Entomologia Experimentalis et Applicata88: 189–193, 1998.© 1998Kluwer Academic Publishers. Printed in the Netherlands.

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Short communication

Inositol in two host plants of Manduca sexta

Nancy Nelson & Elizabeth BernaysDepartment of Entomology and Center for Insect Science, University of Arizona, Tucson, AZ 85721, USA

Accepted: May 11, 1998

Introduction

Inositol is ubiquitous in plants. It is a key structuralcomponent of phospholipids, and it plays importantroles in cell wall formation, osmoregulation, cell sig-naling, and phosphate storage. It is reported to occurfree in foliage cells at concentrations of severalµM/gfresh weight. (Loewus, 1990).

Behavioral and electrophysiological studies showthat caterpillars of many species respond to the sugaralcohol,myo-inositol (see Schoonhoven et al., 1998).Most of the electrophysiological work has focused onthe styloconic sensilla on the galea which are impor-tant in host choice. In most cases, the specificity of thecell responding to inositol is not reported, althoughin several cases it is clearly a different cell from theones responding to sugars, and in some cases thereis a high degree of specificity. For example,Man-duca sextalarvae have one cell in each of the fourstyloconic sensilla that is highly sensitive and specific- 25% of the total numbers of gustatory neurones inthese sensilla (Schoonhoven, 1973). Among many nu-trients and non-nutrients tested on these sensilla, onlyinositol gave a totally predictable and very intense re-sponse, even at levels as low as 0.1 mM (Nelson, 1996;Bernays et al., 1998).

It is not known why some caterpillars should beparticularly responsive to inositol or have such a rela-tively high investment in specific receptors for it. Themajority of insects tested can synthesize inositoldenovo(Dadd, 1977, 1984), but several species of Lepi-doptera require it in the diet (e.g., Horie et al., 1966).M. sexta, did not require inositol in the diet for lar-val development, but without it, female fecundity wasgreatly reduced (Nelson, 1996).

It may be that inositol serves as a general indicatorof plant material. Or, like sugars, it may just indi-cate a usable energy source and for this reason hasbecome phagostimulatory. However, it is difficult to

explain the adaptive significance of such a relativelyhigh investment in neurons apparently dedicated andhighly sensitive to this one compound (Nelson, 1996).Another possibility is that inositol levels may be cor-related with levels of some other critical nutrients suchas protein. This would be of great functional valueto caterpillars in which the need for protein is par-ticularly high (Scriber & Slansky, 1981; Slansky &Scriber, 1985). The evidence to date indicates that in-sects are unable to detect protein directly (Bernays &Chapman, 1994), although there are few critical stud-ies (Schoonhoven et al., 1998). Amino acids stimulatetaste receptors in many species of caterpillars, but theresponses are variable among species (Schoonhoven etal., 1998). Also the levels of free amino acids in plantsare not generally correlated with the level of protein.

This study examines levels of inositol in leaves oftwo host plants ofManduca sexta, tobacco and tomatoin two sets of experiments. First we examined therelationship between inositol concentrations and con-centrations of plant sugars and extractable protein. Inthe second set of experiments we also examined theconcentrations of inositol on the leaf surface, since it isthe leaf surface that is first sampled by a caterpillar andperhaps the surface chemicals influence host choice.In addition, there is evidence that inositol may haveits greatest role as a phagostimulant in the initiation offeeding (Nelson, 1996).

Materials and methods

Plants. Six tomato plants,Lycopersicon esculentum,each with five fully-expanded leaves, were purchasedfrom a local nursery. The amount of protein presentwas measured in one of the two most distal oppositeleaflets from each of the five leaves, while the otherwas used for the measurement of inositol and sugarlevels. For chemical analyses leaflets were individ-

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ually placed in resealable plastic bags and immedi-ately frozen on dry ice. The leaflets were stored at−80 ◦C for up to 3 weeks. For water content, leafletswere quickly weighed, oven dried in open paper bagsovernight at 50◦C, and then reweighed.

