The accumulation of haemolymph proteins and activity of digestive proteinases of grasshoppers (Melanoplus sanguinipes) fed wheat, oats or kochia

Pergamon

0022-1910(94)00116-2

J. Insect Physiol. Vol. 41, No. 5, pp. 425433, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

The Accumulation of Haemolymph Proteins and Activity of Digestive Proteinases of Grasshoppers (Melanoplus sanguinipes) Fed Wheat, Oats or Kochia C. F. HINKS,*

M. A. ERLANDSON*t

Received 26 May 1994; renised 8 September

1994

Lesser migratory grasshoppers, Melanoplus sanguinipes (Fab.), were fed single plant diets of wheat, oats or kochia foliage in the instar preceding sampling, from the 2nd to 5th instars. Haemolymph proteins, tryptic and chymotryptic activity in the gut and total gut protein were determined. Nutritional indices were calculated from data collected from 2nd-instar grasshoppers. In this instar, grasshoppers consumed more than twice as much kochia as either wheat or oats, and produced correspondingly greater amounts of frass. The duration of the instar was in the order wheat < kochia < oats. Approximate digestibility was significantly lower in grasshoppers fed oats than those fed either wheat or kochia. The efficiency of conversion of digested food was significantly lower and the relative consumption rate significantly higher in those fed kochia than either wheat or oats. The efficiency of conversion of ingested food was in the order wheat > oats > kochia. Haemolymph concentrations of both putative apoprotein I and the comigrating complex of apoprotein II and larval storage protein (LSP) subunits (70-80 kDa mol. wt) were significantly higher in grasshoppers fed the standard colony diet of wheat germ, lettuce and wheat in all instars than for those grasshoppers fed single-plant diets. Among grasshoppers fed the single-plant diets, those fed wheat had the highest levels of putative apoprotein/LSP. On an individual grasshopper basis, there was no correlation between the concentrations of apoprotein/LSP in the haemolymph and the levels of gut protein, trypsin or chymotrypsin. Total protein in whole-gut homogenates was significantly higher in kochia-fed grasshoppers in the 2nd instar and significantly lower in those fed oats in the 5th instar. In all instars, grasshoppers fed oats had higher total trypsin activity than those fed wheat; ratios of trypsin activity in oat- vs wheat-fed grasshoppers were 1.8 : 1,1.9 : 1,3.8 : 1 and 1.9 : 1 for the 2nd, 3rd, 4th and 5th instars, respectively. Total chymotrypsin activity was significantly higher in grasshoppers fed oats in the 4th and 5th instars, and in those fed kochia in the 5th instar. The ratios of chymotrypsin activity in oat- vs wheat-fed grasshoppers were 1.5 : 1, 1.0 : 1, 2.5 : 1 and 1.9 : 1 for the 2nd, 3rd, 4th and 5th instars, respectively. The present study confirmed that both the kochia and oats diets were inferior in supporting grasshopper growth, probably because of lower protein acquisition and hypermodulation of digestive proteinases. However, the levels of activity of these enzymes were considerably lower in the earlier instars than those previously reported in adults of this species. The results are discussed in the context of hormonal activity and suggest that protein is allocated differently in nymphs compared to adults. Comparison of the physiological and biochemical responses of grasshoppers to wheat and oats and their variably resistant cultivars provides a valuable model for studying such interactions. h4ehnoplus

Grasshopper Larval storage protein

sanguinipes

Apolipoprotein

Oats Kochia Wheat Nutritional indices

INTRODUCTION Even highly polyphagous migratory grasshopper,

Modulation

Digestive proteinases

display feeding preferences (Mulkern et al., 1962; Chapman, 1990). In native vegetation there is a general correlation between feeding preference and the performance (fitness) parameters-growth, survival and reproduction (Pfadt, 1949; Smith, 1959). The introduction of agriculture has replaced native grasslands in much of North America with croplands dominated by small-grain

grasshoppers such as the lesser Melanophs sanguinipes (Fab.),

*Agriculture Canada Research Station, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2. ~To whom all correspondence should be addressed. 425

