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Growth and development of the Angoumois grain moth, Sitotroga cerealella in an artificial diet
J. InsectPhysiol.,1971, Vol. 17, pp. 109 to 115. PergamonPress. Printed in Great Britain
GROWTH AND DEVELOPMENT OF THE ANGOUMOIS GRAIN MOTH, SITOTROGA CEREALELLA IN AN ARTIFICIAL DIET* G. M. CHIPPENDALE Department of Entomology,
University of Missouri, Columbia, Missouri 65201 (Received 18 May 1970)
Abstract-A
simple method for rearing the Angoumois grain moth, Sitotroga cerealella, in an artificial diet is described. Although the larvae normally feed within grain kernels, they readily became established in pellets containing a mixture of five natural products. An evaluation of the growth rate in this medium revealed a marked sex difference. The males attained a maximum mean larval weight of 7.2 mg at 22 days compared with 12.3 mg for mature females at 24 days. Both sexes were found to pass through four larval instars. A study of the pupation rate showed that males reached 50 per cent pupation a full 5 days before females. While both sexes spent about 6.7 days in the pupal-pharate adult stage, the mean weight of newly-ecdysed female adults was 77 per cent greater than that of the males. INTRODUCTION ALTHOUGH the Angoumois
grain moth, Sitotroga cere&lZu (Olivier), has been recognized for many years as a universally destructive insect of stored and field grain kernels and adapts well to laboratory culture in its natural hosts (FLANDERS, 1929), very little information exists about its developmental physiology. It has been difficult to study the immature stages of the insect because the larvae normally feed entirely within kernels. An artificial diet has been wanting which would allow the easy removal of larvae and pupae for physiological research on this important insect. The present study describes such an artificial medium which meets all the chemical and physical feeding requirements of Sitotroga. This medium contained only five components and was presented to the larvae in pellet form. The growth and development of the insect in this diet were evaluated. It was found that the larvae grew more rapidly than in whole kernel corn or earlier artificial diets (CHIPPENDALE, 1970). A marked sex difference in growth rates was also detected. MATERIALS
AND METHODS
Cdture
The methods used to maintain the stock culture have been previously described (CHIPPENDALE, 1970).
Briefly, larvae were reared in gallon jars partially
filled with
* Contribution from the Missouri Agricultural Experiment Station, Columbia. Journal Series No. 5987. This research was supported in part by a co-operative agreement (12-14100-10,056) from the United States Department of Agriculture. 109
110
G.M.
CHIPPENDALE
field corn (Zea mars> at 30°C and about 70% r.h. in the dark. Newly emerged moths were transferred to quart jars where the females readily oviposited on folded wax paper strips. The egg-laden strips were held in small sealed jars to provide newly hatched larvae for the experiments.
Diet preparation The components of the artificial medium for Sitotroga are listed in Table 1. This diet is a simplified version of an earlier cruder medium which incorporated dog TABLE ~-COMPOSITION
OF A NATURAL
DIBT FORREARING THEANGOUMOIS GRAIN MOTH
PRODUCT
Amount Component Whole wheat flour Corn meal Honey
Amount
63)
Component
38.0 29.3 13.7
Glycerol Brewer’s yeast
64 13.1 5.9
meal, oats, and wheat germ along with the present ingredients (CHIPPENDALE, 1970). To prepare the diet, wheat grains and white degermed corn meal (Quaker) were first individually ground into a powder and then mixed with the other components. The resulting friable medium was easily formed into pellets. A 13 mm piece of aluminium plate containing a series of 5 mm dia. holes was used as a template to make pellets. The diet was packed tightly into the holes and when extruded formed uniform pellets of about 440 mg each. One pellet and two newly hatched larvae were then placed inside a No. 0 transparent gelatine capsule (Parke, Davis and Co., Detroit). The test capsules were held in a vertical position in the holes of a peg board and incubated under the same conditions as the stock culture.