Three tobacco plants,Nicotiana tobacum, weregrown from seed in a greenhouse. Each tobacco planthad between fifteen and twenty leaves. The plantswere divided from the top down into six parts, bydividing the main stem into six, more or less equal,lengths. One leaf from each part was taken for analy-sis. The leaves were cut at the petiole and placed inindividual, resealable plastic bags. The leaves wereimmediately frozen on dry ice and stored at−80 ◦Cfor less than two weeks. The leaves were analyzed forprotein and inositol.

For experiments in which surface concentrations ofsugars and inositol were measured, older plants wereused. Tomato and tobacco plants were grown fromseed in 1 gallon pots in a greenhouse. Each tomatoplant used was 10–12 weeks old, had between 11 and14 fully expanded leaves and were starting to flower.Three leaflets were taken from each leaf of three plantsfor individual analysis. Tobacco plants were 8 to 10weeks old, had between 10 and 15 fully expandedleaves, and had also initiated flowering. Leaves fromfour plants were harvested.

Surface extractions. For surface extraction tobaccoleaves were cut at the base of the petiole (tobacco) orthe petiolule (tomato), and their areas measured usingSigmaScan for the PC. The leaves were then dippedinto 200 ml boiling distilled water for 1 second (Zobel& Brown, 1988), being careful not to dip the cut end.The leaves or leaflets were then immediately placed inindividual zip lock bags and frozen with dry ice. Thesurface-extracted leaflets or leaves to be used for tissueanalysis were stored at−80 ◦C and surface extractswere stored at−4 ◦C for up to three months.

To prepare the surface extracts for HPLC analysis,2 ml aliquots of the surface extract were dried in arotovap (Buchi), and resuspended in 0.2 ml of distilledwater. The solution was run through a PRP-1 columnand the column was washed with an additional 0.2 mlof distilled water.

Tissue extraction procedure for inositol and sugars.The procedure used to extract sugars and inositol fromthe leaf tissue was developed by Adams et al. (1993).Plant tissue was ground with a mortar and pestle inliquid nitrogen. A 50 mg sample was then ground in

a 1.0 ml solution of 12 parts ethanol, 5 parts chloro-form and 3 parts distilled water. Another ml of distilledwater was added to the solution and the solution wasvortexed. After precipitation of solids, 0.2 ml of thesupernatant was dried in a rotovap. This was resus-pended in 0.2 ml distilled water and filtered througha PRP-1 column. The column was washed with anadditional 0.2 ml of distilled water.

HPLC analysis. The analysis of soluble sugars andsugar alcohols was performed using a Dionex (Sunny-vale, CA) HPLC system in conjunction with a HPX-87C Aminex column with H2O at 85◦C equipped witha Gison (Middleton, WI) model 302 pump. Carbohy-drate detection was by pulsed amperometric detection(Dionex Advanced PAD) at 35◦C. For calibrationstandards, various sugars from different sources wereused (Adams et al., 1993).

Protein analysis. A Bradford assay as modified byJones et al. (1989) was used to measure protein levels.The leaves or leaflets were ground in liquid nitrogenand 100 mg of the tissue was then ground in 1 mlof 0.1 N NaOH. The grinding tube and pestle werewashed with an additional ml of NaOH. The sam-ples were vortexed for a few seconds and then leftto extract at room temperature for 30–45 min. Thesupernatant was decanted. Aliquots of 100µl weremixed with 5 ml of Bradford reagent (Bio-Rad). Foreach sample three replicates were run. The absorbanceat 595 nm was recorded using a Milton Roy Spec-tronic 1201 UV spectrophotometer for each aliquotand calibrated against a standard curve made using eggalbumin (Sigma).