426

C. F. HINKS

and M. A. ERLANDSON

cereals, and accompanied by alien weeds. Among the latter, kochia, Kochiu scoparia L., has spread throughout the region and in years of drought often is one of the few remaining green plants. Because this plant is readily grazed by grasshoppers (Olfert et al., 1990a) it was included in previous studies in comparison with wheat and oats (Hinks et al., 1990, 1991). Wheat and oats have been described consistently as suitable and unsuitable diets, respectively, for a wide range of grasshoppers (Carothers, 1923; Hodge, 1933; Pfadt, 1949; Smith et al., 1952; Smith, 1959; Misra, 1962; Pickford, 1962; Hinks et al., 1990, 1991). The foliage of barley (Mulkern et al., 1962) and rye (Gardiner, 1985; Bomar and Lockwood, 199 1) are suitable food for grasshoppers. The question of host-plant suitability, however, is more complex than these studies indicate. Recently, several varieties of wheat have been identified as deleterious to the growth of grasshoppers and grasshopper populations (Hinks et al., 1987; Olfert et al., 1990b). Long-term feeding on introduced crop plants can have serious consequences for grasshopper populations. For example, although alfalfa is readily eaten, grasshoppers feeding on it suffer high mortality and reduced reproductive capacity (Barnes, 1955). Similarly, oats or kochia fed as the sole diet to grasshoppers resulted in population decline (Hinks et al., 1990; Olfert et al., 1990b). Detailed studies of the effects of long-term consumption of oats and kochia, compared to wheat, revealed that these diets caused higher mortality, delayed development and reduced the rate of reproduction (Hinks et al., 1990). It was concluded that when grasshoppers were faced with alien plants, i.e. plants with which they had not co-evolved, the relationship between preference and performance broke down (Hinks et al., 1990). Adult grasshoppers fed on either oats or kochia hypermodulated the digestive proteinases, trypsin and chymotrypsin, but not aminopeptidase or carboxypeptidase (Hinks et al., 1991). Similar hypermodulation in response to dietary proteinase inhibitors was described by Broadway and Duffey (1986) in lepidopterous larvae, and by Burgess et al. (1991) in the black field cricket, TeZeogryZZus commodus (Walker). The consequences of excessive proteinase production resulted in a serious loss of sulphur-containing amino acids, which could be mitigated by supplementing the diet with methionine (Broadway and Duffey, 1986), or improving the quality of the protein (Broadway and Duffey, 1988). Because methionine supplementation of an oats diet failed to mitigate the effects of this diet on grasshoppers, and in vitro inhibition of trypsin and chymotrypsin by oats extracts could not be demonstrated, it was concluded that an explanation for hypermodulation of the proteinases had to be sought elsewhere (Hinks et al., 1991, 1993). Whether the secretion of additional digestive proteinases was a major, or contributing factor in the attritional effects that were observed in grasshoppers fed kochia or oats was not resolved (Hinks et al., 1993). Larocque and Houseman (1990) found that the effects of three proteinase inhibitors in the diet of the European corn

borer, Ostrinia nubialis (Hiibner), were different and that although corn proteinase inhibitor increased digestive proteinase activity, it did not result in weight loss. Among insects, the Orthoptera have an unusually high requirement for protein, of which approximately 50% is deposited in the cuticle (Bernays, 1986). There is evidence that some haemolymph proteins are deposited directly into new cuticle (Peter and Scheller, 1991), but most of the large assemblage of proteins extractable from the cuticle (Nohr and Andersen, 1993), particularly those specific to particular regions of cuticle are synthesized and secreted by hypodermal cells (Willis, 1987). Whatever the source of cuticular proteins, one consequence of dietary factors which interfere with digestion or the accumulation of haemolymph protein is a retardation in growth and development by prevention of the normal acquisition of exoskeletal mass. In the present study, which complements a previous study on adults of M. sanguinipes (Hinks et al., 1991), the capacity of grasshopper nymphs to accumulate haemolymph proteins [apolipoproteins I and II plus larval storage protein (LSP)] and secreted gut proteins (digestive proteinases), when fed nutritionally contrasting diets, was determined. The results are discussed in the context of mechanisms relating to nutrient acquisition, resource allocation and grasshopper development. MATERIALS

Grasshoppers

AND METHODS

and plants

For all experiments, nymphs from a non-diapausing colony of the lesser migratory grasshopper, M. sanguinipes, were used. They were reared according to the method of Ewen and Hinks (1986), with the modifications described by Hinks and Erlandson (1995). Grasshoppers were allowed to feed on lettuce as they hatched, but within 6 h were transferred to one of four diets: Columbus wheat (Triticum aestiuum L.); Cascade oats (Avena sativa L.); kochia (K. scoparia L.); or the standard colony diet consisting of potted wheat seedlings, head lettuce and wheat bran. Grasshoppers sampled in the 3rd to 5th instars were reared on the standard colony diet until the start of ‘the instar preceding that in which they were sampled. They were then reared on each of the single-plant diets until the middle of the succeeding instar. All grasshoppers were provided with fresh plant material ad Zibitum. Plant material for each single-plant diet was grown in a soil-free mix (Stringham, 1971) in growth chambers providing 16 : 8 h light-dark regime at 22 + 1 and 15 + 1°C respectively. Nutritional

indices

Neonate grasshoppers were reared on lettuce and bran until they ceased feeding prior to moulting, and then they were isolated in a cage without food overnight. Freshly moulted grasshoppers were selected the following morning. Ten grasshoppers were used for each diet group. Each grasshopper was weighed and transferred to a

