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Energy difference in lipid and glycogen metabolism of healthy and iridescent virus-infected Heliothis zea (Boddie)
Comp. Biochem. Physiol. Vol. 70B, pp. 179 to 183, 198I Printed in Great Britain. All rights reserved
0305-0491/81/100179-05502.00/0 Copyright © 1981 Pergamon Press Ltd
ENERGY DIFFERENCE IN LIPID AND GLYCOGEN METABOLISM OF HEALTHY AND IRIDESCENT VIRUS-INFECTED HELIOTHIS ZEA (BODDIE) A. C. THOMPSON and P. P. SIKOROWSKI Boll Weevil Research Laboratory, Agricultural Research, Science and Education Administration, USDA, Mississippi State, MS 39762 and Department of Entomology, Mississippi State University, Mississippi State, MS 39762, U.S.A. (Received 11 February 1981) A b s t r a c t - - 1 . Heliothis zea (Boddie) larvae infected with irridescent virus (IV) showed rapid cell dissolution in the fat bodies. 2. Fatty acid accumulation in healthy H. zea was significantly greater (P < 0.001) than in IV-infected larvae. 3. Healthy and IV-infected larvae accumulated glycogen at a rate of 3.86 cal and 0.19 cal/day/insect, respectively. 4. Total protein of healthy and IV-infected larvae showed no significant difference with time.
INTRODUCTION The iridescent viruses (IV) comprise a group of viruses that are pathogenic to Diptera, Lepidoptera, and Coleoptera. The larval fat body appears to be the principal site of virus multiplication, but it also occurs in many other tissues. Iridescent viruses are confined to the class Insecta; the multiplication of the virus is restricted to cytoplasm. Iridescent viruses are large, icosahedron viruses measuring 130-180 m# in dia. In the studies of iridescent viruses, considerable effort has been made to determine the biochemical, biophysical, and morphological properties of these pathogens (Wagner et al., 1974; Carey et al., 1978; Stadelbacher et al., 1978). There is, however, little published information on the effects of viruses u p o n the chemistry and biochemistry of the host insect. Kelly & Tinsley (1974) studied, in vitro, the R N A polymerase activity associated with IV particles isolated from Galleria mellonella (L.) larvae. In another study they investigated the patterns of nucleic acid synthesis in insect cell cultures infected with two IVs (Kelly & Tinsley, 1974). We report here results of studies conducted to establish the time-concentration relationship of fatty acids, glycogen, and protein of the bollworm, Heliothis zea (Boddie), infected with the IV previously isolated from this species (Stadelbacher et al., 1978).
MATERIAL AND METHODS
Isolation of the virus
The IV used in this study was isolated from 2nd-4th instar Heliothis zea larvae. The larvae were triturated in distilled water containing 0.02~o (w/w) streptomycin sulfate and 0.02~ neomycin sulfate. The mixture was centrifuged at 23,000 rpm for 10min. The pellet was resuspended in fresh antibiotic solution and recentrifuged. The virus pellet was then suspended in 10 ml of distilled sterile water. Source of insects Heliothis zea eggs were surface disinfected with 0.2~
179
sodium hypochlorite according to the method of Ignoffo (1966). To produce IV infected insects, we placed the surface-sterilized eggs in the aqueous virus suspension. The IV-contaminated eggs were spread on a piece of filter paper in a sterile petri dish and incubated at 27°C. The control group of eggs was left untreated. After the eggs hatched, the larvae were transferred with a sterile camelhair brush to individual 20-ml plastic cups containing a meridic diet (Berger, 1963). For chemical analysis, infected larvae were compared to control larvae of the same age or instar. For lipid and fatty acid analysis, 5- to 30-day-old IVinfected H. zea larvae were removed from the cups and their age, weight and instar recorded. They were then placed in a mixture of chloroform-methanol (2:1 v/v). For fatty acid analysis, the chloroform-methanol extract of the lipids was washed by the method of Folch et al. (1957). Fatty acid methyl esters were prepared by esterification