Comparison of gravimetric and chromic oxide methods for measuring percentage utilization and consumption of food by phytophagous insects

J. Ins. Physiol., 1964, Vol. 10, $$I. 989 to 995. Pergamon Press Ltd.

Printed in G&at Britain

COMPARISON OF GRAVIMETRIC AND CHROMIC OXIDE METHODS FOR MEASURING PERCENTAGE UTILIZATION AND CONSUMPTION OF .FOOD BY PHYTOPHAGOUS INSECTS A. J. MCGINNIS and R. KASTING Entomology

and Chemistry

Section, Canada Agriculture Alberta, Canada (Received

30 April

Research Station, Lethbridge,

1964)

Abstract-An indirect method using chromic oxide (Cr,O,) for measuring percentage utilization and consumption of dry matter in diets by two species of phytophagous insects is described and results are compared with those of the classical gravimetric method. Fifth-instar larvae of the two-striped grasshopper, Melanoplus bivittutus Say, and fifth-instar larvae of the pale western cutworm, Agrotis mthogoniu Morr., were fed individually. Diets were prepared from lyophilized tissues of the wheat plant to which about 4 per cent of the inert index compound, chromic oxide, was added. Three diets varying widely in nutritive value were compared for both utilization and consumption. When residual food and excreta could be quantitatively recovered, measurements by the two methods generally agreed. However, the chromic oxide procedure for measuring utilization gives more reliable results than did the gravimetric method, is convenient, and can be used even when quantitative recovery of excreta is not possible. INTRODUCTION

GAIN in body weight, rate of development,

quantity of food consumed, and efficiency of food utilization are commonly used to determine nutritional adequacy of diets. Body weights and rate of development can be readily determined for many insects and are used for assessing the nutritional value of their diets, whereas measurements of consumption and utilization are more difficult. Nevertheless, the consumption and utilization of diets have been determined for some insects by classic gravimetric procedures (CROWELL, 1941; MURTHY, 1954; DADD, 1960; HIRANO and ISHII, 1962; WALDBAUER,1962.). At this laboratory, consumption and utilization of foods by the grasshopper, Melanoplus bilituratus Walker (SMITH, 1959), and the pale western cutworm, Agrotis orthogoniu Morr. (MCGINNIS and KASTING, 1959; &STING and MCGINNIS, 1959), were determined gravimetrically. Such measurements probably lack accuracy because of difficulty in obtaining samples for analysis that truly represent the tissues consumed. This problem was decreased by using diets prepared from plant tissues that were lyophilized and ground (MCGINNIS and KASTING, 1960). Because the gravimetric method is 989

990

A. J. MCGINNISANDR. KASTINC

cumbersome, and usually only mg quantities of either food or excreta are available, results are uncertain for insects and a better method would be desirable. Consumption and utilization of food have been measured with vertebrates by incorporating chromic oxide (CraO,), an inert index compound, into the diets (DANSKY and HILL, 1952; MOORE, 1958; CURRIER et RI., 1960). This method requires that the index compound be neither toxic at the concentrations used nor absorbed from the gut. The recent development of a sensitive quantitative method for measuring Cr,O, (MCGINNIS and KASTING, 1964a) has made it possible to investigate this approach for phytophagous insects (MCGINNIS and KASTING, 1964b). This paper compares the gravimetric and chromic oxide methods for determining percentage utilization and consumption of dry matter for individual larvae of the two-striped grasshopper, Melanoplus bivittatus Say, and larvae of the pale western cutworm, Agrotis orthogonia Morr. MATERIALS

AND METHODS

Insects 150-200 first-instar M. bivittatus larvae were confined in a 1 x 1 x 1 ft cage and fed a dry dietary mixture (SMITH, 1952) supplemented with fresh lettuce leaves. The day before the fourth moult forty-five larvae were confined individually in cages consisting of 6 in:lengths of 14 in. diameter glass tubing closed at the top with plastic screen and at the bottom with a tight-fitting cork smoothly covered with waxed paper. Within 24 hr after moulting each larva was weighed and provided with one of the experimental diets. The larvae were maintained at 30 + 1°C and 50 per cent relative humidity. Larvae of the pale western cutworm were reared individually in 60 x 15 mm petri dishes. During the first four instars the larvae were fed fresh lo-day-old leaves of Thatcher wheat. The bottoms of the dishes were covered with moist blotting-paper to delay drying of the plant tissues and the larvae were transferred daily to clean dishes and provided with fresh food. Upon moulting to fifth-instar, each larva was weighed and assigned to one of the experimental diets. During the test period the petri dishes, without blotting-paper, were held in a moist atmosphere in a closed vessel at room temperature. Procedure Each of the three diets described in Table 1 was fed to eleven, twelve, or thirteen grasshopper larvae and ten cutworm larvae. The wheat plant tissues were lyophilized and ground to pass a 40-mesh screen (aperture-042 mm). A paper impregnated with Cr,O, (CORBETT et al, 1960) that was ground to pass a 40-mesh screen was uniformly mixed with other dry components by tumble-mixing for 24-48 hr. Water was thoroughly mixed with the dry components by grinding in a mortar, and each diet was divided into portions of a size that permitted measurements of consumption by individuals. Each portion was wrapped in a pre-weighed piece of aluminium foil and the fresh weight of each package was determined. All packages were stored at - 30°C. Each insect was weighed at the beginning and end