ACCUMULATION

OF HAEMOLYMPH

PROTEINS

AND

16-dram snap-cap polystyrene vial. Each polystyrene vial also contained a 3-dram glass vial filled with water, with the polyethylene stopper slotted to accommodate a length of cereal leaf or whole kochia leaf. Leaf sizes or lengths of each food plant were selected on the basis of a previously determined consumption rate of approximately 80% in 24 h. At 24 h intervals the residual food and frass was collected, oven dried at 75°C for 24 h, then weighed. After feeding ceased at the end of the instar, food was withdrawn and residual leaf fragments, frass and grasshoppers were oven-dried as above. The methods for predicting dry weights and calculating nutritional indices were the same as those described by Hinks et al. (1991) using the formulae given in Reese and Beck (1978). Haemolymph

collection

Haemolymph was collected from unsexed nymphs (15 nymphs for each diet-instar group) 3 days following moulting (i.e. mid-stadium) by severing a metathoracic leg between the femur and the episternum. Using gentle pressure a drop of haemolymph was extruded and collected with a capillary pipette. For each sample collected, the haemocytes were removed by centrifugation at 13,OOOg for 5 min at 4°C and the supernatants stored at -70°C until further use. Electrophoresis

of haemolymph

proteins

The supernatant from haemolymph samples was mixed (1 : 1) with 2X SDS-PAGE sample buffer, electrophoresed on 10% SDS-PAGE separating gels and visualized using the methods described by Hinks et al. (1991). The Coomassie blue-stained gels were analysed on a scanning densitometer (LKB Gelscan XL) and the area (mm’) under peaks corresponding to putative Apo I and Apo II/LSP were determined and statistically analysed using ANOVA. The means were separated using the Waller-Duncan k-ratio t-test at P = 0.05 (SAS Institute, 1989).

DIGESTIVE

PROTEINASES

427

IN M. SANGUINIPES

diet (Hinks et al., 1991). The assay methods used were those of Gooding and Rolseth (1976). Analysis and separation of means was the same as that used for haemolymph proteins.

RESULTS Growth nutritional

indices

Nutritional indices were determined in 2nd-instar grasshoppers (Table 1). In this instar, grasshoppers consumed more than twice as much kochia as either wheat or oats (2.6 : 1 and 2.5 : 1, respectively). Similarly, the weight of frass was much higher on the kochia diet than that produced on wheat or oats (2.6 : 1 and 2.1 : 1, respectively). The higher ratios were reflected in a poorer rate of efficiency of conversion of ingested (ECI) kochia compared to either wheat or oats. Gains in weight were higher in grasshoppers fed wheat and kochia compared to those fed oats, but the differences were not significant. The duration of the instar was in the order wheat < kochia < oats. Approximate digestibility (AD) was significantly lower in grasshoppers fed oats compared to those fed either wheat or kochia. The efficiency of conversion of digested food (ECD) was significantly lower and the relative consumption rate (RCR) was significantly higher in those fed kochia than those fed either wheat or oats. The ECDs were in the order wheat > oats > kochia (Table 1). Haemolymph

proteins

The predominant haemolymph polypeptides from grasshopper nymphs, as detected by SDS-PAGE, were the putative Apo I (216 kDa; Hinks et al., 1991) and the putative Apo II/LSP subunits which co-migrate at approximately 73 kDa (see Fig. 1; Wyatt, 1988,199O). The concentration of putative Apo I and Apo II/LSP subunit bands were quite uniform in the 2nd- to Sth-instar nymphs fed the standard colony diet and sampled

Digestive proteinases Fifteen nymphs sampled from each diet-instar group were starved overnight to eliminate any residual food or faecal material in the gut. Egestion rates for grasshoppers are relatively short (4-7 h) (Mead et al., 1988) and as the soluble protein content of the frass from grasshoppers fed the diets in this study was very low, and we were unable to detect the presence of proteinase inhibitors or tannins which might interfere with protein digestion (unpubl. data) we have assumed that there was complete digestion of soluble plant proteins. Each grasshopper was chilled at 4°C for 30 min, anaesthetized with CO? (to minimize regurgitation of the crop contents), then dissected rapidly and the whole gut removed. Subsequent treatment of gut tissues, including enzyme assays, were done as described previously (Hinks et al., 1991). In the present study only trypsin and chymotrypsin were assayed because the activity of neither aminopeptidase nor carboxypeptidase is influenced by

TABLE 1. Mean feeding data (dry weight in mg) and nutritional indices (f SE) of the lesser migratory grasshoppers fed on one of three plants for the duration of the 2nd instar

Wheat No. of grasshoppers Nutritional data Weight gain* Amount eaten* Weight of frass* Duration of instar Nutritional AD ECD EC1 RCR

Diet Oats

10

(days)

Kochia

9

2.7 12.9 5.4 5.5

f + * +

0.2” 0.9 0.2” 0.4”

2.2 13.3 6.8 8.6

51.3 37.6 21.1 4.9

f * f &

2.2” 3.2” 0.2’ 0.3”

49.4 34.1 16.6 6.4

f f * f

10

0.2” 1.2” 0.8” 0.6b

2.7 33.3 14.1 6.9

_+ 0.3” f 1.6b f 1.7b + 0.3’

57.2 14.5 7.9 13.4

f + f f

index + * f +

1.9b 3.7” 1.6b 0.5”

2.2” 1.7b 0.6” l.lb

*Dry weight in mg. “Means followed by the same letter in each row are not significantly different at the 5% confidence level, Duncan’s multiple range test.

