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Exposure of Ephestia cautella (Wlk.) pupae to carbon dioxide concentrations at different relative humidities: The effect on adult emergence and loss in weight
J. stored Prod. Res., 1974, Vol. 10, pp. 237-241. Pergamon Press. Printed in Great Britain.
EXPOSURE OF EPHESTZA CAUTELLA (WLK.) PUPAE TO CARBON DIOXIDE CONCENTRATIONS AT DIFFERENT RELATIVE HUMIDITIES: THE EFFECT ON ADULT EMERGENCE AND LOSS IN WEIGHT S. NAVARRO Agricultural
and M. CALDERON
Research Organization, The Institute for Technology and Storage of Agricultural Stored Products Research Laboratory, Jaffa, Israel (First received 2 July 1973, and
infinal form
Products,
4 December 1973)
Abstract-The effects on mortality and loss in weight caused by 21, 51 and 88 % carbon dioxide in combination with relative humidities of 20-22, 54-55, and 95-96 % were tested on O-24 hr old Ephestia cautela pupae, at 26°C. Exposure times ranged from 1 to 6 days. At 94-55 %, and 20-22x r.h. pupal mortality was high for all carbon dioxide concentrations used, while at 95 % r.h. total mortality was obtained only at the highest carbon dioxide concentration tested. At carbon dioxide concentrations of 51 and 88 % a 9911% concentration of oxygen was present. At 20-22% and W-55 % r.h. loss in weight of pupae exposed to carbon dioxide concentrations of 21% and higher was very pronounced. At 95-96x r.h., none of the treatments resulted in loss in weight exceeding 16 % after six days of exposure. A 95 % mortality curve represents the interrelated effect of carbon dioxide and relative humidity on the moth pupae. The fumigant effect of carbon dioxide is discussed. INTRODUCTION CARBON
dioxide is widely used in mixture with different fumigants to reduce their flammability risk (MONRO, 1969). COTTON and YOUNG (1929), and JONES (1938) noted that the addition of carbon dioxide to fumigants also increases their lethal effect on insects. Carbon dioxide, in high concentrations, is also used in the laboratory to anaesthetize insects. The exposure of insects to carbon dioxide, and its effect on mortality, oviposition and duration of development were studied by JAY and PEARMAN (1971), LUM and FLAHERTY (1972), and BROOKS (1957) respectively. However, very little is known about the combined effect of carbon dioxide and relative humidity. The latter factor is of special interest when dealing with stored product insects which are known to develop in relatively dry environments (BURSELL, 1964). MATERIALS
AND METHODS
Test insect The test insect was the tropical warehouse moth, Ephestiu cauteffa Wk.). The tests were carried out on O-24 hr old E. cautella pupae which were reared on a mixture of wheatfeed with 12 per cent glycerine by weight (NAVARRO and GONEN, 1970). Insect cultures were kept in a constant temperature and relative humidity room at 26 f 1“C, and 70 f 5 % r.h. Gas compositions Different gas compositions were obtained by preparing mixtures in a g-liter container fed with gases from pressurized cylinders of carbon dioxide, oxygen and nitrogen. These 237
S. NAVARRO and
238
M. CALDERON
mixtures were passed continuously into the test chambers (Erlenmayer flasks of 100 ml capacity) at 10-15 ml/min. The whole apparatus as described by NAVARROand DONAHAYE (1972) was kept in a constant temperature room of 26 & 1°C. Gas samples of 100 ~1 were drawn from the test chambers twice daily and analysed by a gas chromatographic method (NAVARROand DONAHAYE,1972). The gas compositions to which the test insects were exposed are given in Table 1. TABLE 1. AVERAGE GAS CONCENTRATIONS CcWtdh
Treatments
group 1 control
group
2
21% co*
3 51% CO* group
group 4
88% coz
% r.h.
