIT =.2I F +7.49, F 1,55 =26.86, P<.1, r 2 =.32; P intke (I P ), M GIT =.6I P +9.1, F 1,55 =188.46, P<.1, r 2 =.77; C intke (I C ), F 1,55 =.16, P=.693, r 2 =.3]. Locusts of both sexes vried in mss with dietry tretment (tretment: F 4,47 =11.5, P<.1; sex: F 1,47 =16.2, P<.1; tretment sex: F 4,47 =1.49, P=.22; Fig. 2, inset), with those treted on 7P:35C being lighter (P<.2) thn those on ll diets except 35P:7C. All nymphs consumed similr mounts of food (F 4,52 =2.31, P=.71), but s the foods differed in the rtio of P:C, nymphs confined to the 35P:7C diet ingested the most protein nd the lest crbohydrte (Fig. 2). Similr mounts of crbohydrte were ingested by nymphs on both of the high crbohydrte diets nd those llowed to self-select (P>.5). Nymphs regulting their intke of protein nd crbohydrte composed their intke so tht men (±s.e.m.) of 1.9±.6 g crbohydrte ws consumed for every grm of protein (i.e. 1P:1.1C) regrdless of diet piring (ANOVA: P ingested, P=.185; C ingested, P=.38). Locust performnce: rtes of nutrient intke, bsorption nd efficiency of bsorption When feeding on T. ustrlis, locusts tht hd been feeding on either of the diets where protein ws oversupplied reltive to crbohydrte consumed significntly more (~3 4%) thn locusts treted on either of the diets where crbohydrte ws supplied in excess of protein (F 4,47 =6.92, P<.1; Fig. 3A). Locusts tht hd been llowed to self-select their intke of protein nd crbohydrte consumed similr mount of T. ustrlis s locusts on ll other tretments (Fig. 3A). Locusts treted with 35P:7C bsorbed lower rtio of P:C from T. ustrlis thn locusts treted with the two crbohydrte-bised diets becuse they digested crbohydrte more efficiently (4.8±.4%) thn locusts on ll other diet tretments

4 Dry mss (mg per 151 mg crcss) Finl dry mss (mg) 332 The Journl of Experimentl Biology 216 (2) A B,b Trget,b b b P=.2 c P<.1 c Crbohydrte ingested (mg) P<.1 2 b b b,b Trget Protein ingested (mg) C n.s. P=.6 Fig. 2. Protein nd crbohydrte intke over the fifth stdium when the locusts were confined to single diets vrying in the rtio of P:C or llowed to self-select the rtio of protein nd crbohydrte ingested (ʻtrgetʼ). The inset shows the finl dry mss of locusts on ech diet tretment. The P- vlue is for the differences in finl body mss for the five dietry tretments, nd brs with different letters hve significntly different (P<.5) mens (Tukeyʼs HSD test) D.2 n.s. P= Diet (rtio of P:C) Fig. 1. Dry mss lloction to the different regions of the gstrointestinl trct (A) foregut, (B) cec, (C) midgut nd (D) hindgut by locusts fter feeding on diets on which they were ble to self-select protein or crbohydrte (ʻtrgetʼ, 1P:1.1C) or where they were confined to single food vrying in the rtio of P:C. Vlues re ANCOVA djusted mens ± s.e.m. for 151 mg crcss. N=1 12 for ech diet tretment. The P- vlues re those for dietry tretment nd brs with different letters hve significntly different (P<.5) mens (Tukeyʼs HSD test); n.s., P>.5. (27.9±.3%; Fig. 3B, supplementry mteril Tble S2). Protein ws digested with equl efficiency (68.8±.1%) by ll locusts regrdless of tretment (supplementry mteril Tble S2). Experiment 2 Locust performnce: rtes of nutrient intke, bsorption nd efficiency of bsorption Nymphs feeding on 28P:14C nd 14P:28C were sttisticlly similr in mss (femles: 165.1±2.8 mg; mles: 135.8±2.7 mg; tretment diet: F 1,13 =.19, P=.663) nd the tretment diets ffected the msses of the foregut nd midgut cec but not the reminder of the midgut nd hindgut, s found in Experiment 1 (supplementry mteril Tble S1, Fig. S1). In