C. F. HINKS

428

3rd

2nd 1

M

2

and M. A. ERLANDSON

3

4

1

2

4th 3

4

5th

12341234M

205

-A

205 116 97.4 66

116 97.4 66

-B

45

45

FIGURE 1. Electropherograms of haemolymth proteins from 2nd-, 3rd-, 4th- and Sth-instar nymphs of the lesser migratory grasshopper fed the standard colony diet of wheat, lettuce and wheat bran. Equal volumes (0.33 ~1) of haemolymph from four individuals of each instar were electrophoresed on a 4% stacking and 10% separating SDS-PAGE gel and the gel stained with 0.1% Coomassie blue. The molecular weights of standards (M) (Sigma MW-SDS-7OL) are indicated at the side of each panel. The positions of the (A) putative Apo I and (B) putative Apo II/LSP complex bands are indicated.

mid-stadium (Fig. 1, Table 2). However, concentrations of these two major protein bands, Apo I and Apo II/LSP, were very variable among individual grasshoppers fed the single-plant diets at each instar, as illustrated by results from 2nd-instar haemolymph samples (Fig. 2, Table 2); nevertheless, there were discernible trends. Higher concentrations of Apo I were recorded in haemolymph of 2nd- and 3rd-instar grasshoppers fed wheat compared to those fed either oats or kochia, but the differences were negligible in haemolymph of the 4th instar, and although divergent again in the 5th instar, they were not significantly different (Table 2). The general trend was toward a reduced concentration of Apo I as development progressed from the 2nd to the 5th instar (Table 2). The Apo II/LSP complex was by far the most dominant protein band in the haemolymph throughout development (Fig. 1). The concentration of Apo II/LSP varied considerably from instar to instar in grasshoppers fed

TABLE 2. Haemolymph concentrations of (A) putative Apo I and (B) putative Apo II/LSP band (see the text for explanation) in the lesser migratory grasshopper during development, fed one of four diets (standard colony diet, wheat, oats or kochia) for the preceding instar and sampled in the instar tabulated* 2nd instar

3rd instar

4th instar

(A) Putatiue Apo I 12.7 f 1.2” 12.4 k 1.5” 9.3 f 1.9b 6.2 f O.lb 6.6 f 1.3b 5.2 f 0.9’ 6.2 f O.Sb 4.8 f 0.9’

SCD Wheat Oats Kochia

13.0 11.8 6.0 7.7

f + + f

2.5” 1.9” 1.S’ 1.V

SCD Wheat Oats Kochia

194.7 67.7 28.4 20.4

f f + k

(B) Putative Apo II/LSD 1.5” 252.3 f 23.4” 280.3 + 22.1a 60.0 f ll.6b 1l.Y 18.4 f 4.3b 10.2 + 2.lb 26.4 + 6.5c 1.4 11.9 + 1.9b 15.9 f 2.1’ 5.4’

5th instar

12.3 3.4 4.8 2.1

202.6 141.8 80.7 66.2

+ + + +

+ k f f

1.9’ l.Ob l.2b 0.4b

25.5” 22.1b 3 1.Obs 14.4’

*Each value in the main body of the table is the mean k SE of the areas under protein peaks (in mm2 x lO-2), as determined by scanning densitometry of SDS-PAGE gels, of haemolymph samples from 12-15 grasshoppers (0.33 ~1 of haemolymph/sample). “‘Means followed by the same letter in each column are not significantly different at the 5% confidence level, Waller-Duncan’s k-ratio f-test.