SE
AND RELATIVEHUMIDITIES TO WHICH PUPAE WERE EXPOSED
% COz
%N2
20 5 0.02 21 f 0.26 21 f 0.13
22 & 0.23 22 & 0.32 22 + 0.18
58 57 57
51 + 1.39
0 0
0.71
SE
78 78 78
0
55 It 1.5 96 & 1.0
22 f
%02
21 21 21
20 * I.5
21 & 0.04 5s f 044 96 3 0.12
SE
Ephestia
55 & 0.61
51 *
3.08
11 * 0.11 11 & 0.35
95 rt_ 0.00
51 * 1.41
10 r!c 0.29
38 38 39
20 5 0.51 54 i 1.64 95 5 0.49
88 5 0.45 86 & 0.79 89 * 0.47
10 f 0.30 10 & 0.12 9 i_ 0.18
2 4 2
The tests Groups of 10 pupae weighed on an analytical balance and confined in copper mesh no. 80 cages (15 cm dia and 3 cm high) were exposed to the different gas mixtures in the test chambers. After each treatment, the pupae were introduced into 20 ml empty PVC tubes and kept in the culture room until adult emergence was observed. Pupae from which adults failed to emerge within 15 days after the peak of adult emergence from the untreated pupae, were considered as dead. Loss of weight was determined by weighing each group of ten pupae after treatments. As seen in Table 1, in the treatment groups of 51 and 88 % carbon dioxide, the oxygen concentration was 9-11x, while the control and 21 y0 carbon dioxide groups contained 21-22°A of oxygen. Thus, each treatment group was arranged separately in a factorial design (STEEL and TORRIE,1960) in which 3 relative humidity levels (see Table 1) at exposure times of l-5 or 1-6 days were tested. Each treatment was carried out in eight replicates. The significance of the results was tested by analysis of variance. RESULTS Efect on adult emergence From the results presented in Fig. 1 it can be seen that exposure of insects to 21, 51, and 88 % carbon dioxide had the same effect on mortality of insects except when these treatments were carried out at 95 % r.h. Under the effect of high relative humidity, complete mortality
The Effect on Adult Emergence and Loss in Weight *------*0% co,
--m---o 51% co,
---x21%
-0
co,
08%
54-55%
0
I
23456
139
co,
r.h.
95-96
% r.h.
23456
Exposure
time,
days
FIG. 1. The effect of carbon dioxide at different relative humidities on the emergence of adults from pupae of Ephestia cautella.
was only obtained by exposure to 89% carbon dioxide (after 5 days exposure). Adult emergence of the control group was not affected significantly at the different relative humidities employed. Efect an loss in weight The loss in weight of insects exposed to different carbon dioxide and oxygen concentrations in combination with different relative humidities is presented in Fig. 2. At 20-22x and 54-55 oAr.h. exposure of pupae to gas mixtures containing 21% carbon dioxide and .-----•o%co, .-----o 51% co, ----x
c
20-22
60
%r.h.
54-55
/
1
50 -
21%CO,
-.88%CO,
%r.h.
95-96
% r.h.
,X
!'
s
/ E - 40.cr,
!'
f .r
I .I/.
30 -
z 3
p/ .
zo-
IO -
0
*_..* _ ... w..?.--*-~~* iJ;
I I
I I 2
3
I
I
I
4
5~
6
_-
I
I
I
123456
Exposure
I2
time,
3456
days
FIG. 2. The effect of carbon dioxide at different relative humidities on the loss in weight of pupae of Ephestia cautelIa.
240
S.NAVARRO
and M.