both experiments there ws no evidence of the bimodl response reported by Rubenheimer nd Bssil (Rubenheimer nd Bssil, 27) for locusts consuming 14P:28C (Levene s test, Experiment 1, F=.15, P=.962; Experiment 2, F=.82, P=.443). Locusts treted with 28P:14C (with hevier foreguts nd midgut cec) consumed between 2 nd 3% more dry mtter (or 15 25% when clculted s wet mtter) thn locusts fed 14P:28C (with smller foreguts nd midgut cec) (Fig. 4A,B; supplementry mteril Tble S3). The degree to which totl nutrient (P+C) bsorption incresed in locusts fed diet 28P:14C reltive to 14P:28C ws grssspecies dependent (F 2,129 =169.39, P<.1; Fig. 5A). Locusts bsorbed most nutrients when feeding from T. ustrlis followed by T. estivum nd then C. dctylon (Fig. 5A). The three grsses differed chemiclly nd physiclly (Fig. 5B) nd lthough ~9% of the vilble protein ws extrcted from ll the grsses, the efficiency of crbohydrte extrction differed between grsses (~48% for C. dctylon, ~54% for T. estivum nd ~62% for T. ustrlis). Although the P:C rtio bsorbed by locusts from the three grsses differed, there ws no interction between diet tretment nd grss species in totl nutrient (P+C) bsorption (grss tretment interction, F 1,129 =.75, P=.474). Hence, the totl mount of nutrients (P+C) differed with diet tretment (F 1,129 =242.1, P<.1), but not their rtio of bsorption (F 1,129 =.7, P=.791; Fig. 5A). The increse in bsorption of ll nutrients (P+C) cross 24 h by locusts with lrger GIT (fed diet 28P:14C) resulted from the ingestion of more food during the 24 h in which the locusts were feeding on the grsses, rther thn from the differences in the efficiency with which either protein or crbohydrte ws digested (extrcted nd bsorbed) (supplementry mteril Tble S4). The extent to which diet effects were medited by differences in the GIT To estblish the extent to which diet-induced effects were ttributble to differences in the mss of the GIT, we removed the

5 Lrger mels not improved digestibility 333 Crbohydrte ingested (mg dy -1 ) A P>.1 B Diet (rtio of P:C) T. ustrlis 5. b P< Protein ingested (mg dy -1 ) Fig. 3. (A) Totl dry mtter nd (B) protein nd crbohydrte extrcted (digested nd bsorbed) in 24 h by locusts feeding on Themed ustrlis, following genertion of differences in the size of the foregut nd cec. In B, the solid line gives the rtio of P:C in T. ustrlis nd the two dotted lines give the two extremes of the rtios of P:C extrcted from the grss. Vlues re ANCOVA djusted mens ± s.e.m. for 151 mg crcss. N=1 12 for ech diet tretment. The P-vlues re those for dietry tretment, nd brs or symbols with different letters hve significntly different (P<.5) mens (A) or rtios of P:C bsorbed (B) (Tukeyʼs HSD test). effect of body size on both the mss of the foregut plus the midgut cec nd the mount of protein plus crbohdyrte bsorbed by tking the residuls of ANCOVA nlysis (fctors: dietry tretment nd grss). A positive reltionship ws found between the reltive mss of the foregut plus midgut cec (M R,FC ) nd both reltive totl dry mtter intke (I R,DM ) nd reltive nutrient bsorption (A R,PC ) (I R,PC =5.29M R,FC +3.3, F 1,129 =5.8, P=.21; A R,PC =1.24M R,FC +.53; F 1,129 =57.65, P<.1; Fig. 6), regrdless of dietry tretment (I R,PC : F 1,129 =.41, P=.522; A R,PC : F 1,129 =.1, P=.915). The residuls of the foregut nd midgut cec were highly correlted (P<.1), thus these reltionships were significnt when either section of the GIT ws used singly or when combined. Feeding behviour Mel durtions were on verge 27% longer for 28P:14C-treted nymphs (with