Results

Comparison of protein, inositol and sugar levelsin leaf tissue of young tomato plants.The over-all mean concentrations (in mg/g dw) were: protein113±9, inositol 1.2±0.17, sucrose 3.1±0.39 , fruc-tose 5.0±0.85, and glucose 3.4±0.38. All compoundsvaried significantly between plants (Table 1). Within-plant protein concentrations varied with leaf age (Ta-ble 1), with the youngest leaves having highest con-centrations (correlation of protein and leaf age,r =0.67, P<0.001, df 26, two-tailed). Overall, inositollevel was correlated with both protein (rp,i = 0.745,P<0.001) and sucrose levels (rs,i = 0.813, P<0.001),

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Table 1. Differences occurred among the inositol, sucrose, glu-cose and fructose levels in tomato plants. Results of ANOVA areshown below. In all cases degrees of freedom were 5 for the plantand 20 for the residual

Nutrient Plant variation Leaf age variation

F value P value F value P value

Protein 3.806 0.012 3.8 0.015

Inositol 7.311 0.0002 1.2 0.329

Sucrose 5.342 0.002 1.24 0.32

Glucose 4.496 0.006 0.82 0.533

Fructose 4.933 0.004 1.27 0.308

but not with glucose (rg,i = 0.299, P<0. 15) orfructose (rf,i = 0.097, P<0. 65).

Comparison of inositol and protein levels in leaf tissueof young tobacco plants.The mean concentration ofextractable protein was 11.3±1.6 mg/g fw and of in-ositol 0.92±0.12 mg/g fw. The ANOVA did not showa significant plant effect in either protein (F2,15 =0.172, P=0.84) or inositol (F2,15 = 1.77, P=0.203).There were marked differences within plants in pro-tein levels, but not inositol levels (ANOVA, F5,12:protein 23.48, P<0.0001; inositol 2.75, P=0.07).Younger leaves had higher levels of protein than olderleaves (correlation of leaf age and protein,r = 0.67,P=0.002, df 16, 2-tailed). In spite of the fact that inos-itol concentrations did not differ significantly withinor between plants, there was, overall, a positive cor-relation between protein and inositol concentrations(rp,i = 0.503, P=0.03).

Levels of inositol and sugars on the surface oftomato plants in relation to inositol, sugars andprotein within the leaves. On the surface of theleaf inositol was generally more abundant thanany of the sugars at 0.30±0.03 mg/cm2. Su-crose levels were 0.14±0.03 mg/cm2, fructose0.05±0.02 mg/cm2 and glucose 0.04±0.01 mg/cm2.Within the tissues, the mean concentration of proteinwas 5.75±0.22 mg/g fw, inositol 0.76±0.05 mg/g fw,fructose 1.14±.11 mg/g fw, glucose 0.55±0.08 mg/gfw, and sucrose 0.43±0.04 mg/g fw. There was norelation between the level of inositol or a sugar onthe surface of a leaflet and the level found withinthe tissue (inositolr = −0.161, P=0.19; sucroser = 0.022, P=0.86; glucoser = 0.022, P=0.86;fructoser = 0.052, P=0.67).

Levels of inositol on the leaf surface were not cor-related with the levels of extractable protein (r =0.041, P=0.74). Levels of tissue inositol were also notcorrelated with levels of extractable protein (rp,i =−0.177, P=0.15). The leaves near the top of the planthad more protein than those near the bottom.

Comparison of tissue and surface levels of inosi-tol and sugars in tobacco plants with tissue lev-els of protein. On the surface of the leaf in-ositol averaged 0.15 mg/cm2. Sucrose was themost abundant sugar (0.15±0.03 mg/cm2) fol-lowed by fructose (0.08±0.01 mg/cm2) and glucose(0.07±0.01 mg/cm2). In the tissues inositol levelaveraged 0.20±0.02 mg/g fw, while sucrose wasthe most abundant sugar (0.69±0.08 mg/g fw), fol-lowed by glucose (0.54±0.06 mg/g fw) and fructose(0.48±0.07mg/g fw). Protein levels in this study wereconsiderably lower than those in the previous study,averaging 7.9±0.4 mg/g fw.

There was no correlation between the level of sug-ars or inositol on the surface and within the leaf. Therewas no correlation between surface levels of inositoland extractable tissue protein levels (r = −0.432,P=0.03). There was no correlation between tissue in-ositol and protein (rp,i = 0.174, P=0.41), althoughthe leaves near the top of the plant had a higher levelof protein than those near the bottom.

