A quantitative analysis of the osmolytes in the hemolymph of the larval gypsy moth, Lymantria dispar

J. Insect Physiol. Vol.38, No. 11, pp. 823430, Printed in Great Britain. All rights reserved

1992 Copyright 0

0022~1910/92 $5.00 + 0.00 1992 Pergamon Press Ltd

A QUANTITATIVE ANALYSIS OF THE OSMOLYTES IN THE HEMOLYMPH OF THE LARVAL GYPSY MOTH, LYMANTRIA

DISPAR

T. L. PANNAFSECKER, F. ANDREWS and K. W. BEYENBACH Department and Section of Physiology, Cornell University, Ithaca, NY 14853, U.S.A. (Received I6 March 1992; revised 27 May 1992)

Abstract-In spite of the growing number of in vitro studies conducted with the gypsy moth there are no reports which describe its hemolymph composition. For this reason we have conducted a quantitative analysis of the osmolytes in the hemolymph of the fifth-instar gypsy moth larva reared on a wheat-germ diet. The osmotic pressure of hemolymph is 313 mOsm/kg water and the hemolymph pH is 6.6. The dominant inorganic cations are potassium (29.1 mM), magnesium (25.9 mM), calcium (6.8 mM) and sodium (5.0 mM). The dominant inorganic anions are chloride (14.9 mM), sulfate (12.3 mM), and phosphate (5.7 mM). The electrical conductivity of the hemolymph (4.52 ms/cm) is equivalent to the conductivity of a 98.8 mOsm/kg water NaCl solution. Since the sum of the measured hemolymph cations and anions approaches this osmolality, more than 200 mOsm/kg water of hemolymph solutes are non-electrolytes. Among these non-electrolytes we have identified 18 amino acids (112.0 mM), trehalose (21.1 mM), glucose (2.4 mM) and urea (4.8 mM). Organic solutes such as arginine (0.9 mM), histidine (3.9 mM), lysine (13.9 mM), tyrosine (2.5 mM), aspartic acid (0.3 mM), glutamic acid (1.6 mM), organic phosphates (29.6 mM) and uric acid (0.2 mM) are expected to exist in ionized forms at the hemolymph pH. Together the identified electrolytes and non-electrolytes account for nearly 85% of the measured hemolymph osmolality. Key

Word Index:

Gypsy

moth;

hemolymph;

electrolytes;

amino

acids; carbohydrates;

phosphates

INTRODUCTION

Maintenance of appropriate fluid and electrolyte homeostasis in insects is essential for normal development, growth and reproduction. In insects the principal excretory organs involved with maintaining hemolymph osmotic and electrolyte balances are the hindgut, the Malpighian tubule and the rectum (Bradley, 1985). Electrolytes and non-electrolytes are secreted from the hemolymph into the Malpighian tubule lumen via active and passive transport pathways within the epithelium. Water secreted by the tubule carries solutes towards the hindgut and rectum for excretion from the body or reabsorption into the hemolymph. Extensive in vitro studies have shown that the quality and quantity of solute transport and the rate of fluid secreted by the tubule are in large part dependent on peritubular inorganic ion concentrations and proportions (Maddrell, 1977). A variety of other biochemical properties of the peritubular

solution, including osmotic pressure and pH (Nicholson, 1976; Anstee et al., 1979), influence the rate of fluid secretion in vitro. A diverse array of diuretic factors are also known to increase fluid and electrolyte transport, and the capacity of these factors to increase fluid secretion in vitro may in some insects depend on the concentrations of inorganic electrolytes in the peritubular medium (Morgan and Mordue, 1981). Insect Malpighian tubules have in fact been shown to secrete fluid and electrolytes quite readily in simple and diverse salt solutions. However, in order to investigate physiologically relevant fluid and electrolyte transport by the Malpighian tubule in vitro an accurate assessment of hemolymph components is required for the preparation of the appropriate Ringer solution. Since there are no reports describing the hemolymph composition of the gypsy moth we have conducted a comprehensive analysis of the inorganic and organic components of the hemolymph from larval Lymantriu dispar. 823