with boron trifluoride methanol (Metcalfe et al., 1966) and determined quantitatively by flame ionization gas chromatography under the conditions outlined by Thompson & Sikorowski (1979). For glycogen analysis, insects of the desired ages were stored in absolute ethanol at -20°C until analysis (Nettles & Betz, 1965). Individual insects were homogenized in 100 ml of 80~o ethanol and allowed to stand overnight at room temperature. The homogenate was centrifuged and the pellet extracted in 5 ml of a 5~o aqueous trichloroacetic acid (TCA) solution with a polytron ® homogenizer. The extract was centrifuged, the pellet extracted a second time with 5 ml of TCA, and the two TCA extracts were combined. A suitable aliquot of the TCA extract was diluted with 5 vol of 95% ethanol and allowed to stand overnight. After centrifuging, glycogen was determined by the method of Carrol et al. (1956). Total protein was determined according to the method of Bradford (1976) on healthy and IV-infected H. zea. However, no effort was made to determine the contribution of the virus to the total protein of the infected insects. RESULTS Figure la shows the phase contrast photomicrograph of healthy and Fig. lb the electronmicrograph of IV-infected fat lobes of H. zea. In the infected cells
180
A.C. THOMPSONand P. P, SIKOROWSKI
(Fig. lb), there is a total loss of cell differentiation due to the dissolution of the cellular membrane. Also, the cellular organization is lost in the infected fat bodies as shown by the dissipation of the cell nuclei when compared to the healthy cells (Fig. la). Figure 2 shows the fatty acid caloric variation with age of healthy and IV-infected H . z e a larvae. The time required for healthy larvae to reach the 4th and 5th instar was 10.5 + 1.2 days (Thompson & Sikorowski, 1979), and to reach the pupal stage, 10-18 days; the time for IV-infected larvae to reach the same instar was 21 days and none reached the pupal stage (75 larvae used). The majority of infected larvae succumbed to the disease in 21-35 days. Healthy larvae at day 15 had an average fresh wt of 711.4 + 55.7 mg/ insect; infected larvae at the same age averaged 245.3 +_ 59.7 mg/insect. IV-infected larvae at the 5th instar (day 21) had an average fresh wt of 414.8 +_ 53.1 mg/insect, then declined in weight until the prepupal stage (day 31). To eliminate the variation in fresh wt, calculations were made on a per-insect basis (Thompson & Sikorowski, 1980). The rate of development for healthy larvae was twice that for IV-
infected larvae. The rate of accumulation of fatty acids was 124 cal/insect and 52.7 cal/insect for healthy and IV-infected larvae, respectively, over the age period studied. Analysis by t-test showed a highly significant difference in fatty acid calories (P < 0.001) of healthy and IV-infected larvae. Figure 3 shows the relationship of glycogen with age for healthy and IV-infected H . z e a larvae. Healthy and IV-infected larvae accumulated glycogen at a rate of 3.86cal and 0.19 cal/day/insect, respectively. Healthy larvae accumulated 38.6 cal glycogen/insect at the prepupal stage (day 15) and infected larvae 3.99 cal/insect at day 21. Analysis by t-test showed the difference in glyccgen between the two groups to be highly significant (P < 0.001). Table 1 compares the individual fatty acid calories of H . z e a larvae infected on day 6 and day 9 to those of healthy larvae. The larvae infected on day 9 showed the greatest difference in fatty acids, with palmitic (16:0), palmitoleic (16:1), and oleic (18:1) accounting for more than 60~o each over the period studied. However, mortality was higher in the larvae infected on day 6.
Fig. la. Phase contrast photomicrograph of healthy fat lobes of H.
zea
larvae.
Metabolism in iridescent virus infected Heliothis larvae
Fig. lb. Electromicrograph of iridiscent virus-infected fat lobes H. zea larvae.
181
182
A.C. THOMPSONand P. P. SIKOROWSKI
300
ILl Z
T Between S t 0 p e s = - 4 . 2 3
200
[H] [i]
y= -67.19 ÷ 12.38 X J
100
y = - 8 . 0 8 ÷ 1.95X
r O= ~ ~ ~ t
c.)