MEASURING PERCENTAGE UTILIZATIONAND CONSUMPTION OF FOOD

991

of the test period. At 24 hr intervals the excreta produced and the food remaining were separated and quantitatively collected for each insect and fresh food was provided. Food for each insect during the test period was supplied from a single food package. The grasshoppers were fed for 5 days. Cutworm larvae fed the sprout diets were kept on test until they moulted into sixth instar, which averaged 6 days. Those on the pith diet were fed an average of 7 days and none moulted to sixth instar. TABLE I-CONLPOSITION OF DIETS FED TO TWO-STRIPED GRASSHOPPERS AND PALE WESTERN CUTWORMS

Thatcher wheat sprouts* Pith of Golden Ball wheat stems t Cellulose: Cr,O* paper Water

Sprout

Sproutcellulose

Pith

6%)

(8)

k)

10 1.2 20

5 5 l-2 20

10

T2

20

*Prepared

as described by MCGINNIS and KASTING (1960). t Prepared as described~~MCGINNIS and KASTING(~~~~). $ Whatman No. 1 cellulose powder.

Dry matter and Cr,O,

determinations

Dry matter (D.M.) determinations on food and excreta were based on a drying period of 2 hr at 120°C. The percentage D.M. of at least three packages for each diet was averaged and used to calculate the quantity of D.M. initially in each package. The D.M. consumed was determined by difference between the D.M. initially in the package and the combined dry weight of residual food collected from the cage and remaining in the package. CraO, was determined by the method of MCGINNIS and KASTING (1964a). The excreta collected from single insects were dried and then pulverized and mixed in a ‘mortar before sampling for CraO, analysis. The contents of individual food packages were similarly treated. All analyses were done in triplicate. Calculations The procedures described above allowed determination of consumption and percentage utilization by both the gravimetric and the Cr,O,-index procedure. The following relationships were used. Gpavimetpic D.M.

method

consumed

(mg) = D.M. initially in package (mg)-[D.M. remaining package (mg)+residual D.M. in cage (mg)]

in

A. J. MCGINNIS AND R. KA~TING

992

o/o Utilization

D.M.

=

consumed

=

1 _ D.M.

method

D.M.

consumed

(mg) =

o/oUtilization =

consumed

excreted

D.M. O-,0,

(mg) - D.M.

D.M.

(mg)

consumed

(mg)

excreted (mg)

(mg)

1x

1

x 100

100

WW31*Excretax D.M. k-edxoreta)+ a Cr& Insect 13W31*~ood Fh031*~xcreta- FW31*~ood x 1oo (

[Cr2011*Excreta

PS31*3bod ’ 3W4d*Excreta x loo RESULTS

AND

DISCUSSION

The requirement of the index method for a non-toxic inert marker compound was met for both the grasshopper and the cutworm by CraO,. When diets that contained about 4 per cent Cr,O, were fed to either species, consumption was not affected and no toxic effects were observed during the feeding period. Analysis of the insects for Cr,O, after the digestive tracts were emptied showed that none was absorbed from the guts of either species. There was individual variation in intial weight of larvae of M. bivittatus but the utilization values for individuals on any given diet were generally similar (Table 2). TABLE

~-DRY

MATTER

GRASSHOPPER

UTILIZATION

DETERMINED

OF DIETS BY FIFTH-INSTAR

BY THE

GRAVIMETRIC

Insects Diet

Sprout Sprout-cellulose Pith

No.

12 13 11

AND

LARVAE

CHROMIC

OF THE TWO-STRIPED

OXIDE

METHODS

Utilizationl_

Initial wtt

Gravimetric

(mg)

(%)

140 f 27 156+40 158232

56&8 25+8 45 + 5

Cr@, (%I 48 + 1 24+1 44*3

t Mean and standard deviation.

