Sweet problems: insect traits defining the limits to dietary sugar utilisation by the pea aphid, Acyrthosiphon pisum

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1 1395 The Journl of Experimentl Biology 29, Pulished y The Compny of Biologists 26 doi:1.1242/je.2148 Sweet prolems: inset trits defining the limits to dietry sugr utilistion y the pe phid, Ayrthosiphon pisum A. E. Dougls 1, *, D. R. G. Prie 1, L. B. Minto 1, E. Jones 1, K. V. Pesod 1, C. L. M. J. Frnçois 1, J. Prithrd 2 nd N. Boonhm 3 1 Deprtment of Biology (Are 2), University of York, PO Box 373, York, YO1 5YW, UK, 2 Deprtment of Biosienes, University of Birminghm, Edgston, Birminghm, B15 2TT, UK nd 3 Centrl Siene Lortory, Snd Hutton, York, YO41 1LZ, UK *Author for orrespondene (e-mil: ed2@york..uk) Aepted 6 Ferury 26 Plnt phloem sp is n extreme diet for nimls, prtly euse of its high nd vrile sugr ontent. The physiologil nd feeding trits of the pe phid Ayrthosiphon pisum tht define the upper nd lower limits to the rnge of dietry surose onentrtions utilised y this inset were determined priniplly using hemilly defined diets ontining mol l 1 surose. On the diets with.125 mol l 1 nd 1.5 mol l 1 surose, the phids died s lrve within 8 nd 14 dys of irth, respetively. On the other diets, 6 96% of phids developed to dulthood, nd the.5 mol l 1 nd.75 mol l 1 diets supported the highest feundity. The diet with.125 mol l 1 surose ws ingested t 36% of the rte of the.25 mol l 1 surose diet, ut >9% of ingested surose-ron ws ssimilted on oth diets. This suggests tht the lower limit is ditted y the phid feeding response, speifilly, requirement for miniml onentrtion of surose for sustined feeding. The hemolymph osmoti pressure of phids on diets with Summry mol l 1 surose ws up to 68% higher thn on mol l 1 surose diets, ut diet onsumption nd surose-ron ssimiltion ws not redued on the very high surose diets reltive to 1. mol l 1 surose. This suggests tht filure of the osmoregultory pity of the insets on high surose diets my define the upper limit to the rnge of dietry surose utilised y the phids. The men hemolymph osmoti pressure of phids on plnts with phloem sp ontining mol l 1 surose ws 1.61±.63 MP (men ± s.e.m.), not signifintly different from tht (1.47±.59 MP) on diets with mol l 1 surose. It is onluded tht the osmoregultory response of phids to diets nd plnts re omprle, nd, more generlly, tht the feeding nd osmoregultory pilities of the phids re omptile with the phloem sugr levels ommonly enountered y phids feeding on plnts. Key words: phid, Ayrthosiphon pisum, osmoregultion, phgostimultion, phloem sp, surose. Introdution Orgnisms live within their environmentl limits or, more preisely, within multidimensionl envelope defined y the onditions nd resoures omptile with their physiologil pilities (Willmer et l., 2; Begon et l., 25). Outside of this envelope, they disply redued growth, reprodution nd survivorship. A gret mny studies hve determined the environmentl limits of orgnisms, usully with respet to ioti ftors, suh s temperture, irrdine, wter vilility, nd to speifi nutrients or toxins. These dt provide oth vitl informtion for studies of physiologil mehnism nd roust explntions for the eology of mny tx (e.g. Spier nd Gston, 1999; Hohhk nd Somero, 22). In order for environmentl limits to hve rtionl sis nd preditive vlue, reserh should ddress the physiologil proesses defining the environmentl limits s well s tloguing of orgnisml response to environmentl extremes. The physiologil rekdown of orgnisms under extreme onditions ommonly involves multiple proesses nd orgn systems, ut it is generlly underpinned y single, primry proess tht reks down most rpidly or in response to the smllest hnge in onditions. Deteriortion in other proesses is seondry; either more extreme onditions re required for their rekdown or their deteriortion is onsequene of the filure of the primry proess. This study onerns the nutritionl limits for plnt phloem sp feeders. Lrgely euse its nutrient ontent is unlned, phloem sp is n extreme food soure tht is used s the dominnt or sole diet of very few nimls, speifilly insets