Assay procedures The larvae and pupae were easily removed from the pellets because they formed visible feeding cells or tunnels. Since the pigmented testes were readily seen through the larval and pupal cuticle from the third instar onwards, it was possible to obtain separate growth information for the sexes. Growth data were obtained by individually weighing groups of larvae, pupae, and adults of known age and sex on a semimicro balance. The number and duration of larval instars were determined by taking daily measurements of head capsule widths from a large population (DYAR, 1890). The number of insects used in each experiment is recorded with the results. RESULTS
AND DISCUSSION
The larval growth and adult emergence rates of Sitotroga in the artificial diet (Table 1) were determined using a previously described bioassay procedure
GROWTH AND DEVELOPMENT OF SITOTROGA
CERRALELLA
IN AN ARTIFICIAL DIET
111
1970). The larvae grew well in the diet and reached a maximum mean larval weight of 9.7 mg at 25 days with 50 per cent adult emergence occurring at 31 days (Fig. 1). The adults were normal and the females laid viable eggs. Four similar assaysyielded comparable results with 50 per cent adult emergence occurring between 28 and 34 days. Insects reared in this natural product diet invariably had a more uniform growth rate and were generally heavier than those reared in whole kernelsof corn. (CHIPPENDALE,
Age days. 30-C
Adult emergence
Age days.
30°C
FIG. 1. Larval growth and adult emergence curves for Sitotrogu reared in an artificial diet. Each vertical line represents a single standard error.
When separated by sex, the mature male larvae were found to be generally smaller and began to pupate earlier than the females. Separate larval growth and pupation curves were then obtained for both sexes. The larval growth curve (Fig. 2) revealed that significant differences were detected in male and female larval weights beginning on the nineteenth day. The males eventually attained a maximum mean larval weight of 7.2 mg at 22 days compared to 12.3 mg for the mature females at 24 days. Female larvae, therefore, reached maturity about 2 days later and were 70 per cent heavier than their male counterparts. Since the females produce viable eggs without feeding as adults they must accumulate the necessary reserves during their last larval days. A comparison of the pupation curves also revealed a sex difference in growth rates (Fig. 3). The males began to pupate at 17 days and reached 50 per cent pupation as early as 22 days whereas females only began to pupate at 20 days and reached 50 per cent pupation after 27 days. The number of larval instars was then determined by obtaining daily head capsule measurements. A total of 524 larvae was examined between 0 and 24 days. From these data a frequency polygon was constructed by joining the midpoints of
G. M. CHIPPENDALE
112
14
15
Age
16
17
days,
IS
30 oc
I9
20
21
22
23
24
25
FIG. 2. Comparison of male and female growth curves for Sitotrogu larvae reared in an artificial diet. Twenty observations were made at each stage.
IOOgo-
22
23
Age
24
25
days,
26
27
28
29
30
31
32
33
30 ‘C
FIG. 3. Comparison of male and female pupation curves for Sitotrogu larvae reared in an artificial diet. These data were obtained from a population of 41 males and 43 females.
GROWTH ANDDEVELOPMENT OFSITOTROGA
CEREALELLA
IN ANARTIFICIAL DIET 113
the class intervals (0.026 mm) at their respective class frequencies (Fig. 4). Four discontinuous groups were detected, each representing the variation in head capsule width within a single instar (GAINES and CAMPBELL, 1935). Although more variation existed in the head capsule width of third instars than in either first or second instars, all three showed a symmetrical distribution. The distribution of measurements from the fourth instar, however, was slightly skewed because male and female head capsule widths proved to have different distributions. Male head capsules had their highest frequency at O-702 mm, compared with 0.728 mm for those of the females. 0 Males
l
and females
Females
Head
only
capsule
width,
mm
FIG. 4. Frequency polygon depicting four larval instars for Shotrogu reared in an artificial diet.