0
0
5
"
10
r = 0./.8
15
I
I
20
25
I 30
AGE (DAYS) Fig. 2. Variation of fatty acid (F.A.) calories with age in healthy and ifidiscent virus-infected H. zea
larvae.
150 k-O
zLUU~ m 100
[H]
O~--, O~ >-
y = -19.09 ÷ 3.86 x
r = 0,48 ~
[I~ r - - Y = 2.27 0.19 x
0
~, 0
- - w ~ - - " r - - -'a 0
5
10
15
.r=
0.19
i
-7--
J
20
25
30
AGE (DAYS)
Fig. 3. Variation of glycogen with age of healthy and iridescent virus infected H. zea larvae.
There was no significant difference in total protein through day 21 between infected and healthy larvae of the same instar. DISCUSSION
Within the infected fat bodies (Fig. 1B), there is an accumulation of albuminoid-like granules in the trophocytes characteristic of those produced at the time of metamorphosis. Although it was not within the scope of this work to investigate the biochemical properties of this IV, it probably has serological properties similar to those of the IV (Type 21) isolated from H. armiger by Cary et al. (1978). In fact, many consider H. armiger and H. zea to be the same species, differing only in their locale. The efficiency rate for fatty acid caloric accumulation was 84% greater for healthy H. zea than for 1V-infected larvae. This difference amounts to 10.43 cal/day/insect. Wiygul & Sikorowski (private communication) have shown that the efficiency of oxygen consumption by healthy H. zea is 10% greater than for IV-infected H. zea. This difference amounts to 0.046 cal/day/insect. Thus, in healthy H. zea larvae, both lipid accumulation and aerobic oxidation is greater than in IV-infected larvae. Less than 0.4% of
lipid metabolism in healthy H. zea can be accounted for by aerobic metabolism. The average efficiency rate for glycogen accumulation was 95% greater for healthy H. zea than for IV-infected larvae. This difference amounts to 2.67 cal/day/insect, and the difference at any day can be determined by multiplying this value by the day number. If all the oxygen consumed were attributed to glycogen metabolism, only 2% of the glycogen would be undergoing aerobic metabolism, leaving 98% for anaerobic metabolism. Wiygul & Sikorowski (private communication) have shown that a linear regression analysis of healthy and IV infected insect age versus fresh weight have essentially the same slope but differ by a constant value of 275 mg over the period tested. By converting their "oxygen consumed with age" to "calories/insect with age", the difference in weights is eliminated and is comparable to the data in this work. In summary, the chemical changes in the lipids and glycogen occur either by simple hydrolysis to substances not detected by the means used, or by anaerobic metabolism. Hydrolysis of glycogen to the monosaccaride can explain the observed loss, whereas simple hydrolysis of the lipid to the corresponding fatty acids cannot. The energy derived from fatty
Metabolism in iridescent virus infected Heliothis larvae
183
Table 1. Fatty acid (F.A.) calories of healthy and iridescent virus-infected Heliothis zea larvae infected (by injection) at days 6 and 9 and analyzed at days 14 and 21
F.A.
Healthy Larvae
16:0 16:1 18:0 18:1 18:2 18:3
61.6 + 15.0 31.8 + 4.8 0.0 48.8 + 11.4 29.0 + 6.9 6.5 + 1.8
Fatty acid cal. Fresh wt + SE Larvae Larvae Infected day 6 A% Infected day 9 23.7 8.5 4.5 22.8 18.9 5.3
+ 3.2 + 1.6 + 0.2 _+ 3.5 + 2.3 _+ 1.1
acids utilized by the IV-infected larvae a m o u n t s to 5 times the calories derived from glycogen.