2 1396 A. E. Dougls nd others of the order Hemipter, inluding phids, whitefly, plnthoppers nd some penttomid ugs (Dougls, 23). The fous of this pper is the high nd vrile sugr ontent of phloem sp. In mny plnts, surose is the dominnt phloemmoile sugr (Fisher, 2), nd relile reports of its onentrtion in phloem sp rnge from.2 to 1.5 mol l 1, vrying with environmentl onditions, espeilly temperture nd irrdine, ut lso plnt speies nd developmentl ge (Winter et l., 1992; Geiger nd Servites, 1994; Kehr et l., 1998). A seond nutritionl prolem posed y phloem sp is its low essentil mino id ontent whih, in phids, is resolved y symioti teri of the genus Buhner tht provide the inset with supplementry mino ids (Dougls, 1998). Other phloem-feeders possess symioti miroorgnisms (Buhner, 1965) whih, y nlogy to phids, re lso elieved to provide essentil mino ids. The response of phloem-feeding insets to phloem sugrs is physiologilly omplex, involving nutritionl, osmoregultory nd ehviourl omponents. Phloem sugrs re the prinipl ron soure nd respirtory fuel for these insets (Rhodes et l., 1996; Fevy et l., 1999; Slvui nd Crfts-Brndner, 2). They re lso responsile for the high osmoti pressure of phloem sp, up to five times tht of inset ody fluids. Phloem-feeding insets ssimilte only proportion of the ingested sugr, fter hydrolysis y the gut surse to its onstituent monoshrides (Rhodes et l., 1996; Ashford et l., 2). In phids, the osmoti pressure of the remining sugr (whih is voided in honeydew) is redued y gut trnsgluosidse, whih tlyses the polymeristion of the monoshride, espeilly gluose, into oligoshrides in the gut (Fisher et l., 1984; Wlters nd Mullin, 1988; Rhodes et l., 1997; Wilkinson et l., 1997; Ashford et l., 2). In this wy, phids void losing wter from their ody fluids, espeilly their hemolymph, to the gut. The ehviourl spet to the inset response to phloem sugrs rises from their strong feeding response, suh tht the mount of surose ingested does not vry in simple fshion with dietry onentrtion. For exmple, phids require ertin miniml onentrtion of dietry surose for sustined feeding (i.e. surose is phgostimulnt) ut, ove this miniml level, phids ompenste for vrition in dietry onentrtion y feeding fster from diets of lower surose onentrtions (Mittler nd Meikle, 1991; Simpson et l., 1995). The speifi purpose of this study ws to identify the nutritionl, osmoregultory nd ehviourl trits of pe phids, Ayrthosiphon pisum, tht define the upper nd lower limits to the rnge of dietry sugr onentrtions utilised. This reserh ws founded on the exellent understnding of the nutritionl physiology of surose utilistion t the whole orgnism level, espeilly for the pe phid (reviewed y Dougls, 23). Most of the experiments were onduted on phids rered on hemilly defined diets, so tht the dietry inputs were known nd ould e mnipulted preisely, with finl set of experiments tht ompred key results of dietrered phids to phids feeding from plnts. Mterils nd methods The phids nd plnts Prthenogeneti femles of the pe phid Ayrthosiphon pisum (Hrris) lone LL1 were mintined routinely on preflowering Vii f v. The Sutton t 2 C with 18 h:6 h L:D 1 nd irrdine of 1 mol m 2 s photosyntheti tive rdition (PAR). For experiments with phids on hemilly defined diets, the offspring of plnt-rered ptere were trnsferred to sterile diets of formultion A ontining.15 mol l 1 mino ids (Dougls nd Prosser, 1992), nd mintined under the sme onditions s the ultures on plnts. The offspring were ptere. Two experimentl designs were dopted. For studies of life-time performne, 2 replite phids within 12 h of irth were trnsferred individully to eh of seven test diets with.125 mol l 1,.25 mol l 1,.5 mol l 1,.75 mol l 1, 1. mol l 1, 1.25 mol l 1 nd 1.5 mol l 1 surose, nd the diet shets were hnged twie weekly. For nlysis of short-term responses of phids to dietry surose onentrtion, 2-dy-old phids were trnsferred from plnts to diets with.5 mol l 1 surose, nd then 1 replite phids were rered individully on eh of the seven test diets, s ove, from dy 6 to dy 8. For experiments on phids feeding from plnts, preflowering V. f (3 weeks fter sowing) were exposed to three test onditions: (1) 24 h D t 2 C for 36 h; (2) 18 h L (6 mol m 2 s 1 ):6 h D t 2 C for 2 dys; nd (3) 18 h L (6 mol m 2 s 1 ) t 2 C:6 h D t 12 C for 2 dys; with ontrol plnts under stndrd ulture onditions of 18 h L (1 mol m 2 s 1 ):6 h D t 2 C. Aphids rered from dy 2 to dy 6 on the.5 mol l 1 surose diet were trnsferred to these plnts, whih were returned to test onditions for further 2 dys, when the surose ontent of the phloem sp nd the osmoti pressure of the phid hemolymph were quntified (see elow). Aphid performne ssys For the life-time performne experiment, the phids were heked dily from