T. L. PANNABECKER et al.

824 MATERIALS AND METHODS

Experimental animals Gypsy moth eggs were obtained from the USDA Otis Methods Development Center (Otis, MA, U.S.A.). Gypsy moth eggs hatched after being placed in a covered Petri dish at room temperature for 2 days. The larvae were then transferred to a 275 ml plastic cup (10 per cup) containing approx. 75 ml of a wheat-germ diet (Bell et al., 1981). The larvae were provided with this diet ad libitum, under a 16 h light-8 h dark photoperiod, 25-30% r.h. at 23°C. Larvae molted to the fifth instar approx. 20 days after hatching. Hemolymph collection procedure Analyses were conducted on hemolymph obtained from day 5, fifth-instar larvae reared on the wheatgerm diet. The sex of hemolymph donors was not determined. Larvae were anesthetized by chilling on ice for 10 min in a cold room at 3°C. The tip of the second abdominal proleg (the terminal 0.5 mm) was cut with scissors. A 100 ~1 glass capillary was used to collect the hemolymph which flowed from this wound while applying gentle pressure to the abdomen. For compositional analyses the hemolymph was centrifuged at 3°C at 750 g for 10 min (3°C) to remove the hemocytes. Unless otherwise stated, all analyses were conducted with cell-free hemolymph. The supernatant (cell-free hemolymph) was collected and stored on ice or frozen at -20°C. Osmotic pressure, pH and hematocrit determination Osmotic pressures of hemolymph and NaCl solutions were measured with a vapor-pressure osmometer (Model 513OC, Wescor Inc.). Values are reported in units of mOsm solute/kg water. Hemolymph pH values from individual insects were determined with a mini pH electrode and reference electrode (Models MI508 and M1402, Microelectrodes Inc.). The volume of hemolymph occupied by particulate matter was determined in a 10 pl hemolymph sample. Hemolymph was centrifuged for 10 min in a Daman capillary centrifuge. The particulate matter and fluid were observed with a dissecting microscope and the proportion of particulate matter was determined with an ocular micrometer.

Cu, Zn, As, Se, Y, MO, Ag, Cd and Pb. The sensitivity of this technique allowed us to quantitate Na, K, Mg, Ca, Fe, Cu, Zn, P and S in a single hemolymph aliquot from an individual insect. For this analysis hemolymph samples (100-l 50 ~1) were placed into quartz glass tubes and were digested with 200~1 of concentrated nitric acid and 100 ~1 of concentrated perchloric acid at 200°C for 2 h. This material was allowed to cool, 3.7 ml of 5% HCl were added, and it was then analyzed. Chloride concentrations were determined by silver chloride titration with a Buchler-Cutlove chloridometer (Buchler). Inorganic phosphate concentrations were determined with a DACOS chemistry analyzer (Coulter Electronics Inc.) using a modified Daly and Ertingshausen method (1972). Uric acid and urea nitrogen concentrations were determined with a DACOS chemistry analyzer using methods modified from Fossati et al. (1980) and Talke and Schubert (1965), respectively. The concentration of urea nitrogen was converted to urea by multiplying by 2.14 (Woo and Cannon, 1984). Determination of hemolymph conductivity The conductivities of cell-free hemolymph and NaCl solutions were measured with a YSI Model 31 conductivity bridge (Yellow Springs Instruments Co. Inc.). Since the YSI electrode requires a volume of 3 ml, which is greater than that collected from a single gypsy moth, an electrode with a volume of less than 75 ~1 was custom-made. This electrode was manufactured from a glass capillary with the following dimensions: o.d. = 3.9 mm, i.d. = 2.7 mm, length = 7.5 cm (Fig. 1). Two holes (dia 1 mm) were drilled through the glass wall, approx. 6 mm from the lower lip of the capillary. Two platinum wires were passed from the

Elemental composition analysis Cell-free hemolymph was subjected to elemental analysis by inductively coupled argon plasma atomic emission spectrophotometry (ICAP). ICAP has the capability of quantitating the following elements: B, Na, Mg, Al, Si, P, S, K, Ca, V, Cr, Mn, Fe, Co, Ni,

Fig. 1. Measurement of electrical conductivity of microliter fluid volumes.

The osmolytes in gypsy moth hemolymph

825

metric detector; a Rheodyne 9125 non-metal injector, an Advanced Computer Interface with AI-450 chromatography software, and an HPIC-AS6 column. The mobile phase was 200 mM NaOH with isocratic elution, and the flow rate was 1 ml/min (run time was 6 min).