REFERENCES BERGER R. S. (1963) Laboratory techniques for rearing Heliothis species on artificial medium. U.S. Dep. Agric. Res. Serv., pp. 33-84. BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. BiDchem. 72, 248-254. CAREY G. P., LESCOTTT., ROBERTSON J. S., SPENCER L. K. KELLY D. C. (1978) Three africal isolates of small iridescent viruses: Type 21 from Heliothis armigera, Type 23 from Heteronychus arator, and Type 28 from Lethocera columbiae. Virology 84, 40-42. CARROL N. V., LONGLEY R. W. & ROE J. H. (1956) The determination of glycogen in liver and muscle by use of anthrone reagent. J. biol. Chem. 220, 583-593. FOLCH J., LEES M. & SLOANE-STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. J. biol. Chem. 226, 49%509. IGNOFFO C. M. (1966) Insect virus. In Insect Colonization
44 58 -36 21 10
14.9 6.7 4.3 9.9 14.9 6,4
+ 5.5 + 2.3 + 1.1 ___2.7 ___3.6 ___ 1.7
A% 61 65 -66 32 1
and Mass Production (Edited by SMITH C. M.), pp. 511-512. Academic Press, New York. KELLY D. C. & TINSLEY T. W. (1974) Iridescent virus replication: Pattern nucleic acid synthesis in insect cells infected with iridescent virus Types 2 and 6. J. Invert. Pathol. 24, 169-178. METCALFE L. C., SCHMITZ A. A. & BELKER J. R. (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analyt. Chem. 38, 514-515. NETTLES W. C. & BETZ N. L. (1965) Glycogen in the boll weevil with respect to diapause, age, and diet. Ann. Entotool. Soc. Am. 58, 721-726. STADELBACHERE. A., ADAMSJ. R. & FAUST R. M. (1978) A blue-green iridescent virus of the bollworm, Heliothis zea (Boddie). J. Invert. Pathol. 32, 71-76. THOMPSON A. C. & SIKOROWSKI P. P. (1979) Effects of Nosema heliothidis on fatty and amino acids in larvae and pupae of the bollworm, Heliothis zea. Comp. BiDchem. Physiol. 63A, 325--328. THOMPSON A. C. t~ SIKOROWSKIP. P. (1980) Fatty acid and glycogen requirements of Heliothis virescens infected with cytoplasmic polyhedrosis virus. Comp. Biochem. Physiol. 66B, 93-97. WAGNER G. W., PASCHKE J. D., CAMPBELLW. R. & WEBB S. R. (1974) Proteins of two strains of mosquito iridescent virus. Intervirology 3, 97-105.
0305-0491/81/100179-05502.00/0 Copyright © 1981 Pergamon Press Ltd
ENERGY DIFFERENCE IN LIPID AND GLYCOGEN METABOLISM OF HEALTHY AND IRIDESCENT VIRUS-INFECTED HELIOTHIS ZEA (BODDIE) A. C. THOMPSON and P. P. SIKOROWSKI Boll Weevil Research Laboratory, Agricultural Research, Science and Education Administration, USDA, Mississippi State, MS 39762 and Department of Entomology, Mississippi State University, Mississippi State, MS 39762, U.S.A. (Received 11 February 1981) A b s t r a c t - - 1 . Heliothis zea (Boddie) larvae infected with irridescent virus (IV) showed rapid cell dissolution in the fat bodies. 2. Fatty acid accumulation in healthy H. zea was significantly greater (P < 0.001) than in IV-infected larvae. 3. Healthy and IV-infected larvae accumulated glycogen at a rate of 3.86 cal and 0.19 cal/day/insect, respectively. 4. Total protein of healthy and IV-infected larvae showed no significant difference with time.
INTRODUCTION The iridescent viruses (IV) comprise a group of viruses that are pathogenic to Diptera, Lepidoptera, and Coleoptera. The larval fat body appears to be the principal site of virus multiplication, but it also occurs in many other tissues. Iridescent viruses are confined to the class Insecta; the multiplication of the virus is restricted to cytoplasm. Iridescent viruses are large, icosahedron viruses measuring 130-180 m# in dia. In the studies of iridescent viruses, considerable effort has been made to determine the biochemical, biophysical, and morphological properties of these pathogens (Wagner et al., 1974; Carey et al., 1978; Stadelbacher et al., 1978). There is, however, little published information on the effects of viruses u p o n the chemistry and biochemistry of the host insect. Kelly & Tinsley (1974) studied, in vitro, the R N A polymerase activity associated with IV particles isolated from Galleria mellonella (L.) larvae. In another study they investigated the patterns of nucleic acid synthesis in insect cell cultures infected with two IVs (Kelly & Tinsley, 1974). We report here results of studies conducted to establish the time-concentration relationship of fatty acids, glycogen, and protein of the bollworm, Heliothis zea (Boddie), infected with the IV previously isolated from this species (Stadelbacher et al., 1978).