single-plant diets (Table 2). In grasshoppers fed wheat there was a 73% decline in Apo II/LSP in the 3rd instar, a recovery in the 4th instar to almost 90% of that measured in the 2nd instar and a 140% increase in the 5th instar over the preceding instar. Concentrations of Apo II/LSP followed a similar pattern in grasshoppers fed kochia or oats. In all instars, grasshoppers fed wheat had higher levels of Apo II/LSP in their haemolymph than those fed kochia or oats (Table 2). Total protein and digestive proteinases Total protein in whole-gut homogenates was comprised of the luminal enzymes and the protein from the tissues of the gut, and no attempt was made to differentiate the two. By far the greater part of the endopeptidase activity occurs in the luminal fluid (C. F. Hinks, unpubl. observations). Diet had a relatively small effect on total protein. Only in the 2nd and 5th instars were the values significantly different, being higher in grasshoppers fed kochia and lower in those fed oats (Table 3). Total trypsin activity in the guts declined successively from the 2nd to the 3rd and 4th instars, but increased more than 6-fold in the 5th instar in wheat-fed grasshoppers. In oats- and kochia-fed grasshoppers, total trypsin activity also declined in the 3rd instar but increased in the 4th and 5th instars (Table 3). Significantly higher trypsin activity was found in oats-fed grasshoppers compared to those fed either wheat or kochia in all instars, with the exception of those in the 4th instar fed kochia (Table 3). Total chymotrypsin activity followed the same trend as that of trypsin during development in wheat-fed grasshoppers. There was a similar trend in which chymotrypsin values did not exceed those from 2nd-instar grasshoppers until the 5th instar in grasshoppers fed kochia or oats (Table 3). Although total chymotrypsin activity was higher in kochia- or oats-fed grasshoppers than in those fed wheat, the values were significant only

ACCUMULATION

OF HAEMOLYMPH

PROTEINS

AND

DIGESTIVE

PROTEINASES

IN M. SANGUINIPES

429

66.0-

36.0-

14.2-

FIGURE 2. Representative electropherograms of haemolymph proteins from 2nd-instar nymphs of the lesser migratory grasshopper fed wheat (W), oats (0) or kochia (K). Equal volumes (0.33 ~1) of haemolymph from four individuals from each diet-group were electrophoresed on a 4% stacking and 10% separating SDS-PAGE gel and the gel stained with 0.1% Coomassie blue. The molecular weights of standards (S) (Sigma MW-SDS-70L) are indicated on the left-hand side of the panel. The positions of the (A) putative Apo I and (B) Apo II/LSP complex bands are indicated on the right-hand side of the panel.

in 4th- and Sth-instar grasshoppers fed oats, or those in the 5th instar fed kochia (Table 3). DISCUSSION

The three plant species used in the present study represent contrasting diets in terms of palatability and nutritive value (Hinks et al., 1990): wheat alone is suitable, and kochia and oats are inadequate for different reasons. In a number of studies, commencing with the observations of Carothers (1923), oats was demonstrated to be unsuitable for many species of grasshoppers (Pfadt,

1949; Smith, 1959; Pickford, 1962; Hinks et al., 1990). Smith (1959) could find no reason why oats should be nutritionally inadequate and stated that it was simply a matter of insufficient consumption. However, a modern interpretation would take into account secondary plant chemistry in addition to nutritive value. In this context, the phagodeterrent effects of oats were demonstrated by Hinks et al. (1990) in comparative studies which included wheat and kochia. Compensatory feeding, i.e. the property of increasing food intake to meet nutritional requirements, readily occurs in grasshoppers and locusts (McGinnis and Kasting, 1967; Simpson and Abisgold,

TABLE 3. Total protein, trypsin and chymotrypsin activity in total crude gut homogenate from the lesser migratory grasshopper during development, fed wheat, oats or kochia for the preceding instar and sampled in the instar tabulated. The values in the main body of the table are the means f SE (n = 15) of (A) protein content (pg) determined by the Lowry method, (B) trypsin* activity and (C) chymotrypsin* activity 2nd instar

Wheat Oats Kochia

324.7 k 30.1a 303.5 f 27.1” 411.3 k 27.3”

Wheat Oats Kochia

24.3 f 3.5’ 42.9 f 5.1b 16.0 f 2.p

Wheat Oats Kochia

207.1 + 39.3” 305.7 * 35.9 274.6 f 36.6=

3rd instar

4th instar

(A) Total Protein Content 637.5 + 33.9” 896.2 * 69.9” 655.4 + 46.0” 872.2 f 83.9” 693.1 f 37.8” 689.7 + 69.9” (B) Trypsin Activity 12.7 k 4.2” 9.2 f 2.e 23.5 + 3.3b 34.7 f 10.3b 13.1 f 3.1” 28.5 + 7.yb (C) Chymotrypsin Activity 142.9 f 41.6” 100.3 * 17.6” 148.7 f 25.5” 245.5 f 83.7b 154.5 k 23.3” 186.7 f 36.8”

5th instar

3464.3 + 270.2” 2670.9 f 304.3b 3609.2 k 247.2” 58.0 f 11.7” 112.6 + 15.2b 72.6 f 14.4’ 480.6 * 65.9 897.6 _+ 158.2b 896.7 f 158.2b

*One unit of enzyme activity represents the hydrolysis of 1 prnol substrate/min at 30°C; see the text for details. 8,bMeans followed by the same letter in each column are not significantly different at the 5% confidence level, Wailer-Duncan’s k-ratio t-test.