The diets were ranked in the same order with respect to utilization when measured by either the gravimetric or the Cr,O, method. By both methods the average percentage utilization of the three diets was different (P < O*Ol). The results obtained by the two methods were the same for either the sprout-cellulose or the pith diets but were different for the sprout diet (P < 0.01). Based on the values for the sprout and the sprout-cellulose diets, the gravimetric method appears to have overestimated percentage utilization of the former. * pg Cr,O,/mg

D.M.

MEASURING

PERCENTAGE

UTILIZATION AND

CONSUMPTION

With the cutworm, A. orthogonia, the percentage methods for each diet differed (PC 0.01) (Table 3). exception of the high result obtained gravimetrically same order as found for n/r. bivittatus. The pith TABLE

3-DRYMATTBRUTILIZATION CUTWORM DETERMINED

Diet

Sprout Sprout-cellulose GoldenBallpith

No.

10 10 10

993

utilization obtained by the two However, the values, with the for pith, ranked the diets in the diet and resulting fecal pellets

OFDIETSBYFIFTH-INSTARLARVAE BYTHE GRAVIMETRICAND CHROMIC

Insects

OF FOOD

OFTHE PALE WESTERN OXIDE METHODS

Utilization*

Initial wt*

Gravimetric

(mg>

(%>

CraOa (%)

65+21 62+21 67+16

52+2 2452 57*7

41+2 22+2 31*3

* Meanandstandarddeviation.

crumbled more easily than did the other diets and fecal pellets. Normal cutworm activity accelerated crumbling and caused mixing of food and excreta. This circumstance makes complete separation of food and excreta impossible with this diet and probably led to the high value. The percentage utilization for pith determined by the CrsO, method should be more reliable because quantitative recovery of neither food nor excreta is required. A general weakness in use of the gravimetric procedure with insects is the difficulty of accurately determining the amount of dry matter consumed. This is illustrated using actual values obtained with a fifth-instar larva of M. bivittatus fed from a package that contained 1634 mg fresh weight of sprout diet. The D.M. percentages of samples of the food were averaged and used to calculate the amount of D.M. initially present in the package. Depending upon whether the mean plus standard deviation (33.1 per cent), mean (32.5 per cent), or mean minus standard deviation (31.9 per cent) was used in the calculation, the food package contained 541, 531, and 522 mg D.M., respectively. The unconsumed food, which included that left in the package and that recovered from the cage, totalled 403 mg D.M. Hence by difference the quantity consumed by the larva would be 138, 128, or 119 mg for the three calculated D.M. contents. The rt 2 per cent error in D.M. content of the food in the package is increased to & 8 per cent based on the calculated consumption. Of course, if most of the food in the package was consumed by the insect, this error would be reduced. The gravimetric method for measuring utilization of food is slow and cumbersome for routine use with insects and the weaknesses described above further detract from it. In contrast, the CrsO, method is rapid and practical. Moreover, variability among results by the Cr,O, method is lower than among those obtained by the gravimetric method and therefore the former provides a means for measuring differences that could not previously be determined. Furthermore, the Cr,O, method 64

994

A. J. MCGINNIS AND R. KASTING

can be used to measure utilization even when quantitative recovery of food and excreta is not practical. The simplicity of The Cr,O, method combined with these other advantages make it the method of choice. The Cr,O, procedure can also be used to measure food consumption by insects. The quantities of D.M. consumed, determined by the two methods for the grasshopper, agree for the pith and sprout-cellulose diets as indicated by the small

75 0 _a 0, 300-

-

400-

-300.

9 :

_z

-

8

200

E ”

z -

5 ci

0 SPROUT

-I

ci

0 SPROUT

-

’ SPROUT-CELLULOSE

x SPROUT-CELLULOSE

Y?

v PITH

v PITH -

*

100 MG.

200 D.M. GONSUMED

300

400

loo-

0

100 Mt.

~O.w”.>

*oo D.M. CONSUMED

400

300 cGR*“.l

FIG. 1. (A) Dry matter consumption determined by the gravimetric and Cr,O, methods for individual fifth-instar larvae of the two-striped grasshopper. (B) Dry matter consumption determined by the gravimetric and Cr,O, methods for individual f&h-instar larvae of the pale western cutworm.