2.5

Protein and amino acid analysis

01

0

2

4

Conductivity commercial

6

8

10

(mS/cm) electrode

Fig. 2. Relationship between the conductivities of NaCl solutions (20, 40, 60, 80, 100mM) measured with custommade and commercial electrodes (mean + SE). outside through these holes and fastened with epoxy cement to the inside surface. Epoxy was also used to seal the two holes. The platinum wires were then platinized by immersing the electrode in a solution of 1% PtCl and 0.012% Pb-acetate (YSI, personal communication). A current of 1 mA was then passed through the electrode for 5 min, reversing the polarity every 30 s. This procedure deposits a dense, velvetblack layer of colloidal platinum on the wires. The electrode was then washed with running tap water for 15 min, rinsed in distilled water, air-dried and stored until use. To measure conductivity the solution of interest was aspirated to cover the internal platinum wires. A conductivity reading was then taken using the YSI conductivity bridge. Since the surface area of the platinum wires was unknown, the custom-made electrode was calibrated against the commercial electrode with a known cell constant of l/cm (Fig. 2). Pure NaCl solutions consisting of 20, 40, 60, 80 and 100mM NaCl were used for this purpose. Accordingly, the conductivities of gypsy moth cell-free hemolymph were expressed in units of ms/cm. Since a linear relationship exists between the osmolalities and the electrical conductivities of different NaCl solutions (Fig. 3), we estimated the total hemolymph electrolyte concentration from measurements of hemolymph conductivity. Glucose and trehalose determinations

Cell-free hemolymph glucose and trehalose concentrations were determined with a Dionex BioLC 4500i HPLC system consisting of a pulsed ampero-

The total cell-free hemolymph protein content was determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard. For amino acid analysis a 100 ~1 sample of cell-free hemolymph was diluted with 200 ~1 of 30% methanol and 700 ~1 of 20% methanol. A 200 ~1 aliquot of this solution was passed through a SEP-PAK Cl8 cartridge filter. Material was eluted from the SEP-PAK with two 1 ml aliquots of 20% methanol and 1 ml of 30% methanol/O.1 N HCl. The aliquots were dried and reconstituted in 0.5 ml of 0.1 N HCl. Aliquots were analyzed for amino acid content with Waters PICO TAG amino acids analysis system. RESULTS

General physiological characteristics of hemolymph

The hemolymph color was generally blue, green or pale yellow. Infrequently, it was nearly colorless. The biochromes which produce the color are of unknown identity. Periodically, within 15-30 min following collection, the hemolymph began to melanize. The degree of hemolymph melanization was variable. Total particulate matter in the hemolymph comprised 0.6 f 0.2% (mean + SE, n = 6 insects) of the total hemolymph volume. This value is comparable to those reported for hemolymph of other insects

300

2

250

8 g

200

Bl 5 s

150

g 5

100

50

3.9

v.

0

I

5

Conductivity

10

15

20

(mS/cm)

Fig. 3. Relationship between the osmolality and electrical conductivity of pure NaCl solutions (mean & SE).

T. L. PANNABECKER et al.

826

Table 1. Concentrations of inorganic electrolytes in hemolymph from day-S, tifth-instar gypsy moth larva Concentrationa Inorganic electrolytes

(mmol/l)

(mequiv./l)

n

Sodium Potassium Magnesium Calcium Iron, copper, zinc

Cations 5.0 + 0.5 29.1 + 1.8 25.9 + 1.0 6.8 + 0.3 0.2 + 0.0

5.OkO.5 29.1 + 1.8 51.8 + 1.9 13.6kO.6 0.4kO.l

9 9 9 9 9

Total Phosphate Chloride Sulfateb Total

67.0 Anions 5.7 f 0.7 14.9 f 0.7 12.3 & 0.8

9.99 11.4& 1.4 14.9kO.7 24.5 f 1.6

32.9

50.8

6 5 9

“All values represent mean + SE. bDetermined from total elemental sulfur of 12.3 mM.