430

C. F. HINKS and M. A. ERLANDSON

1985) but the low rate of food intake and slow growth of grasshoppers fed oats provide no evidence for compensatory feeding in this case. In view of the limitation phagodeterrency places on the consumption of oats, it is particularly noteworthy that the AD for oats in 2nd-instar grasshoppers was significantly lower than the ADS of either of the alternative diets. This may signify either a poorer nutrient balance in oats compared to wheat or kochia, or the presence of an anti-nutrient. Although the foliage of all green plants contain the nutrients essential to insects (Fraenkel, 1951), the concentration and proportions of nutrients in plants varies widely with species and developmental stage (Slansky and Scriber, 1985). As well, their availability frequently is governed by the presence of various secondary plant chemicals (Slansky, 1992). The nutritional indices reported for 2nd-instar grasshoppers in the present study make an interesting comparison with those previously reported for the adults of the same species (Hinks et al., 1991). In contrast to adults, 2nd-instar grasshoppers consumed significantly more kochia than either wheat or oats. However, the AD for both wheat and oats was substantially lower in 2nd-instar grasshoppers compared to adults, whereas the AD for kochia was slightly higher during the 2nd instar. Similar differences in the AD of five species of grasses were found between Sth-instar nymphs and adults of Locusta migratoria (Hoekstra and Beenakkers, 1976). In this species, the AD was generally higher in Sth-instar nymphs than in adults, but whether the ECDs and ECIs were higher or lower was diet-dependent (Beenakkers et al., 1971). The low digestibility of kochia compared to wheat and oats by adult grasshoppers was explained by Hinks et al. (1991) by the presence of the C4 photosynthetic pathway (Welkie and Caldwell, 1970), whereas the two cereals have the C3 pathway. C4 plants have structural differences in leaf tissue, in the form of thick-walled cells, which render nutrients less accessible to grasshoppers (Caswell and Reed, 1976). The greater digestibility of kochia by 2nd-instar grasshoppers may result from the ability of their smaller mandibles to better triturate kochia leaf tissues, thus partially overcoming the anti-herbivore conformation associated with C4 plants. Notwithstanding the greater efficiency with which 2nd-instar grasshoppers access the nutrients of kochia, it still appears to be a nutrient-poor diet as indicated by significantly lower ECD and EC1 and significantly higher RCR values than either wheat or oats. Secondary chemistry may also be a factor. Compared to poaceous plants, forbs typically have a greater diversity of secondary chemicals (Jones and Firn, 1991), the toxicity of which is strongly influenced by the nutritional value of the plant (Reese, 1979). As the nutrients of kochia may be less accessible to adults than to early-instar nymphs, adults may be denied the mitigating effects of nutrients on the allelochemicals known to be present (Sosulski, 1987, 1988). Diets diluted with cellulose had a lower rate of utilization and were consumed in greater volume in L.

migratoria and Schistocerca gregaria, which more than compensated for any loss in nutrient intake, as measured by rate of growth (Dadd, 1963). Similar experiments by McGinnis and Kasting (1967) on M. sanguinipes, using a substantially greater degree of dilution, resulted in a proportionally greater rate of ingestion, but in this instance there was a moderately decreased rate of growth. These observations formed the basis for the concept of compensatory feeding developed by Simpson and Abisgold (1985) and Simpson and Simpson (1990). High rates of ingestion tend to reduce the efficiency of assimilation (Slansky, 1982) and this is evident in the present study among grasshoppers fed kochia. One explanation for the disparities in values for ECD and EC1 between adults (Hinks et al., 1991) and those for the 2nd instar, is the greater expenditure of energy in adults as a consequence of flight and reproduction. Disparities between immature stages and adults can be interpreted to indicate differing nutritional requirements (Beenakkers et al., 1971). An alternative explanation is that the disparity reflects a general nutritional inadequacy which is apparent because of the differences between nymphs and adults in resource allocation. However, it is difficult to distinguish between this effect and the “transition effect”, discussed by Gordon (1972) and invoked by Hoekstra and Beenakkers (1976) as the explanation for this phenomenon in L. migratoria on certain diets. Differences in rates of conversion of the food to tissue between nymphs and adults of L. migratoriu was attributed by Beenakkers and Broek (1974) to the higher titre of juvenile hormone in the immature stages. Failure to meet the nutritional needs of growth in a temperature/time matrix in the early instars results in expression of a developmental polymorphism, in which the normal 5-instar morph is replaced by one having 6 instars. The induction of such a supernumerary moult in M. sanguinipes can occur in response to low temperature (Pfadt et al., 1979) or nutritionally-inadequate diets (C. F. Hinks, unpubl. observations). As supernumerary moulting is an endocrine-mediated response (Hardie and Lees, 1985) which has to be induced in the early instars (Tanaka, 1981), the difference in capacity to modulate digestive proteinases between the 2nd- to 3rd-instar nymphs and 4th- to Sth-instar nymphs assumes a particular significance. From the data in the present study, it would appear that up to the 4th instar, grasshoppers have a limited capacity to modulate digestive proteinases, but during the 4th and 5th instars the capacity to modulate these enzymes increases. It follows that the preferential allocation of protein to somatic growth and the critical stage in determining moult number may share a common mechanism of endocrine control. Supernumerary moulting has an adaptive value, enabling grasshoppers to develop on diets which would fail to meet their nutritional requirements within the temperature/time constraints referred to above. Because the exoskeletal mass of grasshoppers is very high compared to many other insects and is comprised of