lrger foreguts nd midgut cec) thn for 14P:28Ctreted nymphs (F 1,129 =6.19, P=.14), but intermel durtions did not differ with dietry tretment (F 1,129 =.45, P=.52;,b b b Totl dry mtter ingested (mg dy 1 ) Totl wet mtter ingested (g dy 1 ) 6 A B * *** 28P:14C 14P:28C *** 1.4 *** * *** C. dctylon T. ustrlis T. estivum Grss species Fig. 4. Totl (A) dry nd (B) wet mtter ingested by locusts in 24 h by locusts feeding on Cynodon dctylon, Themed ustrlis or Triticum estivum, following genertion of differences in the size of the foregut nd cec. *P<.5; ***P<.1 (Tukeyʼs HSD test). supplementry mteril Tble S5). Both mel durtion nd time between mels were grss-species specific (Fig. 7; supplementry mteril Tble S4). Locusts spent longer eting mel of T. ustrlis thn the other grsses, nd the longest intermel intervls were found following mels of T. estivum (Fig. 7; supplementry mteril Tble S5). A positive reltionship ws found between the mss of the foregut (M F ) (where the bulk of the mel is stored upon ingestion) nd mel durtion (T mel ), nd between the mss of the foregut plus midgut cec (M FC ) nd totl food intke (I food ) (T mel =.87M F +4.7, F 1,129 =8.6, P=.5; I food =.6M FC +3.98, F 1,129 =21.75, P<.1). No reltionship ws found between intermel intervl nd the mss of the foregut plus midgut cec (F 1,129 =.43, P=.512). We modelled the potentil uptke rtes of protein nd crbohydrte using the durtion of time spent feeding s surrogte for totl dry mtter intke (Fig. 7). We then corrected this for the mount of protein plus crbohydrte ctully bsorbed (% digested) (Fig. 7, inset). The model indictes tht: (1) for ech grss, incresed nutrient bsorption for locusts with hevier GIT occurred becuse of differences in intke rther thn the slight differences in the frequency with which mels were ingested, nd (2) bsorption rtes were highest on T. ustrlis s nymphs took the lrgest mels on verge t the sme frequency s locusts ingesting T. estivum, nd lowest in C. dctylon, on which verge mels were similr in size to those tken from T. estivum but eten less frequently (Fig. 7). Grss chemicl nd biomechnicl properties In Experiment 1, T. ustrlis contined 9.3±.5% protein nd 27.4±1.1% crbohydrte by dry mss (Fig. 3B). Themed ustrlis differed chemiclly between the two experiments, reflecting chnges

6 334 The Journl of Experimentl Biology 216 (2) Crbohydrte (mg dy 1 ) A Trget rtio Fibre:wter (g g 1 ) B Trget rtio C. dctylon T. estivum T. ustrlis Fig. 5. (A) The rtio nd mount of protein nd crbohydrte extrcted (digested nd bsorbed) in 24 h by locusts feeding on Cynodon dctylon (circles), Themed ustrlis (tringles) or Triticum estivum (downwrd-pointing tringles), following genertion of differences in the size of the foregut nd cec. Filled symbols, 28P:14C; open symbols, 14P:28C. N=45 for ech grss nd N=68 for ech diet tretment. (B) Physiochemicl properties of the three grsses. For ll prmeters determined, the grsses were significntly different (P<.5) Protein (mg dy 1 ) C:P (g g 1 ).2. with lef ontogeny given the time between the two experiments. In Experiment 2, the rtio of P:C ws significntly lower (F 1,19 =18.22, P=.1) s the concentrtion of protein ws greter; in ddition, the mount of wter per unit dry mtter ws ~25% higher (F 1,19 =13.13, P=.2). In Experiment 2, ll three grsses differed physiclly nd chemiclly (Tble 1, Fig. 5B). The verge lef mss re of T. ustrlis ws lmost double tht of C. dctylon nd three times tht of T. estivum (F 2,27 =174.25, P<.1), nd T. estivum hd lmost double the wter per unit dry mtter of C. dctylon, with T. ustrlis hving the lest (F 2,34 =637.95, P<.1). Within the dry mtter, the concentrtion of crbohydrte ws the sme in ll grsses (25.9±.7%; F 2,12 =.2, P=.982), but protein (F 2,12 =11.7, P=.2) nd fibre (F 2,12 =5.8, P=.24) vried. Themed ustrlis contined lest protein (16.