irth until they died, nd the dte on whih they developed to dulthood nd the numer of offspring produed per dy were sored. Aphid performne etween dy 6 nd dy 8 ws ssessed y reltive growth rte [RGR: log e (dy-8 mss/dy-6 mss)/2], with eh phid weighed on dy 6 nd dy 8 to the nerest g on Mettler MT5 mirolne. Rdiohemil nlyses of phid feeding nd surose ssimiltion Ten replite 6-dy-old phids were trnsferred individully to 5 l of eh test diet with.146 MBq [ 3 H]inulin nd.148 MBq [ 14 C]surose ml 1 diet on Perspex ring (3.5 m dimeter,.5 m height). Honeydew produed y the feeding phid ws deposited onto 3.5 m GF/C filter (Whtmn, Midstone, Kent, UK) pled eneth eh ring. On dy 8, eh filter ws dded to 4 ml sintilltion fluid (Ultim Gold XR, Perkin Elmer, Boston, MA, USA) nd the 3 H nd 14 C ontents were determined in sintilltion ounter (Tri-Cr, Perkin

3 Sugr utilistion y phids 1397 Elmer, Boston, MA, USA) with preset 3 H/ 14 C dul windows nd quenh urve. The men of ounts otined from filters of two phids feeding from non-rdiotive diets of the sme formultion ws sutrted from experimentl dt. Inulin is not degrded or ssimilted y pe phids (unpulished results), nd the volume of diet ingested nd dietry surose-derived ron ingested nd egested were lulted from the 3 H nd 14 C ontents of filters, s desried y Wright nd oworkers (Wright et l., 1985) nd Dougls nd oworkers (Dougls et l., 21). The surose-ron ssimilted ws otined y sutrtion of surose-ron egested from tht ingested, nd the ssimiltion effiieny ws quntified s the proportion of ingested surose-ron tht ws ssimilted. Osmoti pressure determintions Eh of 1 replite groups of five 6-dy-old phids were strved for 2 h to ensure their guts were evuted nd then trnsferred to either test diet or the xil surfe of lef of plnt suspended over 3.5 m Petri dish filled with 99% n-hexdene (Fisher, Loughorough, Leis, UK). Honeydew droplets relesed from the phids snk elow the surfe of the hexdene. On dy 8, two honeydew smples were olleted from eh dish into pulled miropipettes, with smll volume of hexdene either side of the honeydew to prevent evportion of the smple. In prllel, hemolymph smples were olleted from phids on eh test diet. The phid ody ws held under wter-sturted light white oil (Sigm-Aldrih, Poole, Dorset, UK) to prevent ny evportion, nd one hindleg ws mputted y single, shrp pull on the leg with foreps. The hemolymph droplet tht exuded from the stump ws olleted in pulled miropipette, etween two droplets of oil. The honeydew nd hemolymph smples were stored t 8 C prior to nlysis. The osmolrity of.5.5 nl smples ws determined y freezing point depression (Mlone nd Tomos, 1992) lirted ginst.6 mol l 1 NCl stndrd. Quntifition of Buhner symioti teri DNA from 1 individul 8-dy-old phids from eh test diet ws extrted y the method of Cenis et l. (Cenis et l., 1993), with minor modifition for single inset nlysis. Reltime PCR (TqMn ) retions were set up in 96-well retion pltes. Cyling nd dt olletion were performed using n ABI Prism 79HT Sequene Detetion System (Applied Biosystems, Wrrington, UK). The primers nd proe, designed for 12 p region of the dnk gene of Buhner APS (GenBnk ession numer D88673) with the Primer Express TM softwre (Applied Biosystems), were: forwrd primer TGT-AAA-TCC-AGA- TGA-AGC-TGT-AGC-AG; reverse primer ACC-CAT-AGT- TTC-AAT-TCC-TAG-GGA; nd proe CAG-GGA-GGA- GTT-CTC-TCT-GGT-GAT-GTT-AAA-GAC-GTC-T. (The nuleotide numers of sequene D88673 orresponding to 5 nuleotide of the forwrd nd reverse primers nd the proe were 192, 1132 nd 1215, respetively.) For the proe, the 5 terminl reporter dye ws FAM (6-roxyfluoresein) nd the 3 quenher dye ws TAMRA (tetr-methylroxyrhodmine). The proe nd primers were supplied y MWG- Bioteh AG (Eerserg, Germny). The retion mixture onsisted of 1 PCR uffer (1 mmol l 1 Tris HCl ph 8.1 t 25 C, 5 mmol l 1 KCl,.1% geltin), 5.5 mmol l 1 MgCl 2,.2 mmol l 1 eh of datp, dgtp, dctp nd dutp,.5 l Rox Referene Dye (Invitrogen Ltd, Pisley, UK),.3 mol l 1 eh of forwrd nd reverse primers,.1 mol l 1 fluoresene-lelled proe,.625 i.u. Hot Tq DNA Polymerse (BioGene Ltd, Kimolton, Cmridgeshire, UK), nd templte DNA in totl volume of 25 l. The yling onditions were: 95 C for 1 min followed y 4 yles of 95 C for 15 s nd 6 C for 1 min. Vlues of threshold yle (C T : the yle t whih signifint inrese in fluoresene ours) elow 4 were tken s positive result. Stndrd urves were generted with seril dilutions of the templte, synthesised nd