ACCUMULATION

OF HAEMOLYMPH

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up to 50% protein (Bernays, 1986), grasshoppers have a correspondingly high demand for dietary protein during development. In contrast to the larvae of holometabolous insects, therefore, grasshopper nymphs require food with a lower water content and a more concentrated supply of nutrients. The principal protein requirements of developing grasshopper nymphs are for somatic growth, but protein also is needed for the production of digestive enzymes and the peritrophic membrane, both of which are naturally lost in the formation of the food bolus and its passage and elimination as a fecal pellet. The presence of polyphenols in plants can stimulate an increased production of peritrophic membrane, which preferentially binds polyphenols (Bernays et al., 1989). Similarly, proteinase inhibitors stimulate the increased production of digestive proteinases (Broadway and Duffey, 1986; Burgess et al., 1991). Whether, in either case, the loss of protein results in retarded growth depends on the quality and quantity of protein available in the diet (Broadway and Duffey, 1988; Burgess et al., 1991). The persistent hypermodulation of digestive proteinases is potentially detrimental to grasshoppers feeding on oats (Hinks et al., 1991). This and other observations were synthesized into a model of protein allocation taking into account the major protein “sinks” in grasshoppers, and plant factors which sequentially influence the processes involved in protein acquisition (Hinks et al., 1993). However, the extent to which the hypermodulation of digestive proteinases results in loss of protein remains uncertain. The countercurrent system which operates in insect guts conserves solutes (Dow, 1986). Therefore, in the absence of proteinase inhibitors, the additional enzyme secreted into the gut lumen in response to dietary oats and kochia should simply accumulate. This may indeed be the case, because hypermodulation of enzymes was accompanied by increased volume of crop fluid, which in part accounts for the the higher level of enzyme activity (Hinks, unpub. observations). Although distension of the crop results in stimuli which terminate the meal (Bernays and Simpson, 1982) the relatively large volume of fluid retained in the crop by grasshoppers feeding on kochia does not appear to affect frequency of feeding or meal size. The retention of a similarly large volume of crop fluid by grasshoppers feeding on oats, therefore, can be discounted as an explanation for its low rate of consumption. Apo I and II are ubiquitous proteins and integral components of the high-density lipophorin which plays a key role in lipid transport in the haemolymph of the larval stages of insects (Kanost et al., 1990). There is evidence also for a similar role in the transport of cholesterol, cuticular hydrocarbons and other hydrophobic molecules, including xenobiotics (Chino et al., 1974; Katase and Chino, 1984; Haunerland and Bowers, 1986; Shapiro, 1989). In adult grasshoppers the primary function of lipophorin is that of a reusable shuttle, in large measure to transport lipid from reserves to the flight muscles. In grasshopper nymphs Apo I and II would function similarly as components of lipophorin in the transporIP41,&-D