(Mullins, 1983). The structure of the particulate material was qualitatively assessed by viewing prepared specimens with a compound microscope. Judging from their cellular structure these particles were probably circulating hemocytes. Hemolymph (not cell free) from gypsy moth larvae had a pH of 6.6 k 0.1 (n = lo), and an osmotic pressure of 3 13 f 5 mOsm/kg water (n = 18). The electrical conductivity of cell-free hemolymph was 4.52 + 0.06 mS/cm (n = 11). As shown in Fig. 3 this conductivity is equivalent to the conductivity of an NaCl solution having an osmolality of 98.8 & 1.3 mOsm/kg water. Therefore the concentration of hemolymph electrolytes was nearly 100 mM, accounting for approx. 30% of the osmotic pressure measured in gypsy moth hemolymph. Quantitation of inorganic hemolymph components

Magnesium, potassium, calcium and sodium were the principal inorganic cations measured in gypsy moth hemolymph (Table 1). From a total of 24 elements analyzed iron, copper and zinc were the only other cations detected in significant amounts. The combined concentration of these elements was 0.2 + 0 mM (n = 9). Magnesium and potassium together accounted for more than 80% of the total concentration of inorganic cations. Sodium and calcium contributed almost equally to the remaining 20%. Inorganic anions included chloride, phosphate and sulfate (Table 1). Chloride was the dominant anion identified in our analysis and accounted for nearly 45% of the total molar concentration of inorganic anions. The sulfate concentration was calculated from the concentration of elemental sulfur. The sulfur-containing free amino acid methionine was present (see below), cysteine was absent, and taurine

was not measured. exists in non-sulfate

Therefore a portion form.

of sulfur

Quantitation of organic hemolymph components

The concentrations of non-protein organic solutes are shown in Table 2. Gypsy moth hemolymph contained 29.6 f 2.1 mM organic phosphate (mean t_ SE). This was calculated by subtracting the mean inorganic phosphate concentration (5.7 mM) from the total phosphate concentration (35.3 + 2.1 mM, n = 9). The assumption was made that each organic phosphate molecule has just one phosphorus atom. The equivalent concentration of organic phosphate is 59.2 + 4.2 mequiv./l, assuming a mean valence of -2. Uric acid was present at a concentration of 0.2 k 0.1 mM (n = 3), and with a pK, of approx. 5.75 would be almost completely ionized at pH 6.6 (Gutman and Yu, 1968). The concentration of urea was 4.8 f. 0.7 mM (n = 5). The hemolymph glucose concentration was 2.4 f 0.5 mM (n = 3) and the trehalose concentration was 21.2 & 3.1 mM (n = 3). Eighteen amino acids were identified and quantitated by amino acid analysis (Table 3). The total concentration of amino acids was 112.0 mM. On the basis of this concentration amino acids account for approx. 30% of the hemolymph osmotic pressure of 313 mOsm/kg. A proportion of the amino acids will be positively or negatively charged at pH 6.6. On the basis of the pK, of the R groups, histidine will be partially ionized (with 3.7mequiv./l as cations) whereas tyrosine (2.5 mequiv./l), arginine (0.9 mequiv./l) and lysine (13.9 mequiv./l) will be fully ionized with positive charges. Aspartic acid (0.3 mequiv./l) and glutamic acid (1.6 mequiv./l) will be fully ionized with negative charges. The remaining amino acids will be predominantly neutral. Several additional u.v.-absorbing compounds eluted from the HPLC column during amino acid analysis but did not co-elute with any of the standards. Therefore, additional but unidentified amino acids may be present. The total protein concentration of cell-free hemolymph is 1.83 + 0.52 g/l00 ml hemolymph (n = 4).

Table 2. Concentrations of nonprotein organic solutes in hemolymph from day-5, fifth-instar gypsy moth larva Solute Phosphate Trehalose Glucose Urea Uric acid

Concentration (mmol/l)

n

29.6 &-2.1 21.2 + 3.1 2.4 + 0.5 4.8 f 0.7 0.2 * 0.0

9 3 3 5 3

“All values represent mean + SE.

The osmolytes in gypsy moth hemolyrnph

827

Table 3. Amino acid concentrations in hemolymph from day-5 fifth-instar gypsy moth larva Amino acid electrolyte concentration at pH 6.6 Amino acid Arginine Histidine” Lysine Tyrosine Aspartic acid Glutamic acid Alanine Asparagine Glutamine Glycine Isoleucine Leucine Methionine Phenylalanine Prolined Serine Threonine Tyrosine Valine

Amino acid concentrationa (mM) 0.9 + 0.2 18.5 f 1.0 13.9 f 0.3 2.5 f 1.3 0.3 f 0.2 1.6 f 1.2 2.0 * 0.3 7.5 + 1.1 25.8 + 8.2 7.0 + 3.0 l.OkO.2 1.1 + 0.3 2.1 + 0.5 0.9 + 0.1 12.1 * 1.9 5.4k2.1 4.2 + 0.2 2.5 + 1.3 5.2 + 0.2 112.0