deviations of actual results from the theoretical 1 : 1 relationship (Fig. 1A). However, the values determined gravimetrically for the sprout diet are consistently high and it is suggested that this is because the initial D.M. content of the food was overestimated. For the cutworm the Cr,O, method gave consistently lower values than did the gravimetric method (Fig. 1B) but results of the two methods for the sprout diets generally agreed within 10 per cent; differences were greater between results of the two methods for the pith diet. Measurements of consumption by either the gravimetric or the Cr,O, method require that residual food and excreta be quantitatively separated. Hence errors in sorting would give inaccurate results by both methods and, particularly for the pith diet, could account for the deviations. The Cr,O, method offers advantages, however, since fewer weighings are needed and also because it avoids the errors of the gravimetric method discussed earlier. REFERENCES CORBETTJ. L., GREZENHALGH J_ F. D., MCDONALD I., and FLORENCEE. (1960) Excretion of chromium sesquioxide administered as a component of paper to sheep. Brit. J. iW&. 14. 289-299.

MEASURING PERCENTAGE UTILIZATIONAND CONSUMPTION OF FOOD

995

CROWELLH. H. (1941) The utilization of certain nitrogenous and carbohydrate substances by the southern armyworm, Prodenia eridania Cram. Ann. ent. Sot. Amer. 34, 503-512. CURRIERA., KITTS W. D., and COWAN I. McT. (1960) Cellulose digestion in the beaver (Castor canadensis). Canad. J. Zool. 38, 1109-I 116. DADD R. H. (1960) Observations on the palatability and utilization of food by locusts, with particular reference to the interpretation of performances in growth trials using synthetic diets. Ent. exp. appl. 3, 283-304. DANSKY L. M. and HILL F. W. (1952) Application of the chromic oxide indicator method to balance studies with growing chickens. J. Nutr. 47, 449-459. HIRANO C. and ISHII S. (1962) Utilization of dietary carbohydrates and nitrogen by rice stem borer larvae, under axenic conditions. Ent. exp. appl. 5, 53-59. KASTING R. and MCGINNIS A. J. (1959) Nutrition of the pale western cutworm, Agrotis orthogonia Morr. (Lepidoptera: Noctuidae). II. Dry matter and nitrogen economy of larvae fed on sprouts of a hard red spring and a durum wheat. Canad. J. Zool. 37, 713-720. MCGINNIS A. J. and KASTING R. (1959) Nutrition of the pale western cutworm, Agrotis ovthogonia Morr. (Lepidoptera: Noctuidae). I. Effects of underfeeding and artificial diets on growth and development, and a comparison of wheat sprouts of Thatcher, T&cum aestivum L., and Golden Ball, T. durum Desf., as food. Canad. J: Zool. 37, 259-266. MCGINNIS A. J. and KASTING R. (1960) Nutrition of the pale western cutworm, Agrotis orthogoniu Morr. (Lepidoptera: Noctuidae). III. Lyophilized sprouts and leaves of wheat as a basal diet for larvae, and effects of supplementation with L-leucine. Canad. J. Zool. 38, 585-592. MCGINNIS A. J. and KA~TING R. (1961) Comparison of tissues from solid- and hollowstemmed spring wheats during growth. I. Dry matter and nitrogen contents of pith and wall and their relation to sawfly resistance. Canad. J. Plant Sci. 41, 469-478. MCGINNIS A. J. and KA~TINGR. (1964a) Calorimetric analysis of chromic oxide used to study food utilization by phytophagous insects. J. A&c. Food Chem. 12, 259-262. MCGINNIS A. J. and KASTINGR. (1964b) Chromic oxide indicator method for measuring food utilization in a plant feeding insect. Science 144, 1464-1465. MOOREJ. H. (1958) The effect of diurnal variations in composition of the faeces of pigs on the determination of digestibility coefficients by the chromium oxide method. Brit. J. Nutr. 12, 24-34. MURTHY M. H. V. (1954) Studies on the nutrition of the silkworm (Bombyx mori L.). Thesis, Mysore University. SMITH D. S. (1959) Utilization of food plants by the migratory grasshopper, Melanoplus bilituratus (Walker) (Orthoptera: Acrididae), with some observations on the nutritional value of the plants. Ann. ent. Sot. Amer. 52, 674-680. SMITH R. W. (1952) Another method of rearing grasshoppers (Orthoptera) in the laboratory. Canad. Ent. 84, 269-271. WALDBAUERG. P. (1962) The growth and reproduction of maxillectomized tobacco homworms feeding on normally rejected non-solanaceous plants. Ent. exp. appl. 5, 147-158.