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tation of lipids, which under a favourable dietary regime would be from the mid-gut to the fat body, a storage site for lipids. Evidently, this is the direction of the process in wheat-fed grasshoppers, for most individuals had well-developed fat body, often strongly pigmented yellow or orange. In contrast, the fat body from both oats- and kochia-fed grasshoppers most frequently was sparse and pallid. In these grasshoppers reserves of both fat and proteins appear to be declining, presumably because neither diet provides a positive balance after meeting the requirements for energy, for protein allocated to somatic growth and the production of additional digestive enzymes. LSPs are some of the more predominant haemolymph proteins in insects. In locusts, the haemolymph titre of LSP (a hexameric protein of 410 kDa made up of 75 kDa subunits) rises by day 3 of the 5th instar and reaches a peak by day 6 (de Kort and Koopmanschap, 1987,199l). The regulation of LSP gene expression appears to be repressed by juvenile hormone and its concentration in the haemolymph may be closely tied to the moulting cycle of nymphs (Wyatt, 1990). Thus, the titre of LSP is usually low after moulting and accumulates beginning in the early to mid portion of the stadium. For many insects, including the locust, Apo II and the subunits of the hexameric storage proteins [such as LSP, the persistent storage protein (PSP) and the cyanoprotein] co-migrate in the 7&80 kDa mol. wt size range when analysed by SDS-PAGE (Wyatt, 1990). Thus, the determination of the effects of diet and dietary components on the concentrations of each of these proteins will require different approaches for their separate quantification. However, some general observations can be made regarding the effect of diet on haemolymph proteins in the present study of M. sanguinipes. As all of the haemolymph samples were taken in mid-instar some comparisons can be made. Grasshoppers taken from the colony which had access to wheat germ, lettuce and wheat plants-a favourable diet in terms of lipid, carbohydrate and protein-had consistent and high haemolymph titres of Apo I and Apo II/LSP as determined by SDS-PAGE (Fig. 1 and Table 2). The Apo I and Apo II/LSP haemolymph concentrations were lower when grasshopper nymphs were fed on single-plant diets but the effects of the kochia and oats diets were particularly evident in terms of lower Apo II/LSP concentrations (Table 2). This could indicate that under these diet regimes the grasshopper nymphs were unable to synthesize as much storage protein (probably the major contributor to the Apo II/LSP polypeptide band) as in the colony-fed grasshoppers. In Lepidoptera, concentrations of arylphorin (a major haemolymph storage protein) have been related to feeding and nutrient availability (Kanost et al., 1990). M. sunguinipes fed on various diets may also make a useful model for the study of the effect of diet on the allocation of resources to storage proteins. As well, the apparent failure to accumulate additional lipid reserves and perhaps their withdrawal from existing reserves in

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grasshoppers fed oat and kochia diets raises questions concerning the regulation of lipophorin levels. On an individual basis, there was no correlation between quantities of apoprotein/LSP in the haemolymph and the levels of gut enzyme activity among the grasshoppers at any nymphal instar. A high degree of variability among individuals was noted in all parameters measured on the single-plant diets. From these observations it can be assumed that a number of independently variable factors determine gut enzyme activity and concentrations of haemolymph proteins. Nevertheless, on two of the diets studied, increased enzyme activity could be associated with protein attrition, and reduced concentrations of apoprotein/LSP could be associated with attrition of the fat body. It is becoming increasingly important to understand insect/plant interactions at the biochemical level as technologies are developed to engineer resistance to insects in cultivated plants. However, in most interactions knowledge of the dynamics of nutrition and secondary plant chemistry is fragmentary. Comparison of the physiological and biochemical responses of grasshoppers to wheat and oats and their variably resistant cultivars (Hinks et al., 1987; Olfert et al., 1990b) provides a valuable model for studying such interactions. REFERENCES Barnes 0. L. (1955) Effect of food plants on the lesser migratory grasshopper. J. Econ. Ent. 48, 119-124. Beenakkers A. M. T. and Broek A. T. M. van den (1974) Influence of juvenile hormone on growth and digestion in fifth instar larvae and adults of Locusta migratoria. J. Insect Physiol. 20, 1131-l 142. Beenakkers A. M. T., Meisen M. A. H. Q. and Scheres J. M. J. C. (1971) Influence of temperature and food on growth and digestion in fifth instar larvae and adults of Locusta. J. Insect Physiol. 17, 871-880. Bernays E. A. (1986) Evolutionary contrasts in insects: nutritional advantages of holometabolous development. Physiol. Ent. 11, 377-382. Bernays E. A. and Simpson S. J. (1982) Control of food intake. A&. Insect Physiol. 16, 59-118. Bernays E. A., Cooper Driver G. and Bilgener M. (1989) Herbivores and plant tannins. Adv. ecol. Res. 19, 263-302. Bomar C. M. and Lockwood J. A. (1991) Development and dietary effects on consumption of wheat bran by laboratory reared Melanoplussanguinipes (F.) (Orthoptera: Acrididae). J. Kansas Ent. Sot. 64,295-299. Broadway R. M. and Duffey S. S. (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J. Insect Physiol. 32, 827-833. Broadway R. M. and Duffey S. S. (1988) The effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. J. Insect Physiol. 34, 111-l 17. Burgess E. P. J., Stevens P. S., Keen G. K., Laing W. A. and Christeller J. T. (1991) Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus. Ent. Exp. Appl. 61, 123-130. Carothers E. E. (1923) Notes on the taxonomy, development and life history of certain Acrididae (Orthoptera). Trans. Am. Ent. Sac. 49, l-24. Caswell H. and Reed F. (1976) The indigestibiltiy of C4 bundle sheath cells by grasshoppers. Oecologia (Berlin) 26, 151-156. Chapman R. F. (1990) Food selection. In Biology of Grasshoppers (Eds Chapman R. F. and Joem A.), pp. 39-72. Wiley, New York.

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Acknowledgements-We thank D. Hupka, M. Matheson, L. Braun and K. Moore for technical assistance, and R. Underwood for photography. This study was supported in part by the Western Grains Research Foundation.