Discussion

General comparison between series.The two dif-ferent series of experiments yielded different resultsin many respects. Also, comparison is complicatedby the fact that in measurements for young tomatoplants concentrations of nutrients were related to dryweight, whereas all others were on a fresh weight ba-sis. In studies with the older plants the leaves were firstdipped into hot water to remove the surface inositoland sugars, so that these would not have been in-cluded in the analysis of the whole leaf tissue. Perhapsmost importantly, the second sets of plants were older,apparently ‘pot-bound’, and at the flowering stage.

With respect to tomato, rough comparisons be-tween the two sets of experiments indicated that, inolder plants, protein concentrations were approxi-mately 50% of those found in younger plants as ex-pected (Slansky & Scriber, 1985), while inositol levelswere much higher in the older than in the youngerplants. The three sugars measured were generally sim-

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ilar in the younger and older plants. With respect totobacco, protein levels in older plants were again abouthalf those found in younger plants. Inositol levels werealso lower in older plants (approximately one thirdof those found in young plants). In all experiments,younger leaves always had the higher levels of pro-tein. The levels of all three sugars and of inositol were,however, more variable, and no consistent patterns inrelation to leaf age were observed.

In both tomato and tobacco, positive correlationswere found between inositol and protein levels inleaves of plants that were young and preflowering. Intomato, sucrose level was also correlated with proteinlevel. Where plants were older, flowering and appar-ently ‘pot-bound’, and where the sugars were firstwashed off the leaf surface, there was no correlationbetween inositol and protein levels. Neither were therecorrelations between levels of any of the sugars and theprotein levels.

The lack of correlation between the levels of sug-ars found on the surface and within the tissue wasunexpected but may be partly explained by possibledifferences in the rates of leaching of the chemicals tothe leaf surface (Tukey, 1971). Surface levels of inosi-tol were extremely high in relation to surface levels ofother sugars monitored. One potential reason for this isthat inositol is an important precursor for pectic com-ponents in cell walls and perhaps a larger proportion ofinositol is external to the cell and more readily leachedto the surface than the sugars, which are presumed tobe mainly intracellular or in the phloem.

It should be noted that the highest concentrationsof inositol detected in the leaf tissues were of the orderof 1 mM. This is considerably lower that the concen-trations typically used in electrophysiological studiesto date. In many of the studies mentioned in the intro-duction, concentrations of 50 or 100 mM were used.Styloconic sensilla ofManduca sexta, however, havebeen shown to respond to concentrations of 0.1 mMand to show a very rapid increase in firing rate asconcentrations are increased to 1 mM (Bernays et al.,1998). Further studies are needed with other species ofcaterpillars so that a measure of sensitivity at relevantplant concentrations can be made and a more detailedexamination of the adaptive significance, if any, can beundertaken.

Another interesting point to note for insect nu-trition generally, is that inositol concentrations werefound to be of the same order of magnitude as glu-cose, fructose and sucrose. SinceManduca sextacanutilize inositol as an energy source (Nelson, 1996) it

may sometimes be a significant proportion of the totalutilizable leaf carbohydrates ingested.

In conclusion, levels of sugars and inositol on thesurfaces of the leaves could not predict tissue lev-els of these compounds, or of protein. A relationshipbetween tissue inositol and protein/sugar levels wasfound in healthy young preflowering plants, but notolder plants. Further studies are needed with bettercontrol over plant age and growing conditions to de-termine if these trends are widespread, and whether, ingeneral, it would be possible for insects to use inositolas a cue for less readily detected nutrients.

Acknowledgments

We particularly thank Dr Richard Jensen and PatriciaAdams for help with the sugar and inositol analyses.Thanks are also due to Betty Estesen for help with theinsects, and to Stacey Brenner for help with the plantpreparation. The work was funded in part by a plantinsect interactions training grant from the NationalScience Foundation/ U.S.Department of Agriculture/Department of Energy.

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