Four larval instars probably represent the minimum number for this species when reared under optimal conditions. CROMBIE(1943) reared a small group of larvae on wheat flour and after taking twenty body measurements also concluded that the insect had four larval instars. MILLS (1965), on the other hand, used counts of cast mandibles as his criterion and detected four to seven instars for larvae reared in various kernels. He concluded that the larvae which had lower growth rates passed through more instars than the faster-growing ones. Mean head capsule widths were plotted against age to determine the duration of each instar. Fig. 5 depicts a uniform progression through the larval instars. Larvae ecdysed into the second instar on the fifth day and into the third instar on the eighth and ninth days. They then ecdysed into the fourth instar on the fourteenth and fifteenth days, depending on their sex. This last larval ecdysis was essentially complete by the fifteenth day for the males and sixteenth day for the females. The values obtained during the fourth instar again show that the females had a slightly larger mean head capsule width than the males. In a f&l experiment, separate weight loss data were obtained for both sexes during metamorphosis (Fig. 6). The results show that the mean weight loss of males (3.11 mg or 47 per cent) during the transition from larva to adult was
114
G. M. CHIPPENDALE 0.6 -
@Males and females 0 Males only
E a.7 -
l
Females
.mE*:r:
only //
go-6
&*
P
0’.
u o.s/*‘;gf
i to.4
-
0
/
P c 0.3 -
e/e;.P
6 ii 0.2 /
+-o,e-e 0.1
0
I 2
, 4
I
I
I
I
/
t
I
I
I
I
6
6
IO
12
14
I6
I6
20
22
24
Age days,
30-c
FIG. 5. Relationship between age and mean width of head capsule as a means for estimating the duration of each instar. Values obtained from 524 observations.
12 II IO s6-
Males
Females
FIG. 6. Bar chart summarizing the mean weight losses for the sexes during metamorphosis. The number of insects weighed is encircled in each bar. ML, mature larvae; NEP, newly ecdysed pupae ( < l-day-old) ; NEA, newly ecdysed adults ( < 1 -day-old).
GROWTHANDDEVELOPMENT OF SITOTROGA
CERl3ALELL.A IN AN
ARTIFICIALDIET
115
proportionally the same as for the females (5.57 mg or 48 per cent). The newly ecdysed adult weight ratio between the sexes (O-6) was therefore identical to similar mature larval and pupal weight ratios. The mean difference in weight between the male and female newly ecdysed adults (2.66 mg) approximates the weight of the ovaries of a mature female adult. No sex differences in the duration of the pupalpharate adult stage were detected. Its mean duration calculated from 113 observations was 6.7 days for both males and females. The present results have revealed new insights into the growth and development of the Angoumois grain moth. Since the insect was reared in a homogeneous medium under controlled conditions the observed sex differences can be attributed to inherent differences in male and female growth rates. These differences account for some of the variation in growth rates observed when the species is reared in its natural hosts (MILLS and WILBUR, 1967). Other main sources of growth variation in whole kernels apparently result from the location of larval feeding sites. For example, MILU (1965) f ound that larvae which fed in both the germ and endosperm of wheat kernels during their early instars matured faster than others which only fed in endosperm during the same period. AcRnowZe&emenf-I thank Mrs. EDITH MCKENZIE for her skilled technical assistance, and Mr. JOHN BROWNwho drew the figures.
REFERENCES CHIPPENDALEG. M. (1970) Development of artificial diets for rearing the Angoumois grain moth. J. econ. Ent. 63,844-848. CROMBIIIA. C. (1943) The development of the Angoumois grain moth Sitotroga cer.euZelZu (Oliv.). iVuture, Lond. 152,246. DYAR H. G. (1890) The number of moults of lepidopterous larvae. Psyche, Cumb. 5, 420422. FLANDERSS. E. (1929) The production and distribution of Trichogramma. J. econ. Ent. 22, 245-248. GAINERJ. C. and CAMPBELLF. L. (1935) Dyar’s rule as related to the number of instars of the corn earworm, Heliothis obsoletu (Fab.), collected in the field. Ann. ent. Sot. Am. 28, 445-461. MILLS R. B. (1965) Early germ feeding and larval development of the Angoumois grain moth. J. econ. Ext. 58,220-223. MILLS R. B. and WILBUR D. A. (1967) Radiographic studies of Angoumois grain moth development in wheat, corn, and sorghum kernels. y. econ. ht. 60,671-677.