±.6%) nd the most fibre (39.9±.8%), nd T. triticum hd the most protein (27.5±.5%) nd lest fibre (36.8±.8%), with C. dctylon hving intermedite mounts (protein: 25.7±.5%; fibre: 38.8±.5%; Tble 1). This resulted in the digestible nutrients, protein nd crbohydrte, being most concentrted in T. estivum nd most dilute in T. ustrlis (Fig. 5B). DISCUSSION This study demonstrtes tht incresing the mss of the GIT serves to mximize the uptke of ll nutrients rther thn redress nutrient imblnces. Locusts confined to diets in which protein ws supplied in higher thn optiml concentrtion reltive to crbohydrte hd hevier GITs thn locusts either llowed to self select their protein nd crbohydrte intkes or confined to diets with surplus of crbohydrte reltive to protein. The GIT ws hevier becuse of increses in the msses of the foregut nd midgut cec (Fig. 1). These chnges were ssocited with 2 4% increse in bsorption of nutrients over 24 h when feeding from three biomechniclly nd chemiclly different grsses (Fig. 4). Although the efficiencies with which protein nd crbohydrte were bsorbed were grss-species specific (Fig. 5A), greter bsorption of mcronutrients occurred becuse locusts with hevier GIT ingested lrger mels, which were bsorbed with the sme efficiency nd with no chnge in the time food ws retined in the GIT (Fig. 7). Consequently, increses to the mss of the foregut nd midgut cec on high P:C diet led to the bsorption of limiting crbohydrte t the potentil cost of supplying the tissues with even more protein (Fig. 5A). The dry mss of the GIT is often used s mesure of size or cpcity (e.g. Yng nd Joern, 1994b; Ny et l., 27; Sørensen et l., 21). However, interpreting increses in dry mss in terms of djustments to the cpcity of the GIT is problemtic becuse the reltionship between dry mss nd volume is not strightforwrd. In vertebrtes, increses to both the surfce re (e.g. Dykstr nd Krsov, 1992) nd thickness of the tissues hve been found to ccompny incresed mss of the GIT (reviewed by Strck, 25). In the present study, increses in mss of the foregut most likely represented volumetric increse rther thn chnges to the thickness of the foregut tissue. This follows from the close ssocition seen between the extent of chnge in foregut mss on high-protein diet nd the similr proportionl increses in mss of food consumed nd verge mel durtion. We suggest tht the commensurtely incresed mss of midgut cec llowed these lrger mels to be digested nd bsorbed t the sme rte s on lower-protein diets. Previous work hs shown tht the nterior rms of the cec cn vry gretly in mss with diet nd development, while the mss of the posterior cecl rms nd ventriculus remin reltively constnt (Chpmn, 1988). Incresed mss of the nterior cecl rms is likely to enhnce both the ctivities of digestive enzymes nd the rtes of nutrient bsorption. The precise nture of GIT remodelling is thought to determine the reltionship between the extrction of nutrients nd the rte t P+C bsorbed residuls Foregut + cec residuls Fig. 6. Locusts with lrger gstrointestinl trcts thn were explined by body size hd corresponding increse in nutrient bsorption tht ws lso not explined by body size. This cn be seen by plotting the residuls of ANCOVA nlysis (with dietry tretment nd grss s fctors nd body size the covrite).