Total

Cations (meqniv./l)

Anions (mequiv./l)

R groupb

0.9 + 0.2 3.7kO.2 13.9kO.3 2.5* 1.3 -

0.3 + 0.2 1.6 & 1.2 -

12.48 6.0 10.53 10.07 3.86 4.25 -

21.0

1.9

PK, of

-

“Amino acid concentration values represent mean + SE, n = 3 animals. bIonization constants of ionized R groups were obtained from Wheast (1972). from the relationship Electrolyte concentrations were determined pH =pK, + log [base]/[acid]. ‘P-Alanine may not be resolved from histidine. dl-Methyl histidine and 3-methyl histidine may not be resolved from proline. Assuming

a molecular

weight equal to that of albu-

mins (approx. 69,000 Da) and an average protein valence of - 10, the anionic protein equivalent concentration was approx. 2-3 mequiv./l.

DISCUSSION

Electrolyte composition of gypsy moth hemolymph We have assessed the electrolyte composition of gypsy moth hemolymph by two methods: (1) as a function of the hemolymph electrical conductivity, relative to the conductivity of known NaCl solutions and, (2) by inorganic elemental analysis. The conductivity analysis indicates that the total hemolymph electrolyte concentration is equivalent to approx. 98.8 mOsmo1 NaCl/kg water. Our inorganic elemen-

would

balance

the

cations

and

anions

to

near

electroneutrality. Other charged organic solutes may include amino acids which, at pH 6.6 would make a net contribution to cations (Table 3). Hemolymph urates and proteins would make a small net contribution

to anions

Non-electrolyte

(see Results).

composition of gypsy moth hemo-

lymph The total non-electrolyte composition of gypsy moth hemolymph was determined as the difference between the total solute concentration (osmolality) and the total electrolyte concentration, as estimated from the conductivity analysis. The total concentration of non-electrolytes is approx. 214.2 mM

tal analysis identified concentrations of cations and anions that, in summation (99.9 mM, Table 4) are in close agreement to this concentration. Therefore, the

Table 4. Summary of osmolyte concentrations in hemolymph from day-5, fifth-instar avusy moth larva

total electrolytes may be accounted for largely by the cations sodium, potassium, magnesium and calcium and by the anions phosphate, chloride and sulfate. From our hemolymph elemental analysis we have estimated a total of 99.9 mequiv./l inorganic cations and 50.8mequivJl inorganic anions (Table 1). The inclusion of organic phosphate (59.2 mequiv./l)

Total solutes Total electrolytes Inorganic elemental analysis Cations Anions Conductivity analysis Total non-electrolytes Total inorganic solutes Total organic solutes

Component

Concentration 3 13 + 5 mOsmol/kg water

67.0 mM 32.9 mM 98.8 mmol NaCl/kg water 214.2 nnnol/kg water 99.9 mM 170.2 mM

T. L. PANNABECKERet al.

828

(Table 4). Non-electrolytes account for nearly 68% of the total solutes in gypsy moth hemolymph. These solutes are organic compounds consisting predominantly of amino acids (112mM). Other organic non-electrolytes which we have identified include trehalose (21.2 mM), glucose (2.4 mM) and urea (4.8 mM). UnidentiJied osmolytes of gypsy moth hemolymph

The total concentration of identified hemolymph solutes in gypsy moth larvae is 269.2 mM. On the basis of this solute concentration and the hemolymph osmotic pressure of 313 mOsm/kg water, more than 85% of the osmotically active solutes have been accounted for in our qualitative and quantitative analyses. The components which remain unaccounted for include primarily non-electrolytes. These missing osmolytes are likely to include uncharged organic molecules such as carbohydrates, polyols, amino acids and amino sugars (Wyatt, 1961; Mullins, 1983). Significant concentrations of organic acid anions have been identified in hemolymph of lepidopteran larvae (Wyatt, 1961; Chamberlin, 1989) and they would also likely be present in gypsy moth hemolymph. These include primarily the carboxylic acids citrate, malate, succinate, for-mate, a-ketoglutarate and pyruvate. Bicarbonate will make a small contribution to the total anions however insect hemolymph is in general not believed to play a significant role in carbon dioxide transport (Wyatt, 1961; Mullins, 1983). Comparison lepidopteran