CALDERON
100 -
/’
,’
90 a0
s
-
70 -
.$ 602c
50-
$ 40._ 5 c+! 30 -
/,
20 -
/' /'
IO /
/' I
I
2
345
IIl!1I
I
IO Carbon
dloxlde,
20
I
I
lllll
30 4050
too
%
FIG. 3. The combined effect of carbon dioxide and relative humidity in producing a lethal environment for Ephestiu cautello pupae after 4 days exposure. The line represents the LT95 for the pupae.
above resulted in significant loss in weight, and the rates of loss are very close to each other. These curves differ significantly from the control group. At 95 % r.h. none of the treatments resulted in loss in weight exceeding 16 % after six days of exposure. These values are close to those obtained with the control group. DISCUSSION
Only recently has the interdependence of the effects of carbon dioxide and ambient relative humidity on insect mortality been investigated (PEARMAN and JAY, 1970; JAY et al., 1971). The results in this work clarify this combined effect on E. caufella pupae. All carbon dioxide concentrations tested prevented adult emergence after exposure for 5 days at the two lower relative humidity levels. At the high relative humidity level, the 21 % carbon dioxide concentration failed to cause complete mortality even after six days exposure. However, at 95 % r.h. under the effect of 89 % carbon dioxide a significant lethal effect was recorded in the presence of 9 % oxygen concentration. This oxygen level was found to be sufficient for survival and emergence of E. cuutellu pupae (unpublished results). The data obtained in this work together with results obtained previously in which a 4.3 % carbon dioxide level was also tested (NAVARRO and CALDERON, 1973), permitted calculation of a curve for the combined concentrations of carbon dioxide and relative humidity causing 95 % mortality at the end of four days of exposure (Fig. 3). The data incorporated in this curve were extrapolated from LT (lethal time) curves obtained by probit analysis (FINNEY, 1964) of the pupal mortality data derived from the experimental treatments. Figure 3 illustrates the close interaction between the two factors and may serve as a model from which the approximate conditions for application of the controlled atmosphere method for insect control can be predicted. The effect of exposure to carbon dioxide concentrations at different relative humidities on the loss in weight of insects was discussed by NAVARRO and CALDERON(1973). From the
The Effect on Adult Emergence and Loss in Weight
241
present work it can be deduced that at all carbon dioxide concentrations tested the lower the relative humidity the greater the loss of water by the insects. We assume that this loss of weight eventually results in insect mortality when a certain critical level has been attained. The data obtained in this study show that loss in weight figures resulting from exposure to different carbon dioxide concentrations of 21% and higher, differ very little from each other. This indicates that at a given relative humidity there is a certain threshold value for carbon dioxide concentration beyond which the effect on loss in weight does not increase. The role of relative humidity on the effect of exposure to carbon dioxide concentrations is strongly pronounced at its higher level; at 95% relative humidity even the 89% carbon dioxide concentration did not result in loss in weight to the calculated critical level of 30 % for E. cuutella pupae (NAVARRO and CALDERON, 1973). In this case insect mortality resulting from the treatments cannot be attributed to loss of water which is considered to be the causative factor at the lower relative humidities. We suggest that the carbon dioxide itself acts as a fumigant at high concentrations causing a lethal effect on insects. The basis of the above results should contribute to evaluations on the feasibility of using carbon dioxide concentrations for insect control, taking into consideration ambient relative humidity. Acknowledgements-The authors are grateful to Mrs. M. CARMELand Mr. A. AZRIELI for their efficient technical assistance during this study. This work forms a part of a Ph.D. thesis of the senior author under the supervision of Prof. I. HARPAZof the Hebrew University, Faculty of Agriculture, Rehovot. REFERENCES BROOKS,M. A. (1957) Growth-retarding effect of carbon dioxide anaesthesia on the German cockroach. J. lnsecr Physiol. 