gel-purified y MWG-Bioteh AG, nd dnk opy numer ws estimted from the stndrd urve. The dt were normlised to totl DNA ontent per smple, determined y NnoDrop ND-1 spetrophotometer (LTeh Interntionl, Ringmer, Est Sussex, UK), to ontrol for ny vrition in effiieny of DNA extrtion. Anlysis of free mino ids of phids Ten replite dy-8 phids from eh test diet were weighed individully nd then hnd-homogenised in.1 ml ie-old 8% methnol. The homogente ws entrifuged t 5 g for 5 min t 4 C, nd the superntnt ws stored t 2 C. Amino ids were seprted y reverse-phse HPLC following derivtistion with o-phthldildehyde (Jones et l., 1981) using Hewlett-Pkrd HP11 Series utosmpling LC system with C 18 ZORBAX TM Elipse XDB-C8 olumn nd fluoresene detetion. The mino ids were quntified y omprison to the AA-S-18 stndrd mino ids (Sigm) supplemented with tryptophn, sprgine nd glutmine. Surse ssy Ten individul 8-dy-old phids from eh test diet were homogenised in 2 l 5 mmol l 1 Hepes ph % (v/v) Triton X-1, nd then entrifuged t 1 g for 5 min. The surse tivity of the superntnt ws ssyed y the method of Dhlqvist (Dhlqvist, 1984) using regents from the Sigm Dignostis gluose ssy kit with the hromogen o- dinisidine t 125 g ml 1. One unit of tivity is defined s the mount of enzyme tht releses 1 mol gluose from surose per minute t 37 C, nd the tivities were normlised to phid protein, s determined y the BCA protein mirossy (Piere Biotehnology, Rokford, Illinois, USA), ording to the mnufturer s instrutions, with ovine serum lumin s stndrd. Totl phid surse tivity ws dopted s n index of gut surse tivity following preliminry experiments tht ssigned >98% of the totl surse tivity to the gut (D.R.J.P., unpulished dt). Quntifition of phloem sp surose Phloem sp smples of Vii f were olleted y styletomy of dult ptere using proedure modified from

4 1398 A. E. Dougls nd others the method of Fisher nd Frme (Fisher nd Frme, 1984). A feeding phid in suitle position ws seleted y exmintion under disseting mirosope. The pltinum needle of miroutery unit (Synteh CA-5, Hilversum, Netherlnds) ws positioned with miromnipultor, nd the stylets were severed y high frequeny pulse from the miroutery unit. A 2 m dimeter Perspex ring with greseoted ruer se ws lipped to the lef round the severed stylets nd immeditely filled with wter-sturted light white minerl oil (Sigm), into whih the phloem sp exuded from the stylet stump. The phloem sp ws olleted into miropillry tue kfilled with minerl oil. Susequently, susmples of known volume were prepred using onstrition pipettes, working under oil to prevent evportion, nd then diluted into 1 l distilled wter nd stored t 8 C prior to nlysis. To determine the surose ontent, eh 1 l smple ws hydrolysed to ompletion with.1 i.u. invertse (Sigm I- 454) in 5 mmol l 1 sodium ette uffer, ph 4.5 t 37 C for 3 min, nd the gluose produed ws determined y the Sigm Dignostis gluose ssy kit, following the mnufturer s instrutions, ut with o-dinisidine t 1 g ml 1, with gluose stndrds. Sttistil nlysis Dt sets were nlysed y prmetri tests [t-test, nlysis of vrine (ANOVA) or lest squres regression], following onfirmtion tht they were normlly distriuted (Anderson- Drling test) with homogenous vrines (Brtlett s test). This required logrithmi or rsine-squre root trnsformtions where indited. For nlyses of phid feeding rte nd surose-ron ssimiltion, initil phid weight ws inluded in the ANOVA s ovrite. Tukey s honestly signifint differene method ws used for pirwise omprisons ontriuting to signifint ANOVA differenes; nd men vlues tht were not signifintly different (P>.5) y this test re indited y the sme supersript letter in figures nd tles. Exeptionlly, no trnsformtion onduted yielded homogenous vrines for phid lifespn, nd this ws nlysed y the nonprmetri Kruskl Wllis test. Results Aphids on hemilly defined diets The first experiments quntified the lifetime performne of the pe phids on hemilly defined diets of different surose onentrtions. Aphids lived for etween 3 nd 31 dys (Fig. 1A), nd the medin lifespn vried signifintly with dietry surose (Kruskl Wllis: H 6 =73.69, P<.1). At lest hlf the phids on diets with surose onentrtions in the rnge mol l 1 lived for t lest 15 dys, ut phid lifespn on the diets with lowest nd highest surose onentrtions ws severely urtiled, with ll the phids ded y dy 8 on the.125 mol l 1 diet nd y dy 14 on the 1.5 mol l 1 diet. At the dily soring of this experiment, most of the phids were