MATERIAL AND METHODS
Isolation of the virus
The IV used in this study was isolated from 2nd-4th instar Heliothis zea larvae. The larvae were triturated in distilled water containing 0.02~o (w/w) streptomycin sulfate and 0.02~ neomycin sulfate. The mixture was centrifuged at 23,000 rpm for 10min. The pellet was resuspended in fresh antibiotic solution and recentrifuged. The virus pellet was then suspended in 10 ml of distilled sterile water. Source of insects Heliothis zea eggs were surface disinfected with 0.2~
179
sodium hypochlorite according to the method of Ignoffo (1966). To produce IV infected insects, we placed the surface-sterilized eggs in the aqueous virus suspension. The IV-contaminated eggs were spread on a piece of filter paper in a sterile petri dish and incubated at 27°C. The control group of eggs was left untreated. After the eggs hatched, the larvae were transferred with a sterile camelhair brush to individual 20-ml plastic cups containing a meridic diet (Berger, 1963). For chemical analysis, infected larvae were compared to control larvae of the same age or instar. For lipid and fatty acid analysis, 5- to 30-day-old IVinfected H. zea larvae were removed from the cups and their age, weight and instar recorded. They were then placed in a mixture of chloroform-methanol (2:1 v/v). For fatty acid analysis, the chloroform-methanol extract of the lipids was washed by the method of Folch et al. (1957). Fatty acid methyl esters were prepared by esterification with boron trifluoride methanol (Metcalfe et al., 1966) and determined quantitatively by flame ionization gas chromatography under the conditions outlined by Thompson & Sikorowski (1979). For glycogen analysis, insects of the desired ages were stored in absolute ethanol at -20°C until analysis (Nettles & Betz, 1965). Individual insects were homogenized in 100 ml of 80~o ethanol and allowed to stand overnight at room temperature. The homogenate was centrifuged and the pellet extracted in 5 ml of a 5~o aqueous trichloroacetic acid (TCA) solution with a polytron ® homogenizer. The extract was centrifuged, the pellet extracted a second time with 5 ml of TCA, and the two TCA extracts were combined. A suitable aliquot of the TCA extract was diluted with 5 vol of 95% ethanol and allowed to stand overnight. After centrifuging, glycogen was determined by the method of Carrol et al. (1956). Total protein was determined according to the method of Bradford (1976) on healthy and IV-infected H. zea. However, no effort was made to determine the contribution of the virus to the total protein of the infected insects. RESULTS Figure la shows the phase contrast photomicrograph of healthy and Fig. lb the electronmicrograph of IV-infected fat lobes of H. zea. In the infected cells
180
A.C. THOMPSONand P. P, SIKOROWSKI
(Fig. lb), there is a total loss of cell differentiation due to the dissolution of the cellular membrane. Also, the cellular organization is lost in the infected fat bodies as shown by the dissipation of the cell nuclei when compared to the healthy cells (Fig. la). Figure 2 shows the fatty acid caloric variation with age of healthy and IV-infected H . z e a larvae. The time required for healthy larvae to reach the 4th and 5th instar was 10.5 + 1.2 days (Thompson & Sikorowski, 1979), and to reach the pupal stage, 10-18 days; the time for IV-infected larvae to reach the same instar was 21 days and none reached the pupal stage (75 larvae used). The majority of infected larvae succumbed to the disease in 21-35 days. Healthy larvae at day 15 had an average fresh wt of 711.4 + 55.7 mg/ insect; infected larvae at the same age averaged 245.3 +_ 59.7 mg/insect. IV-infected larvae at the 5th instar (day 21) had an average fresh wt of 414.8 +_ 53.1 mg/insect, then declined in weight until the prepupal stage (day 31). To eliminate the variation in fresh wt, calculations were made on a per-insect basis (Thompson & Sikorowski, 1980). The rate of development for healthy larvae was twice that for IV-