to hemolymph larvae

composition

of

other

A literature survey of the composition of hemolymph from 11 species of fifth-instar lepidopteran larvae is presented in Table 5. Qualitatively, the hemolymph composition of the gypsy moth is remarkably similar to hemolymph of other lepidopterans. The inorganic ion concentrations in hemolymph of the gypsy moth, and their relative proportions, are qualitatively similar to the ion concentrations and proportions in hemolymph from other lepidopterans. In comparison to vertebrate blood and to hemolymph of insects from less phylogenetically advanced orders the inorganic electrolytes make a relatively small contribution to the total solute concentration (Mullins, 1983). As shown in Table 5, the sodium : potassium and sodium : magnesium ratios are generally less than unity for lepidopterans. Consequently, divalent cations contribute significantly to the total complement of inorganic cations. Potassium

The osmolytes

in gypsy moth hemolymph

and magnesium combined typically account for SO-75% of the total inorganic cations. The dominant inorganic anion in lepidopteran hemolymph is chloride. Phosphate makes a minor contribution to the inorganic anion complement. There are few reports of the sulfate concentrations although our studies on gypsy moth suggest that sulfate may be an important anion. For the species listed in Table 5, chloride will balance the charges of only lo-36% of the inorganic cations. Therefore, for these Lepidoptera organic anions must play a major role in preserving hemolymph electroneutrality. The principal organic solutes which have been measured in lepidopteran hemolymph include phosphate, trehalose and amino acids, with amino acids contributing the largest share (Table 5). These organic solutes are predominantly non-electrolytes and their total concentration is generally equal to or higher than total inorganic solutes. Relatively high concentrations of organic phosphates have been reported for at least two other lepidopteran species and it is likely that organic phosphates comprise a major proportion of the total phosphorus reported for Heliothis virescens and Manduca sexta (Table 5). The carboxylic acid anion composition of insect hemolymph has been relatively unstudied although it is likely that along with organic phosphates they play an important role in balancing the inorganic cations to electroneutrality. Organic phosphates in insects are primarily acid soluble with minor phospholipid and phosphoprotein components (Wyatt, 1961). The biochemical nature of the organic phosphates in gypsy moth hemolymph has not been fully investigated, however qualitative estimates of glucose-6-phosphate dehydrogenase and u-glycerophosphate dehydrogenase have been previously reported (Brown and Mazzone, 1977). The amino acid and trehalose concentrations in gypsy moth hemolymph are qualitatively similar to the concentrations reported for other lepidopterans and both lie near the lower end of the reported range of concentrations for the species shown in Table 5. The pH of lepidopteran larval hemolymph is sightly acidic. Gypsy moth hemolymph lies within the reported pH range of 6.4-6.7. Quantitative variations in hemolymph osmotic pressure have been reported for lepidopterans (Florkin and Jeuniaux, 1974). The osmotic pressure of gypsy moth hemolymph lies near the middle of the broad range of reported osmotic pressures. The hemolymph protein concentration among lepidopteran species varies significantly (Table 5). Notable fluctuations in protein concentrations occur during ontogeny, especially immediately prior to pupation (Chen, 1983) and may account for the high

829

variability. In support of this argument, we found the mean protein concentration in hemolymph of late fifth-instar larvae (data not shown) to be nearly two-fold higher than the concentration measured in day 5 animals. The data in Table 5 indicate that there are few distinct quantitative differences between the hemolymph composition of gypsy moth and that of other lepidopterans as a whole. Compared to other lepidopterans the total concentration of inorganic solutes is slightly lower in the gypsy moth. Particularly notable are the relatively low sodium and chloride concentrations in gypsy moth hemolymph. In general these differences may reflect the wide variety of analytical techniques employed, as well as the physiological status of the donor insect. Nutritional influences and environmental factors may also underlie some of this variability (Florkin and Jeuniaux, 1974). Acknowledgements-The authors would like to thank Dr Andv Chen (USDA. College Station) for the carbohydrate analyses, Ra‘he Haggeman-(YSI Inc:, Yellow Springs) for advice on conductivity cell construction, and Dr John Wooten for enlightening discussions. Inorganic and organic solute analyses were conducted at Cornell University by the Clinical Pathology Laboratory, the Spectrophotometry Laboratory in the Department of Fruit and Vegetable Science, and the Biotechnology Analytical Facility. This work was supported by USDA Grant No. 89-37250-4519 to Dr Pannabecker.

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