oserved tthed to the diet shets on ll Numer of live phids Cumultive no. offspring phid 1 Reltive growth rte Time (dys) Dietry surose (mol l 1 ) for A nd B Time (dys) Dietry surose onentrtion (mol l 1 ) Fig. 1. Aphid performne on diets of different surose onentrtion. (A) Lifetime survivorship. (B) Cumultive reprodutive output per live phid. (C) Reltive growth rte of 6- to 8-dy-old finl instr lrve tht hd een rised from dy 2 to dy 6 on diet with.5 mol l 1 surose (mens ± s.e.m.). Vlues tht re not signifintly different (P>.5) re indited y the sme letter. diets exept.125 mol l 1 surose, for whih mny of the phids were moving round the ge or settled on the ge wll wy from the diet shet. All the phids on diets with.125 mol l 1 nd 1.5 mol l 1 surose died s lrve. Between 12 nd 18 of the 2 phids on eh of the other diets developed to dulthood on dy 9 to dy 16. Lrviposition rtes were highest on.5 mol l 1 nd.75 mol l 1 surose diets, with totl feundity of offspring per live phid, nd were depressed y 6 7% on.25 mol l 1 nd 1. mol l 1 diets (Fig. 1B). Just two phids on the 1.25 mol l 1 surose diet lrviposited, produing one nd three offspring, respetively. A B C

5 Sugr utilistion y phids 1399 The seond experimentl design ddressed the impt of dietry surose on the performne of phids tht hd een rised from dy 2 to dy 6 on.5 mol l 1 surose. All the phids were in the finl lrvl stdium on dy 6. They survived the 2-dy experiment to dy 8. Aphid reltive growth rte (RGR; Fig. 1C) vried signifintly with dietry surose (F 6,63 =14.15, P<.1). The post ho nlysis reveled signifintly lower RGR, first, on.125 mol l 1 surose diet thn on ll the other diets nd, seond, on the Surose-ron ingested (μmol) Diet volume ingested (μl) Assimiltion effiieny ,,, Dietry surose onentrtion (mol l 1 ) Fig. 2. Diet ingestion nd ssimiltion y 6- to 8-dy-old pe phid lone LL1 on diets of different surose onentrtion. (A) Volume of diet ingested (mens ± s.e.m.). (B) Dietry surose-ron ingested (losed symols) nd ssimilted (open symols) (mens ± s.e.m.). (C) Assimiltion effiieny [1 (moles ssimilted/moles ingested]. Vlues tht re not signifintly different (P>.5) re indited y the sme letter., A B, C 1.25 mol l 1 nd 1.5 mol l 1 diets ompred to.5 1. mol l 1 diets. Diet onsumption y the 6- to 8-dy-old phids vried nerly sixfold with dietry surose onentrtion (Fig. 2A), nd the vrition ws sttistilly signifint (ANOVA for logtrnsformed dt: F 6,53= 7.87, P<.1). The volume ingested inresed progressively with deresing dietry surose from 1. mol l 1 to.25 mol l 1, for whih the men volume ingested, 2.23 l, ws the highest men vlue otined. Aphids on the.125 mol l 1 diet ingested just 36% of the volume onsumed y those on the.25 mol l 1 diets nd similr mounts to phids on mol l 1 diets. In supplementry nlysis, the RGR ws regressed on the volume of food ingested y eh phid. A signifint regression with positive slope ws otined for the phids on the.125 mol l 1 diet (F 1,4 =26.71, P=.7, r 2 =87.%) (Fig. 3) ut for no other diet (dt not shown), suggesting tht feeding rte is prtiulrly importnt determinnt of growth rte on the.125 mol l 1 diet. Fig. 2B shows the solute mounts of surose-ron ingested nd ssimilted; nd the ssimiltion effiieny is shown in Fig. 2C. All three mesures of sugr utilistion vried signifintly with dietry surose onentrtion [ANOVA for surose-ron ingested: F 6,53 =8.32, P<.1; surose-ron ssimilted: F 6,53 =9.48, P<.1; ssimiltion effiieny (rsin-squre root trnsformed dt): F 6,53 =22.31, P<.1]. The phids feeding on.25 mol l 1 nd.5 mol l 1 diets ssimilted signifintly more surose thn those on.75 nd 1. mol l 1 diets nd omprle mounts to the phids on the 1.25 mol l 1 surose diet; nd this result ould e ttriuted to omintion of high feeding rtes nd ssimiltion effiieny on the.5 mol l 1 nd espeilly.25 mol l 1 diets. Although the phids on.125 mol l 1 diet ssimilted more thn 9% of the surose ingested, their low feeding rtes ment tht the solute mount of surose they ingested ws less thn 2% of tht for phids on the.25 mol l 1 diet. The phids on 1.25 mol l 1 nd 1.5 mol l 1 diets ssimilted more thn those on.75 mol l 1 nd 1. mol l 1 diets. For phids on diets with.125 mol l 1 to 1. mol l 1 surose (nd osmoti pressure of.9 4. MP), the men osmoti pressure of the hemolymph rnged etween 1.35 nd Reltive growth rte (g g 1 dy 1 ) log 1 (μl diet ingested) Fig. 3. Regression of phid reltive growth rte on volume of diet ingested on the.125 mol l 1 surose diet.