1, 76-84. BURSELL,E. (1964) Environmental aspects: humidity. In The physiology of insectu. (Ed. ROCKSTEIN,M.), Vol. 1, pp. 323-361. Academic Press. New York. COTTON,R. T. and YOLJNG,H. D. (1929) The use of carbon dioxide to increase the insecticidal efficacy of fumigants. Proc. ent. SOL-.Wash. 31,97-102. FINNEY,D. J. (1964) Probit Analysis. Cambridge University Press, London. JAY, E. G., ARBOGAST,R. T. and PEARMAN,G. C. (1971) Relative humidity: its importance m the control of stored product insects with modified atmospheric gas concentrations. J. storedprod. Res. 6.325-329. JAY, E. G. and PEARMAN,G. C. (1971) Susceptibility of two species of Tribofium (Coleoptera: Tenebrionidae) to alterations of atmospheric gas concentrations. J. storedprod. Res. 7,181-186. JONES,R. M. (1938) Toxicity of fumigant-COz mixtures to the red flour beetle. J. econ. Ent. 31, 298-309. LUM, P. T. M. and FLAHERTY,B. R. (1972) Effect of carbon dioxide on production and hatchability of eggs of Plodiu interpunctella (Lepidoptera: Phycitidae). J. econ. Ent. 65, 976-977. MONRO, H. A. U. (1969) Manual of fumigation for insect control. F.A.O. Agricultural studies No. 79. Rome. 381 pp. NAVARRO,S. and CALDERON,M. (1973) Carbon dioxide and relative humidity: interrelated factors affecting the loss of water and mortality of Ephestiu cauteha (Wlk.). (Lepidoptera: Phycitidae). Israel J. Ent. 8, 143-152. NAVARRO,S. and DONAHAYE,E. (1972) An apparatus for studying the effects of controlled low pressures and compositions of atmospheric gases on insects. J. stored Prod. Res. 8, 223-226. NAVARRO,S. and GONEN, M. (1970) Some techniques for laboratory rearing and experimentation with Ephestiu cautellu (Wlk.) (Lepidoptera, Phycitidae). J. stored Prod. Res. 6, 187-189. PEARMAN,(3. C. and JAY, E. G. (1970) The effect of relative humidity on the toxicity of carbon dioxide to Tribolium castaneum in peanuts. J. Georgia ent. Sot. 51, 6-64. STEEL,R. Cf. D. and TORRIE,J. H. (1960) Principles undprocadures in sratistics. McGraw-Hill, New York. p. 481.
EXPOSURE OF EPHESTZA CAUTELLA (WLK.) PUPAE TO CARBON DIOXIDE CONCENTRATIONS AT DIFFERENT RELATIVE HUMIDITIES: THE EFFECT ON ADULT EMERGENCE AND LOSS IN WEIGHT S. NAVARRO Agricultural
and M. CALDERON
Research Organization, The Institute for Technology and Storage of Agricultural Stored Products Research Laboratory, Jaffa, Israel (First received 2 July 1973, and
infinal form
Products,
4 December 1973)
Abstract-The effects on mortality and loss in weight caused by 21, 51 and 88 % carbon dioxide in combination with relative humidities of 20-22, 54-55, and 95-96 % were tested on O-24 hr old Ephestia cautela pupae, at 26°C. Exposure times ranged from 1 to 6 days. At 94-55 %, and 20-22x r.h. pupal mortality was high for all carbon dioxide concentrations used, while at 95 % r.h. total mortality was obtained only at the highest carbon dioxide concentration tested. At carbon dioxide concentrations of 51 and 88 % a 9911% concentration of oxygen was present. At 20-22% and W-55 % r.h. loss in weight of pupae exposed to carbon dioxide concentrations of 21% and higher was very pronounced. At 95-96x r.h., none of the treatments resulted in loss in weight exceeding 16 % after six days of exposure. A 95 % mortality curve represents the interrelated effect of carbon dioxide and relative humidity on the moth pupae. The fumigant effect of carbon dioxide is discussed. INTRODUCTION CARBON