infected larvae. The rate of accumulation of fatty acids was 124 cal/insect and 52.7 cal/insect for healthy and IV-infected larvae, respectively, over the age period studied. Analysis by t-test showed a highly significant difference in fatty acid calories (P < 0.001) of healthy and IV-infected larvae. Figure 3 shows the relationship of glycogen with age for healthy and IV-infected H . z e a larvae. Healthy and IV-infected larvae accumulated glycogen at a rate of 3.86cal and 0.19 cal/day/insect, respectively. Healthy larvae accumulated 38.6 cal glycogen/insect at the prepupal stage (day 15) and infected larvae 3.99 cal/insect at day 21. Analysis by t-test showed the difference in glyccgen between the two groups to be highly significant (P < 0.001). Table 1 compares the individual fatty acid calories of H . z e a larvae infected on day 6 and day 9 to those of healthy larvae. The larvae infected on day 9 showed the greatest difference in fatty acids, with palmitic (16:0), palmitoleic (16:1), and oleic (18:1) accounting for more than 60~o each over the period studied. However, mortality was higher in the larvae infected on day 6.
Fig. la. Phase contrast photomicrograph of healthy fat lobes of H.
zea
larvae.
Metabolism in iridescent virus infected Heliothis larvae
Fig. lb. Electromicrograph of iridiscent virus-infected fat lobes H. zea larvae.
181
182
A.C. THOMPSONand P. P. SIKOROWSKI
300
ILl Z
T Between S t 0 p e s = - 4 . 2 3
200
[H] [i]
y= -67.19 ÷ 12.38 X J
100
y = - 8 . 0 8 ÷ 1.95X
r O= ~ ~ ~ t
c.)
0
0
5
"
10
r = 0./.8
15
I
I
20
25
I 30
AGE (DAYS) Fig. 2. Variation of fatty acid (F.A.) calories with age in healthy and ifidiscent virus-infected H. zea
larvae.
150 k-O
zLUU~ m 100
[H]
O~--, O~ >-
y = -19.09 ÷ 3.86 x
r = 0,48 ~
[I~ r - - Y = 2.27 0.19 x
0
~, 0
- - w ~ - - " r - - -'a 0
5
10
15
.r=
0.19
i
-7--
J
20
25
30
AGE (DAYS)
Fig. 3. Variation of glycogen with age of healthy and iridescent virus infected H. zea larvae.
There was no significant difference in total protein through day 21 between infected and healthy larvae of the same instar. DISCUSSION
Within the infected fat bodies (Fig. 1B), there is an accumulation of albuminoid-like granules in the trophocytes characteristic of those produced at the time of metamorphosis. Although it was not within the scope of this work to investigate the biochemical properties of this IV, it probably has serological properties similar to those of the IV (Type 21) isolated from H. armiger by Cary et al. (1978). In fact, many consider H. armiger and H. zea to be the same species, differing only in their locale. The efficiency rate for fatty acid caloric accumulation was 84% greater for healthy H. zea than for 1V-infected larvae. This difference amounts to 10.43 cal/day/insect. Wiygul & Sikorowski (private communication) have shown that the efficiency of oxygen consumption by healthy H. zea is 10% greater than for IV-infected H. zea. This difference amounts to 0.046 cal/day/insect. Thus, in healthy H. zea larvae, both lipid accumulation and aerobic oxidation is greater than in IV-infected larvae. Less than 0.4% of
lipid metabolism in healthy H. zea can be accounted for by aerobic metabolism. The average efficiency rate for glycogen accumulation was 95% greater for healthy H. zea than for IV-infected larvae. This difference amounts to 2.67 cal/day/insect, and the difference at any day can be determined by multiplying this value by the day number. If all the oxygen consumed were attributed to glycogen metabolism, only 2% of the glycogen would be undergoing aerobic metabolism, leaving 98% for anaerobic metabolism. Wiygul & Sikorowski (private communication) have shown that a linear regression analysis of healthy and IV infected insect age versus fresh weight have essentially the same slope but differ by a constant value of 275 mg over the period tested. By converting their "oxygen consumed with age" to "calories/insect with age", the difference in weights is eliminated and is comparable to the data in this work. In summary, the chemical changes in the lipids and glycogen occur either by simple hydrolysis to substances not detected by the means used, or by anaerobic metabolism. Hydrolysis of glycogen to the monosaccaride can explain the observed loss, whereas simple hydrolysis of the lipid to the corresponding fatty acids cannot. The energy derived from fatty
Metabolism in iridescent virus infected Heliothis larvae
183
Table 1. Fatty acid (F.A.) calories of healthy and iridescent virus-infected Heliothis zea larvae infected (by injection) at days 6 and 9 and analyzed at days 14 and 21
F.A.