6 14 A. E. Dougls nd others Osmoti pressure (MP) Dietry surose onentrtion (mol l 1 ) 1.75 MP, with no onsistent trend in reltion to dietry surose onentrtion; ut it inresed progressively with higher dietry surose onentrtions to 2.3 MP on the 1.5 mol l 1 surose diet (of osmoti pressure 5.8 MP; Fig. 4). The vrition in osmoti pressure with dietry surose ws sttistilly signifint (ANOVA: F 6,63 =2.86,.5>P>.1), with signifintly elevted men vlue on the 1.5 mol l 1 diet. The hemolymph osmoti pressure of the phid lone used here ws somewht greter thn vlues of ~1. MP otined in our previous nd ongoing reserh on different pe phid lones (Wilkinson et l., 1997; Krley et l., 25) (E.J., unpulished dt), inditive of intrspeifi vrition. Elevted osmoti pressure of the hemolymph of pe phids hs een desried previously under two onditions: () posymiosis, i.e. the experimentl elimintion of the symioti teri, Buhner sp., tretment tht uses n inrese in free mino id levels nd onsequent inrese in hemolymph osmoti pressure (Wilkinson et l., 1997); nd () inhiition of the phid gut surse, whih results in inhiition of oth oligoshride synthesis in the gut nd the linked redution in osmoti pressure of the gut ontents, using the pssge of wter from ody fluids to the gut (Krley et l., 25). Further experiments were therefore onduted to estlish the undne of Buhner, the onentrtion of free mino ids nd the surse tivity in the phids rered on diets of different surose onentrtion. The opy numer of the Buhner gene dnk, n index of Buhner undne, vried signifintly with dietry surose (ANOVA: F 6,61 =4.46, P=.1) nd ws signifintly depressed in phids on 1.25 nd 1.5 mol l 1 surose reltive to phids on lower dietry surose onentrtions (Fig. 5A). The free mino id ontent of the phids lso vried signifintly with dietry surose onentrtion (ANOVA: F 6,62 =7.99, 1.75 Fig. 4. Osmoti pressure of the hemolymph of 8-dy-old phids rered on either hemilly defined diets of different surose onentrtion (irles) or on plnts (tringles) with phloem sp surose onentrtion s shown in Tle 1. Vlues re mens ± s.e.m. Men vlues tht re not signifintly different (P>.5) re indited y the sme letter. nmol free mino ids mg 1 phid 1 6 no. dnk opies ng 1 totl DNA Dietry surose onentrtion (mol l 1 ) Fig. 5. Aundne nd funtion of symioti teri Buhner sp. in 8-dy-old phids on diets of different surose onentrtion. (A) Numer of opies of Buhner gene dnk normlised to totl DNA ontent. (B) Free mino id ontent normlised to phid mss. Vlues re mens ± s.e.m. Men vlues tht re not signifintly different (P>.5) re indited y the sme letter. P<.1), with levels in the phids on.125 mol l 1 surose diets signifintly greter thn on 1. mol l 1 surose (Fig. 5B). The dt do not support the predition (ove) tht redued Buhner density on mol l 1 diets is ompnied y elevted free mino id ontent. A seond hrteristi of posymioti phids with high totl free mino id titres is depressed essentil mino id ontent s proportion of the totl free mino ids (Prosser nd Dougls, 1991). The essentil mino ids were, on verge, 29 33% of the totl free mino ids in the diet-rered phids nd were not redued in phids on high surose diets (dt not shown). The surse tivity of the phids vried signifintly with dietry surose (F 6,63 =8.23, P<.1), nd ws elevted on diets ontining 1. mol l 1 nd 1.25 mol l 1 surose reltive to lower dietry onentrtions (Fig. 6). Aphids on plnts Phloem sp ws olleted y styletomy from Vi f rered under the ontrol nd three test tretments given in the Mterils nd methods. The surose onentrtion in the phloem sp vried signifintly ross the tretments (Tle 1). The 6-dy-old phids trnsferred to these plnts settled redily nd fed, produing honeydew. At the end of the experiment, the osmoti pressure of their hemolymph (Fig. 4) did not vry, A B