dioxide is widely used in mixture with different fumigants to reduce their flammability risk (MONRO, 1969). COTTON and YOUNG (1929), and JONES (1938) noted that the addition of carbon dioxide to fumigants also increases their lethal effect on insects. Carbon dioxide, in high concentrations, is also used in the laboratory to anaesthetize insects. The exposure of insects to carbon dioxide, and its effect on mortality, oviposition and duration of development were studied by JAY and PEARMAN (1971), LUM and FLAHERTY (1972), and BROOKS (1957) respectively. However, very little is known about the combined effect of carbon dioxide and relative humidity. The latter factor is of special interest when dealing with stored product insects which are known to develop in relatively dry environments (BURSELL, 1964). MATERIALS
AND METHODS
Test insect The test insect was the tropical warehouse moth, Ephestiu cauteffa Wk.). The tests were carried out on O-24 hr old E. cautella pupae which were reared on a mixture of wheatfeed with 12 per cent glycerine by weight (NAVARRO and GONEN, 1970). Insect cultures were kept in a constant temperature and relative humidity room at 26 f 1“C, and 70 f 5 % r.h. Gas compositions Different gas compositions were obtained by preparing mixtures in a g-liter container fed with gases from pressurized cylinders of carbon dioxide, oxygen and nitrogen. These 237
S. NAVARRO and
238
M. CALDERON
mixtures were passed continuously into the test chambers (Erlenmayer flasks of 100 ml capacity) at 10-15 ml/min. The whole apparatus as described by NAVARROand DONAHAYE (1972) was kept in a constant temperature room of 26 & 1°C. Gas samples of 100 ~1 were drawn from the test chambers twice daily and analysed by a gas chromatographic method (NAVARROand DONAHAYE,1972). The gas compositions to which the test insects were exposed are given in Table 1. TABLE 1. AVERAGE GAS CONCENTRATIONS CcWtdh
Treatments
group 1 control
group
2
21% co*
3 51% CO* group
group 4
88% coz
% r.h.
SE
AND RELATIVEHUMIDITIES TO WHICH PUPAE WERE EXPOSED
% COz
%N2
20 5 0.02 21 f 0.26 21 f 0.13
22 & 0.23 22 & 0.32 22 + 0.18
58 57 57
51 + 1.39
0 0
0.71
SE
78 78 78
0
55 It 1.5 96 & 1.0
22 f
%02
21 21 21
20 * I.5
21 & 0.04 5s f 044 96 3 0.12
SE
Ephestia
55 & 0.61
51 *
3.08
11 * 0.11 11 & 0.35
95 rt_ 0.00
51 * 1.41
10 r!c 0.29
38 38 39
20 5 0.51 54 i 1.64 95 5 0.49
88 5 0.45 86 & 0.79 89 * 0.47
10 f 0.30 10 & 0.12 9 i_ 0.18
2 4 2
The tests Groups of 10 pupae weighed on an analytical balance and confined in copper mesh no. 80 cages (15 cm dia and 3 cm high) were exposed to the different gas mixtures in the test chambers. After each treatment, the pupae were introduced into 20 ml empty PVC tubes and kept in the culture room until adult emergence was observed. Pupae from which adults failed to emerge within 15 days after the peak of adult emergence from the untreated pupae, were considered as dead. Loss of weight was determined by weighing each group of ten pupae after treatments. As seen in Table 1, in the treatment groups of 51 and 88 % carbon dioxide, the oxygen concentration was 9-11x, while the control and 21 y0 carbon dioxide groups contained 21-22°A of oxygen. Thus, each treatment group was arranged separately in a factorial design (STEEL and TORRIE,1960) in which 3 relative humidity levels (see Table 1) at exposure times of l-5 or 1-6 days were tested. Each treatment was carried out in eight replicates. The significance of the results was tested by analysis of variance. RESULTS Efect on adult emergence From the results presented in Fig. 1 it can be seen that exposure of insects to 21, 51, and 88 % carbon dioxide had the same effect on mortality of insects except when these treatments were carried out at 95 % r.h. Under the effect of high relative humidity, complete mortality
The Effect on Adult Emergence and Loss in Weight *------*0% co,
--m---o 51% co,
---x21%
-0
co,
08%
54-55%
0
I
23456
139
co,
r.h.