Healthy Larvae
16:0 16:1 18:0 18:1 18:2 18:3
61.6 + 15.0 31.8 + 4.8 0.0 48.8 + 11.4 29.0 + 6.9 6.5 + 1.8
Fatty acid cal. Fresh wt + SE Larvae Larvae Infected day 6 A% Infected day 9 23.7 8.5 4.5 22.8 18.9 5.3
+ 3.2 + 1.6 + 0.2 _+ 3.5 + 2.3 _+ 1.1
acids utilized by the IV-infected larvae a m o u n t s to 5 times the calories derived from glycogen.
REFERENCES BERGER R. S. (1963) Laboratory techniques for rearing Heliothis species on artificial medium. U.S. Dep. Agric. Res. Serv., pp. 33-84. BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analyt. BiDchem. 72, 248-254. CAREY G. P., LESCOTTT., ROBERTSON J. S., SPENCER L. K. KELLY D. C. (1978) Three africal isolates of small iridescent viruses: Type 21 from Heliothis armigera, Type 23 from Heteronychus arator, and Type 28 from Lethocera columbiae. Virology 84, 40-42. CARROL N. V., LONGLEY R. W. & ROE J. H. (1956) The determination of glycogen in liver and muscle by use of anthrone reagent. J. biol. Chem. 220, 583-593. FOLCH J., LEES M. & SLOANE-STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. J. biol. Chem. 226, 49%509. IGNOFFO C. M. (1966) Insect virus. In Insect Colonization
44 58 -36 21 10
14.9 6.7 4.3 9.9 14.9 6,4
+ 5.5 + 2.3 + 1.1 ___2.7 ___3.6 ___ 1.7
A% 61 65 -66 32 1
and Mass Production (Edited by SMITH C. M.), pp. 511-512. Academic Press, New York. KELLY D. C. & TINSLEY T. W. (1974) Iridescent virus replication: Pattern nucleic acid synthesis in insect cells infected with iridescent virus Types 2 and 6. J. Invert. Pathol. 24, 169-178. METCALFE L. C., SCHMITZ A. A. & BELKER J. R. (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analyt. Chem. 38, 514-515. NETTLES W. C. & BETZ N. L. (1965) Glycogen in the boll weevil with respect to diapause, age, and diet. Ann. Entotool. Soc. Am. 58, 721-726. STADELBACHERE. A., ADAMSJ. R. & FAUST R. M. (1978) A blue-green iridescent virus of the bollworm, Heliothis zea (Boddie). J. Invert. Pathol. 32, 71-76. THOMPSON A. C. & SIKOROWSKI P. P. (1979) Effects of Nosema heliothidis on fatty and amino acids in larvae and pupae of the bollworm, Heliothis zea. Comp. BiDchem. Physiol. 63A, 325--328. THOMPSON A. C. t~ SIKOROWSKIP. P. (1980) Fatty acid and glycogen requirements of Heliothis virescens infected with cytoplasmic polyhedrosis virus. Comp. Biochem. Physiol. 66B, 93-97. WAGNER G. W., PASCHKE J. D., CAMPBELLW. R. & WEBB S. R. (1974) Proteins of two strains of mosquito iridescent virus. Intervirology 3, 97-105.