7 Sugr utilistion y phids 141 Surse tivity (m i.u. mg 1 protein) Dietry surose onentrtion (mol l 1 ) Fig. 6. Surse tivity of 8-dy-old phids rered on diets of different surose onentrtion. Vlues re mens ± s.e.m. Men vlues tht re not signifintly different (P>.5) re indited y the sme letter. signifintly with plnt tretment (F 3,27 =.43, P>.5). The overll men osmoti pressure of the hemolymph of phids rered on plnts, 1.61±.63 MP (men ± s.e.m., N=31), did not differ signifintly for the overll men vlue for phids rered on diets with mol l 1 surose, t 1.47±.59 MP (N=3; t 58 =1.52, P>.5). Disussion To investigte the limits to the dietry surose onentrtions utilised y pe phids, this study foused minly on the responses of finl-instr lrve over 2 dys. This experimentl design enled the primry proesses underpinning poor performne on diets with extreme surose onentrtions of.125 mol l 1 nd 1.5 mol l 1 (Fig. 1) to e identified without interferene from non-speifi seondry effets ssoited with generl mlise tht re expeted to rise over longer time-sles. In the experimentl design, only dietry surose onentrtion ws vried, resulting in vrition of the rtio of surose to ll other dietry onstituents. In prtiulr, the surose:mino id rtio rnged from.83:1 (the.125 mol l 1 surose diet) to 1:1 (the 1.5 mol l 1 surose diet). Other experiments (Dougls et l., in press) hd demonstrted tht phid performne ws not driven entirely y surose:mino id rtio. Speifilly, phid performne differed etween diets of different surose ontent (.4 nd 1. mol l 1 ) ut the sme surose:mino id rtio (6.4:1). Despite this, the possiility nnot e exluded tht vrition in the rtio of surose to other nutrients my hve influened the responses of phids in this study. The entrl role of surose phgostimultion in defining the lower limit of dietry surose utilised y phids hs een suggested y erly studies, espeilly of Mittler nd Ddd (Mittler nd Ddd, 1963) nd Srivstv nd Aulir (Srivstv nd Aulir, 1971). This interprettion is onfirmed mply y the evidene from this study tht the phids on the.125 mol l 1 surose diet performed very poorly euse they ingested food slowly. Speifilly, the volume of diet ingested y these phids ws less thn hlf of tht on the.25 mol l 1 diet (Fig. 2A), nd phid performne ws strongly orrelted with the volume of diet ingested on the diet with.125 mol l 1 surose ut on no other diet (Fig. 3). The low feeding rte on this diet presumly reflets the high energeti ost of mintining high feeding rtes for little nutritionl dvntge. It is most unlikely tht impirment of post-ingestive proesses ontriuted to the poor performne of the phids on the.125 mol l 1 diet euse, first, the phids ssimilted >9% of the surose-ron ingested (Fig. 2C) nd, seond, they mintined the osmoti pressure of their hemolymph (1.5 MP) despite the osmoti hllenge of diet with lower osmoti pressure (.9 MP) (Fig. 4). The phids lso performed poorly on diets with mol l 1 surose (Fig. 1), even though they fed suffiiently to ingest more surose thn hieved y the phids on 1. mol l 1 surose diet nd hd n ssimiltion effiieny greter thn phids on.75 mol l 1 nd 1. mol l 1 diets (Fig. 2). The one potentilly deleterious physiologil response identified for phids on the high surose diets ws the elevted osmoti pressure of their hemolymph (Fig. 4). Inreses in hemolymph osmoti pressure hve een reported previously, of 3% for phids with elevted free mino id ontents linked to elimintion of the symioti teri (Wilkinson et l., 1997) nd of 5% for phids lking gut surse tivity (Krley et l., 25). However, further experiments (Figs 5, 6) showed tht the phids on high surose diets hd neither elevted free mino id titres nor sustntilly depressed surse tivity. It is, therefore, very prole tht the osmoti pressure of the phid hemolymph ws very high euse the osmoti pressure of the high surose diets exeeded the osmoregultory pility of the phids. One possiility is tht the levels of surose-derived monoshride in the gut of phids on the high surose diet were greter thn the sugr Tretment Tle 1. Conentrtion of surose in phloem sp of Vii f plnts Surose onentrtion (mol l 1 ) of phloem sp (1) 24 h D t 2 C for 36 h.37±.29 Control: 18 h L (1 mol m 2 s 1 ):6 h D t 2 C.65±.38 (2) 18 h L (6 mol m 2 s 1 ):6 h D t 2 C for 2 dys.85±.67 (3) 18 h L (6 mol m 2 s 1 ) t 2 C:6 h D t 12 C for 2 dys.97±.85 ANOVA (fter logrithmi trnsformtion) F 3,28 =24.75, P<.1 Vlues re men ± s.e.m., N=8 Men vlues tht re not signifintly different (P>.5) re indited y the sme letter.