95-96
% r.h.
23456
Exposure
time,
days
FIG. 1. The effect of carbon dioxide at different relative humidities on the emergence of adults from pupae of Ephestia cautella.
was only obtained by exposure to 89% carbon dioxide (after 5 days exposure). Adult emergence of the control group was not affected significantly at the different relative humidities employed. Efect an loss in weight The loss in weight of insects exposed to different carbon dioxide and oxygen concentrations in combination with different relative humidities is presented in Fig. 2. At 20-22x and 54-55 oAr.h. exposure of pupae to gas mixtures containing 21% carbon dioxide and .-----•o%co, .-----o 51% co, ----x
c
20-22
60
%r.h.
54-55
/
1
50 -
21%CO,
-.88%CO,
%r.h.
95-96
% r.h.
,X
!'
s
/ E - 40.cr,
!'
f .r
I .I/.
30 -
z 3
p/ .
zo-
IO -
0
*_..* _ ... w..?.--*-~~* iJ;
I I
I I 2
3
I
I
I
4
5~
6
_-
I
I
I
123456
Exposure
I2
time,
3456
days
FIG. 2. The effect of carbon dioxide at different relative humidities on the loss in weight of pupae of Ephestia cautelIa.
240
S.NAVARRO
and M.
CALDERON
100 -
/’
,’
90 a0
s
-
70 -
.$ 602c
50-
$ 40._ 5 c+! 30 -
/,
20 -
/' /'
IO /
/' I
I
2
345
IIl!1I
I
IO Carbon
dloxlde,
20
I
I
lllll
30 4050
too
%
FIG. 3. The combined effect of carbon dioxide and relative humidity in producing a lethal environment for Ephestiu cautello pupae after 4 days exposure. The line represents the LT95 for the pupae.
above resulted in significant loss in weight, and the rates of loss are very close to each other. These curves differ significantly from the control group. At 95 % r.h. none of the treatments resulted in loss in weight exceeding 16 % after six days of exposure. These values are close to those obtained with the control group. DISCUSSION
Only recently has the interdependence of the effects of carbon dioxide and ambient relative humidity on insect mortality been investigated (PEARMAN and JAY, 1970; JAY et al., 1971). The results in this work clarify this combined effect on E. caufella pupae. All carbon dioxide concentrations tested prevented adult emergence after exposure for 5 days at the two lower relative humidity levels. At the high relative humidity level, the 21 % carbon dioxide concentration failed to cause complete mortality even after six days exposure. However, at 95 % r.h. under the effect of 89 % carbon dioxide a significant lethal effect was recorded in the presence of 9 % oxygen concentration. This oxygen level was found to be sufficient for survival and emergence of E. cuutellu pupae (unpublished results). The data obtained in this work together with results obtained previously in which a 4.3 % carbon dioxide level was also tested (NAVARRO and CALDERON, 1973), permitted calculation of a curve for the combined concentrations of carbon dioxide and relative humidity causing 95 % mortality at the end of four days of exposure (Fig. 3). The data incorporated in this curve were extrapolated from LT (lethal time) curves obtained by probit analysis (FINNEY, 1964) of the pupal mortality data derived from the experimental treatments. Figure 3 illustrates the close interaction between the two factors and may serve as a model from which the approximate conditions for application of the controlled atmosphere method for insect control can be predicted. The effect of exposure to carbon dioxide concentrations at different relative humidities on the loss in weight of insects was discussed by NAVARRO and CALDERON(1973). From the
The Effect on Adult Emergence and Loss in Weight
241