GROWTH AND DEVELOPMENT OF THE ANGOUMOIS GRAIN MOTH, SITOTROGA CEREALELLA IN AN ARTIFICIAL DIET* G. M. CHIPPENDALE Department of Entomology,
University of Missouri, Columbia, Missouri 65201 (Received 18 May 1970)
Abstract-A
simple method for rearing the Angoumois grain moth, Sitotroga cerealella, in an artificial diet is described. Although the larvae normally feed within grain kernels, they readily became established in pellets containing a mixture of five natural products. An evaluation of the growth rate in this medium revealed a marked sex difference. The males attained a maximum mean larval weight of 7.2 mg at 22 days compared with 12.3 mg for mature females at 24 days. Both sexes were found to pass through four larval instars. A study of the pupation rate showed that males reached 50 per cent pupation a full 5 days before females. While both sexes spent about 6.7 days in the pupal-pharate adult stage, the mean weight of newly-ecdysed female adults was 77 per cent greater than that of the males. INTRODUCTION ALTHOUGH the Angoumois
grain moth, Sitotroga cere&lZu (Olivier), has been recognized for many years as a universally destructive insect of stored and field grain kernels and adapts well to laboratory culture in its natural hosts (FLANDERS, 1929), very little information exists about its developmental physiology. It has been difficult to study the immature stages of the insect because the larvae normally feed entirely within kernels. An artificial diet has been wanting which would allow the easy removal of larvae and pupae for physiological research on this important insect. The present study describes such an artificial medium which meets all the chemical and physical feeding requirements of Sitotroga. This medium contained only five components and was presented to the larvae in pellet form. The growth and development of the insect in this diet were evaluated. It was found that the larvae grew more rapidly than in whole kernel corn or earlier artificial diets (CHIPPENDALE, 1970). A marked sex difference in growth rates was also detected. MATERIALS
AND METHODS
Cdture
The methods used to maintain the stock culture have been previously described (CHIPPENDALE, 1970).
Briefly, larvae were reared in gallon jars partially
filled with
* Contribution from the Missouri Agricultural Experiment Station, Columbia. Journal Series No. 5987. This research was supported in part by a co-operative agreement (12-14100-10,056) from the United States Department of Agriculture. 109
110
G.M.
CHIPPENDALE
field corn (Zea mars> at 30°C and about 70% r.h. in the dark. Newly emerged moths were transferred to quart jars where the females readily oviposited on folded wax paper strips. The egg-laden strips were held in small sealed jars to provide newly hatched larvae for the experiments.
Diet preparation The components of the artificial medium for Sitotroga are listed in Table 1. This diet is a simplified version of an earlier cruder medium which incorporated dog TABLE ~-COMPOSITION
OF A NATURAL
DIBT FORREARING THEANGOUMOIS GRAIN MOTH
PRODUCT
Amount Component Whole wheat flour Corn meal Honey
Amount
63)
Component
38.0 29.3 13.7
Glycerol Brewer’s yeast
64 13.1 5.9
meal, oats, and wheat germ along with the present ingredients (CHIPPENDALE, 1970). To prepare the diet, wheat grains and white degermed corn meal (Quaker) were first individually ground into a powder and then mixed with the other components. The resulting friable medium was easily formed into pellets. A 13 mm piece of aluminium plate containing a series of 5 mm dia. holes was used as a template to make pellets. The diet was packed tightly into the holes and when extruded formed uniform pellets of about 440 mg each. One pellet and two newly hatched larvae were then placed inside a No. 0 transparent gelatine capsule (Parke, Davis and Co., Detroit). The test capsules were held in a vertical position in the holes of a peg board and incubated under the same conditions as the stock culture.