7 Lrger mels not improved digestibility 335 Mel durtion (min) Corrected mel durtion (min) Time (min) T. ustrlis T. estivum C. dctylon 28P:14C 14P:28C Time (min) T. ustrlis T. estivum C. dctylon Fig. 7. Using the durtion of time spent feeding s surrogte for intke, we modelled the potentil uptke rtes nd then corrected this for the mcronutrient (P+C) content nd the percentge digested (inset) to model the rtes of mcronutrient bsorption. Although we did not mesure mel size, when the mel durtion is djusted for mcronutrient content digested, this is highly correlted with mcronutrient bsorption over the 24 h the locusts te the grsses (Fig. 4C), s illustrted by the verticl double-heded rrows. The regulr rrows (thick, locusts ingesting 14P:28C; thin, locusts ingesting 28P:14C) indicte the nth mel nd demonstrte tht differences in nutrient bsorption for T. ustrlis nd T. triticum cn only hve been due to differences in mel sizes, s intermel durtions were the sme (s indicted by the horizontl double-heded rrows). which this occurs. The flow of food through the GIT is considered function of GIT volume, the mobility of the substrte, the initil mount of substrte, the rte of enzymtic hydrolysis (i.e. how quickly cn the substrte be digested) nd subsequent bsorption. Modelling the GIT s tube shows tht volume cn be incresed by incresing the length nd/or dimeter. It is thought tht lengthening the GIT will increse the efficiency of nutrient bsorption, s the surfce re to volume rtio is mintined. Consequently, intke rte is mintined, but s ech mel tkes longer to pss through the GIT, greter proportion of nutrients cn be extrcted. However, if the increse is to the dimeter, then the time mel is retined should lso increse proportiontely or, if retention time remins constnt, then the efficiency of bsorption should decline (Sibly, 1981; Yng nd Joern, 1994; Rubenheimer nd Simpson, 1996; Krsov nd Hume, 1997; Rubenheimer nd Simpson, 1998). For ech grss species, regrdless of mel size, similr proportion of ingested nutrients were bsorbed in the sme mount of time (Fig. 7). It is difficult to envisge the presumed chnge in volume occurring becuse the foregut grew longer, s the size of the locusts from both tretments ws the sme nd the foregut is n unfolded tube. Thus, the most likely explntion is tht the foregut incresed in dimeter, nd tht digestive efficiency nd intermel durtions were similr regrdless of mel sizes becuse greter surfce re in the cec llowed for increses to the production of digestive enzymes, nutrient trnsporters nd/or surfce re for pssive nutrient bsorption. Little is known regrding the regultion of bsorption in insects; however, studies on mmmls hve shown tht intestinl nutrient trnsporters re regulted positively by substrte levels in the diet (Dimond nd Krsov, 1987). Physicl plsticity of the GIT hs been well described for mny tx (e.g. Hume, 1989; Krsov nd Hume, 1997; Strck, 25; Ny et l., 27) in response to chnges in either energy demnd or energy density in the diet, nd is thought to be wy of blncing the expensive costs of mintining the GIT ginst returns. In mmmls it is believed tht the GIT ccounts for ~25% of digested energy (McBride nd Kelly, 199; Cnt et l., 1996), nd thus ny upregultion is only justified if ccompnied by pyoff in clories (Krsov nd Dimond, 1983). While this is clerly the cse for nimls following period of strvtion (e.g. Secor nd Dimond, 1998; Hume et l., 22) or when fttening up for migrtion (e.g. vn Gils et l., 28), enlrgement of the GIT when dietry nutrients re supplied in sub-optiml rtios hs been difficult to interpret using energy-bsed cost benefit rguments (Rubenheimer nd Bssil, 27; Sørensen et l., 21). A previous study by Rubenheimer nd Bssil (Rubenheimer nd Bssil, 27) found tht locusts ingesting protein-bised diets (28P:14C) hd hevier GITs thn locusts feeding on ner optiml diet (21P:21C). Locusts ingesting slightly crbohydrte-bised diet (14P:28C) over the entire fifth stdium showed bimodl response, with pproximtely hlf of the insects hving GITs similr in mss to locusts ingesting n optiml diet nd hlf hving hevier GITs, similr to locusts on protein-bised diet. Although some insects prtition protein nd crbohydrte digestion into different regions of the GIT (Terr nd Ferreir, 212), we found n increse in the msses of the foregut nd midgut cec only for locusts ingesting protein-bised diets (Fig. 1). Similr to the results of Rubenheimer nd Bssil (Rubenheimer nd Bssil, 27), in the present study, incresed mss of the GIT ws ssocited with incresed protein intke, but we did not find similr increse, nor bimodl response, in locusts fed crbohydrte-bised diet (14P:28C). The functionl outcomes of increses in the mss of prts of the GIT when protein-rich/crbohydrte-poor diets re eten hve been difficult to interpret. Does the increse in size fcilitte the incresed uptke of the deficient nutrient, i.e. compenstory response, or is the response counter-compenstory, with the GIT growing in response to excess protein intke nd, s consequence, re tissues re supplied with even more of this excess nutrient (Rubenheimer nd Bssil, 27; Sørensen et l., 28)? Our results show tht the incresed mss of the foregut nd cec led to n increse in intke Tble 1. Physiochemicl properties of the three grsses used in Experiment 2 Cynodon dctylon Themed ustrlis Triticium estivum P Protein (%) 25.7±.5 b 16.± ±.5 b.2 Soluble crbohydrte (%) 25.9± ± ± Wter:dry mtter (g g 1 ) 4.89± ±.9 b 7.9±.16 c <.1 Cell wll (%) 38.8± ± ±.8 b.24 Lef mss re (mm 2 g 1 ) 25.1± ±1.6 b 16.5±.3 c <.1 Dt re mens ± s.e.m. Different letters indicte mens tht re significntly different (P<.5, Tukeyʼs post hoc comprison). Bold P-vlues indicte significnt differences between grsses.