8 142 A. E. Dougls nd others polymeristion pity of the gut trnsgluosidse, leving the osmoti pressure of the gut ontents sustntilly greter thn tht of the ody fluids. This would use the net movement of wter from the phid ody fluids to the gut lumen, proess whih n e orreted only y the tive trnsport of wter in the reverse diretion. The inresed ssimiltion effiieny of phids on mol l 1 surose diets my reflet heightened demnd for dietry ron s respirtory fuel in response to this osmoti stress. A seond effet of high dietry surose ws the redued undne of opies of the dnk gene of the symioti terium Buhner (Fig. 5). This index of Buhner undne provides n urte mesure of the numer of teril genomes euse dnk is single-opy gene (Shigenou et l., 2), ut not neessrily of the numer of teril ells, euse the genome opy numer per Buhner ell is lrge nd vrile (Komki nd Ishikw, 1999). Although further reserh is required to estlish the full implitions of the results in Fig. 5, the onsequene of redued numers of Buhner genomes ould e sustntil euse of the importne to the phid of Buhner s soure of essentil mino ids, whih re in short supply in the phid diet of phloem sp (Dougls, 1998). Generlly, hemilly defined diets offer more lned mix of mino ids thn phloem sp, nd udget nlysis of lone LL1 on the diet formultion used here indites tht just two mino ids derived from Buhner, methionine nd phenyllnine, re required for sustined growth (A.E.D., unpulished dt). The redution in Buhner genome opy numer on the high surose diets is therefore likely to depress performne. Over the 2-dy timesle of the experiment, however, it did not trnslte into the onsequene of impired Buhner funtion elieved to result in inresed hemolymph osmoti pressure: redued onentrtion of limiting essentil mino ids in the free mino id pool, using depressed protein synthesis nd ssoited umultion of other non-limiting mino ids in the free mino id pool (Prosser nd Dougls, 1991; Wilkinson et l., 21). In summry, the min onlusion of this study is tht the lower nd upper limits to the dietry surose onentrtions utilised y pe phids re shped primrily y different proesses: ehviourl response, speifilly redued feeding refleting the importne of surose s phgostimulnt, for the lower limit; nd osmoregultory filure for the upper limit. A further issue is the relevne of these results to phids feeding on their nturl diet of plnt phloem sp. It is widely reognised tht, lthough phids perform less well on hemilly defined diets thn on some plnts, their physiologil responses, espeilly in short-term studies, re similr on the two food soures (e.g. Rhodes et l., 1996; Simpson et l., 1995; Fevy et l., 1999; Dougls et l., 21) so tht results n e extrpolted from diets to plnts with some onfidene. This is illustrted y dt in this study. The lifetime reprodutive output, up to 17 offspring per phid, on the diets ws muh redued ompred to the 6 8 offspring on the stndrd ulture plnt Vii f (unpulished dt); ut phids rered on diets with surose onentrtions of mol l 1 mintined rodly uniform hemolymph osmoti pressures tht mthed the vlues otined for phids rered on plnts with similr phloem surose ontents (Fig. 4). In this study, the phids performed well in the short-term on diets with surose ontents of mol l 1, spnning the full rnge of phloem surose onentrtions of mol l 1 otined y environmentl perturtions under lortory onditions in this study (Fig. 1C; Tle 1). Furthermore, the osmoregultory response of phids to diets nd plnts were omprle (Fig. 4). This suggests tht the feeding nd osmoregultory pilities of the phids re omptile with the phloem sugr levels ommonly enountered y phids feeding on plnts. However, it would e premture to onlude tht the surose onentrtion in phloem sp is invrily omptile with phid physiology nd ehviour, i.e. tht phids re perfetly dpted to the full rnge of surose ontent of plnts. The phloem surose ontent of plnts in the field my e more vrile thn in the lortory. In prtiulr, field plnts generlly experiene higher light levels thn n e generted under lortory onditions; nd styletomy smples of some plnts exposed for short periods to light intensities pprohing nturl dylight yielded phloem sp with 1.5 mol l 1 surose (K.V.P., unpulished results). This rises the possiility tht, under ertin irumstnes individul sieve elements, ertin plnt prts or even entire plnts my hllenge the osmoregultory pity of phids, rendering them unsuitle s food soure. We thnk Dr A. J. Krley for helpful omments on the mnusript. This reserh ws funded y BBSRC grnt 87/S16725 nd the DEFRA Seedorn Fund of the Centrl Siene Lortory. Referenes Ashford, D. A., Smith, W. A. nd Dougls, A. E. (2). Living on high sugr diet: the fte of surose ingested y phloem-feeding inset, the pe phid Ayrthosiphon pisum. J. Inset Physiol. 46, Begon, M., Townsend, C. R. nd Hrper, J. L. (25). Eology: From Individuls to Eosystems. Oxford: Blkwell. Buhner, P. (1965). Endosymioses of Animls with Plnt Miro-orgnisms. Chihester: John Wiley & Sons. Cenis, J. L., Perez, P. nd Fereres, A. (1993). Identifition of phid (Homopter, Aphidide) speies nd lones y rndom mplified polymorphi DNA. Ann. Entomol. So. Am. 86, Dhlqvist, A. (1984). -gluosidses (dishridses). In Methods of Enzymti Anlysis (ed. H. U. Bergmeyer), pp Weinheim: Verlg Chemie. Dougls, A. E. (1998). Nutritionl intertions in inset-miroil symioses. Annu. Rev. Entomol. 43, Dougls, A. E. (23). Nutritionl physiology of phids. Adv. Inset Physiol. 31, Dougls, A. E. nd Prosser, W. A. (1992). Synthesis of the essentil minoid tryptophn in the pe phid (Ayrthosiphon pisum) symiosis. J. Inset Physiol. 38, Dougls, A. E., Minto, L. B. nd Wilkinson, T. L. (21). Quntifying nutrient prodution y the miroil symiosis in n phid. J. Exp. Biol. 24, Dougls, A. E., Frnois, C. L. M. J. nd Minto, L. B. (in press). Fulttive

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