present work it can be deduced that at all carbon dioxide concentrations tested the lower the relative humidity the greater the loss of water by the insects. We assume that this loss of weight eventually results in insect mortality when a certain critical level has been attained. The data obtained in this study show that loss in weight figures resulting from exposure to different carbon dioxide concentrations of 21% and higher, differ very little from each other. This indicates that at a given relative humidity there is a certain threshold value for carbon dioxide concentration beyond which the effect on loss in weight does not increase. The role of relative humidity on the effect of exposure to carbon dioxide concentrations is strongly pronounced at its higher level; at 95% relative humidity even the 89% carbon dioxide concentration did not result in loss in weight to the calculated critical level of 30 % for E. cuutella pupae (NAVARRO and CALDERON, 1973). In this case insect mortality resulting from the treatments cannot be attributed to loss of water which is considered to be the causative factor at the lower relative humidities. We suggest that the carbon dioxide itself acts as a fumigant at high concentrations causing a lethal effect on insects. The basis of the above results should contribute to evaluations on the feasibility of using carbon dioxide concentrations for insect control, taking into consideration ambient relative humidity. Acknowledgements-The authors are grateful to Mrs. M. CARMELand Mr. A. AZRIELI for their efficient technical assistance during this study. This work forms a part of a Ph.D. thesis of the senior author under the supervision of Prof. I. HARPAZof the Hebrew University, Faculty of Agriculture, Rehovot. REFERENCES BROOKS,M. A. (1957) Growth-retarding effect of carbon dioxide anaesthesia on the German cockroach. J. lnsecr Physiol. 1, 76-84. BURSELL,E. (1964) Environmental aspects: humidity. In The physiology of insectu. (Ed. ROCKSTEIN,M.), Vol. 1, pp. 323-361. Academic Press. New York. COTTON,R. T. and YOLJNG,H. D. (1929) The use of carbon dioxide to increase the insecticidal efficacy of fumigants. Proc. ent. SOL-.Wash. 31,97-102. FINNEY,D. J. (1964) Probit Analysis. Cambridge University Press, London. JAY, E. G., ARBOGAST,R. T. and PEARMAN,G. C. (1971) Relative humidity: its importance m the control of stored product insects with modified atmospheric gas concentrations. J. storedprod. Res. 6.325-329. JAY, E. G. and PEARMAN,G. C. (1971) Susceptibility of two species of Tribofium (Coleoptera: Tenebrionidae) to alterations of atmospheric gas concentrations. J. storedprod. Res. 7,181-186. JONES,R. M. (1938) Toxicity of fumigant-COz mixtures to the red flour beetle. J. econ. Ent. 31, 298-309. LUM, P. T. M. and FLAHERTY,B. R. (1972) Effect of carbon dioxide on production and hatchability of eggs of Plodiu interpunctella (Lepidoptera: Phycitidae). J. econ. Ent. 65, 976-977. MONRO, H. A. U. (1969) Manual of fumigation for insect control. F.A.O. Agricultural studies No. 79. Rome. 381 pp. NAVARRO,S. and CALDERON,M. (1973) Carbon dioxide and relative humidity: interrelated factors affecting the loss of water and mortality of Ephestiu cauteha (Wlk.). (Lepidoptera: Phycitidae). Israel J. Ent. 8, 143-152. NAVARRO,S. and DONAHAYE,E. (1972) An apparatus for studying the effects of controlled low pressures and compositions of atmospheric gases on insects. J. stored Prod. Res. 8, 223-226. NAVARRO,S. and GONEN, M. (1970) Some techniques for laboratory rearing and experimentation with Ephestiu cautellu (Wlk.) (Lepidoptera, Phycitidae). J. stored Prod. Res. 6, 187-189. PEARMAN,(3. C. and JAY, E. G. (1970) The effect of relative humidity on the toxicity of carbon dioxide to Tribolium castaneum in peanuts. J. Georgia ent. Sot. 51, 6-64. STEEL,R. Cf. D. and TORRIE,J. H. (1960) Principles undprocadures in sratistics. McGraw-Hill, New York. p. 481.