8 336 The Journl of Experimentl Biology 216 (2) nd nutrient bsorption. This is counter-compenstory effect in terms of mintining nutritionl homeostsis, i.e. the nutritionl conditions triggering the increse in mss of the GIT result in the tissues being supplied with even more of the nutrient (protein) in excess of requirements (Fig. 5A, results for C. dctylon nd T. estivum). It seems counterintuitive tht n niml would increse investment in n orgn tht results in incresed uptke of nutrient surplus to requirements with potentil reductions in fitness (Rubenheimer et l., 25; Boersm nd Elser, 26; Lee et l., 28). However, it hs been rgued tht insects best scertin their nutritionl sttus post-bsorptively, nd thus it is dvntgeous to rpidly bsorb nd then subsequently excrete surpluses rther thn pssing excess nutrients through the digestive trct (Simpson nd Rubenheimer, 1993). If this ws the cse, then the GIT should be bigger when crbohydrte ws supplied in excess reltive to protein, nd this did not occur (Fig. 1). Our results might be tken to imply tht the long-term cost of ingesting surplus protein is smller thn the cost of ingesting too little crbohydrte. However, it is not cler from existing dt why this is, s L. migrtori ppers to hve very limited cpcity to convert excess protein to crbohydrte vi the demintion of proteins (Rubenheimer nd Simpson, 23). Locusts feeding on the 35P:7C foods digested nd bsorbed greter proportion of ingested crbohydrte from T. ustrlis thn locusts from ll other tretments (Fig. 3B), result consistent with our previous findings where protese ctivity decresed when locusts were ingesting 35P:7C diet (Clissold et l., 21). This differentil relese of digestive enzymes occurred in response to very shortterm nutrient imblnces, over period of time tht is too short to induce detectble chnges in the mss of the cec (Clissold et l., 21). The over-ingestion of either protein or crbohydrte with respect to the other resulted in reduced ctivity of the digestive enzyme for the excess substrte; i.e. when 7P:35C diet ws eten, mylse ctivity declined, nd when 35P:7C ws ingested, α- chymotrypsin ctivity ws reduced (Clissold et l., 21). Interestingly, in the present study, differences in digestibility only occurred when protein nd crbohydrte were very imblnced with respect to requirements (35P:7C) (Fig. 3B), nd we detected no differences in the efficiency with which protein or crbohydrte were digested nd bsorbed when the locusts were feeding on slightly protein/crbohydrte-imblnced foods (Fig. 3B, Fig. 5A). However, the msses of the foregut nd cec incresed in response to the ingestion of even slight excesses of protein (i.e. 28P:14C) (Fig. 1). Thus, it ppers tht modultion of digestive enzymes occurs rpidly in response to extreme nutrient imblnces, while chnges in mss pper to occur more slowly nd only in response to the overingestion of protein reltive to crbohydrte. Knowledge of how the GIT functions is n essentil component of understnding how behviourl nd physiologicl mechnisms re coordinted by nimls in reltion to the ecologicl niche they occupy (Simpson et l., 21). We suggest tht future studies need to consider ny chnges to digestive function with regrd to the optiml requirements of n orgnism, together with knowledge of the orgnism s current nutritionl sttus nd the supply of nutrients from ingested food (i.e. the rtio of P to C bsorbed rther thn tht in the ingest). ACKNOWLEDGEMENTS We thnk Tim Dodgson, Nz Sorn nd members of the Behviour, Physiology nd Ecology lbortory for generl ssistnce nd locust rering. FUNDING S.J.S. ws supported by n Austrlin Reserch Council Lurete Fellowship. REFERENCES Behmer, S. T. (29). 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