Assay procedures The larvae and pupae were easily removed from the pellets because they formed visible feeding cells or tunnels. Since the pigmented testes were readily seen through the larval and pupal cuticle from the third instar onwards, it was possible to obtain separate growth information for the sexes. Growth data were obtained by individually weighing groups of larvae, pupae, and adults of known age and sex on a semimicro balance. The number and duration of larval instars were determined by taking daily measurements of head capsule widths from a large population (DYAR, 1890). The number of insects used in each experiment is recorded with the results. RESULTS
AND DISCUSSION
The larval growth and adult emergence rates of Sitotroga in the artificial diet (Table 1) were determined using a previously described bioassay procedure
GROWTH AND DEVELOPMENT OF SITOTROGA
CERRALELLA
IN AN ARTIFICIAL DIET
111
1970). The larvae grew well in the diet and reached a maximum mean larval weight of 9.7 mg at 25 days with 50 per cent adult emergence occurring at 31 days (Fig. 1). The adults were normal and the females laid viable eggs. Four similar assaysyielded comparable results with 50 per cent adult emergence occurring between 28 and 34 days. Insects reared in this natural product diet invariably had a more uniform growth rate and were generally heavier than those reared in whole kernelsof corn. (CHIPPENDALE,
Age days. 30-C
Adult emergence
Age days.
30°C
FIG. 1. Larval growth and adult emergence curves for Sitotrogu reared in an artificial diet. Each vertical line represents a single standard error.
When separated by sex, the mature male larvae were found to be generally smaller and began to pupate earlier than the females. Separate larval growth and pupation curves were then obtained for both sexes. The larval growth curve (Fig. 2) revealed that significant differences were detected in male and female larval weights beginning on the nineteenth day. The males eventually attained a maximum mean larval weight of 7.2 mg at 22 days compared to 12.3 mg for the mature females at 24 days. Female larvae, therefore, reached maturity about 2 days later and were 70 per cent heavier than their male counterparts. Since the females produce viable eggs without feeding as adults they must accumulate the necessary reserves during their last larval days. A comparison of the pupation curves also revealed a sex difference in growth rates (Fig. 3). The males began to pupate at 17 days and reached 50 per cent pupation as early as 22 days whereas females only began to pupate at 20 days and reached 50 per cent pupation after 27 days. The number of larval instars was then determined by obtaining daily head capsule measurements. A total of 524 larvae was examined between 0 and 24 days. From these data a frequency polygon was constructed by joining the midpoints of
G. M. CHIPPENDALE
112
14
15
Age
16
17
days,
IS
30 oc
I9
20
21
22
23
24
25
FIG. 2. Comparison of male and female growth curves for Sitotrogu larvae reared in an artificial diet. Twenty observations were made at each stage.
IOOgo-
22
23
Age
24
25
days,
26
27
28
29
30
31
32
33
30 ‘C
FIG. 3. Comparison of male and female pupation curves for Sitotrogu larvae reared in an artificial diet. These data were obtained from a population of 41 males and 43 females.
GROWTH ANDDEVELOPMENT OFSITOTROGA
CEREALELLA
IN ANARTIFICIAL DIET 113
the class intervals (0.026 mm) at their respective class frequencies (Fig. 4). Four discontinuous groups were detected, each representing the variation in head capsule width within a single instar (GAINES and CAMPBELL, 1935). Although more variation existed in the head capsule width of third instars than in either first or second instars, all three showed a symmetrical distribution. The distribution of measurements from the fourth instar, however, was slightly skewed because male and female head capsule widths proved to have different distributions. Male head capsules had their highest frequency at O-702 mm, compared with 0.728 mm for those of the females. 0 Males
l
and females
Females
Head
only
capsule
width,
mm
FIG. 4. Frequency polygon depicting four larval instars for Shotrogu reared in an artificial diet.
Four larval instars probably represent the minimum number for this species when reared under optimal conditions. CROMBIE(1943) reared a small group of larvae on wheat flour and after taking twenty body measurements also concluded that the insect had four larval instars. MILLS (1965), on the other hand, used counts of cast mandibles as his criterion and detected four to seven instars for larvae reared in various kernels. He concluded that the larvae which had lower growth rates passed through more instars than the faster-growing ones. Mean head capsule widths were plotted against age to determine the duration of each instar. Fig. 5 depicts a uniform progression through the larval instars. Larvae ecdysed into the second instar on the fifth day and into the third instar on the eighth and ninth days. They then ecdysed into the fourth instar on the fourteenth and fifteenth days, depending on their sex. This last larval ecdysis was essentially complete by the fifteenth day for the males and sixteenth day for the females. The values obtained during the fourth instar again show that the females had a slightly larger mean head capsule width than the males. In a f&l experiment, separate weight loss data were obtained for both sexes during metamorphosis (Fig. 6). The results show that the mean weight loss of males (3.11 mg or 47 per cent) during the transition from larva to adult was
114
G. M. CHIPPENDALE 0.6 -
@Males and females 0 Males only
E a.7 -
l
Females
.mE*:r:
only //
go-6
&*
P
0’.
u o.s/*‘;gf
i to.4
-
0
/
P c 0.3 -
e/e;.P
6 ii 0.2 /
+-o,e-e 0.1
0
I 2
, 4
I
I
I
I
/
t
I
I
I
I
6
6
IO
12
14
I6
I6
20
22
24
Age days,
30-c
FIG. 5. Relationship between age and mean width of head capsule as a means for estimating the duration of each instar. Values obtained from 524 observations.
12 II IO s6-
Males
Females
FIG. 6. Bar chart summarizing the mean weight losses for the sexes during metamorphosis. The number of insects weighed is encircled in each bar. ML, mature larvae; NEP, newly ecdysed pupae ( < l-day-old) ; NEA, newly ecdysed adults ( < 1 -day-old).
GROWTHANDDEVELOPMENT OF SITOTROGA
CERl3ALELL.A IN AN
ARTIFICIALDIET
115
proportionally the same as for the females (5.57 mg or 48 per cent). The newly ecdysed adult weight ratio between the sexes (O-6) was therefore identical to similar mature larval and pupal weight ratios. The mean difference in weight between the male and female newly ecdysed adults (2.66 mg) approximates the weight of the ovaries of a mature female adult. No sex differences in the duration of the pupalpharate adult stage were detected. Its mean duration calculated from 113 observations was 6.7 days for both males and females. The present results have revealed new insights into the growth and development of the Angoumois grain moth. Since the insect was reared in a homogeneous medium under controlled conditions the observed sex differences can be attributed to inherent differences in male and female growth rates. These differences account for some of the variation in growth rates observed when the species is reared in its natural hosts (MILLS and WILBUR, 1967). Other main sources of growth variation in whole kernels apparently result from the location of larval feeding sites. For example, MILU (1965) f ound that larvae which fed in both the germ and endosperm of wheat kernels during their early instars matured faster than others which only fed in endosperm during the same period. AcRnowZe&emenf-I thank Mrs. EDITH MCKENZIE for her skilled technical assistance, and Mr. JOHN BROWNwho drew the figures.
REFERENCES CHIPPENDALEG. M. (1970) Development of artificial diets for rearing the Angoumois grain moth. J. econ. Ent. 63,844-848. CROMBIIIA. C. (1943) The development of the Angoumois grain moth Sitotroga cer.euZelZu (Oliv.). iVuture, Lond. 152,246. DYAR H. G. (1890) The number of moults of lepidopterous larvae. Psyche, Cumb. 5, 420422. FLANDERSS. E. (1929) The production and distribution of Trichogramma. J. econ. Ent. 22, 245-248. GAINERJ. C. and CAMPBELLF. L. (1935) Dyar’s rule as related to the number of instars of the corn earworm, Heliothis obsoletu (Fab.), collected in the field. Ann. ent. Sot. Am. 28, 445-461. MILLS R. B. (1965) Early germ feeding and larval development of the Angoumois grain moth. J. econ. Ext. 58,220-223. MILLS R. B. and WILBUR D. A. (1967) Radiographic studies of Angoumois grain moth development in wheat, corn, and sorghum kernels. y. econ. ht. 60,671-677.