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The effect on the mortality of adult Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae), Sitophilus granarius (L.) (Coleoptera: Curculionidae) and Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) of interrupting low oxygen exposures with periods of elevated oxygen
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J. storedProd. Res. Vol. 32, No. 3, pp. 187-194. 1996 Crown Copyright 0 1996 Published by Elsevier Science Ltd Printed in Great Britain 0022-474X/96 $15.00 + 0.00 SOO22-474X(%)OOO29-X
The Effect on the Mortality of Adult Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae), Sitophilus granarius (L.) (Coleoptera: Curculionidae) and Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) of Interrupting Low Oxygen Exposures with Periods of Elevated Oxygen SIMON T. CONYERS* and CHRISTOPHER
H. BELL
Central Science Laboratory, Ministry of Agriculture, Fisheries and Food, London Road, Slough, Berks SL3 7HJ, U.K. (Accepted 1 July 1996)
Abstract-A pbospbine-resistant (R) and a laboratory-susceptible (S) strain of Cryptolestes ferrugineus (Stephens) and malathion-resistant (R) and a laboratory-susceptible (S) strain of Sitopkilus granarius (L.) and Oryzaephilus surinumensis (L.) were exposed as adults to a controlled atmosphere generated from mixing nitrogen and compressed air to give an oxygen content of 1%. These exposures all took place at 20°C and 50% r.b. They lasted for 4, 6 and 8 d for C. ferrugineus and S. granarius, and for 2,4 and 6 d for 0. surinamensis. Simultaneously, similar exposures were carried out wbicb included up to three periods at bigber oxygen levels (3, 5 or 10%) during the course of tbe exposure to 1% oxygen. These periods with increased oxygen concentratious lasted for 8 (only C. ferrugineus) or 16 b (all tbree species). There was no significant difference (P > 0.05) in the response of the two strains of C. ferrugineus to the atmosphere so furtber analysis was carried out using pooled data of both straius. However, there was a significant difference (Z’ < 0.05) between tbe strains of the other two species. Tbis ditierence was not tbe same for botb species as tbe R strain was more tolerant than the S strain of S. granarius whereas the reverse was true of 0. surinamensis. Adult C. ferrugikeus of either strain appeared the most tolerant to the 1% oxygen atmosphere, followed by the R strain of S. granarius, tben its S straiu and 6nally tbe S and tben tbe R strain of 0. surinamensis. Mortality of all species increased as tbe exposure period increased. Comporisous of mortalities between the continuous 1% exposures and interruptions witb bigber oxygen levels produced similar patterns of response for the three species, even when tbeir tolerances to tke atmosphere differed. Tbere was increased survival as tke level of oxygen during the interruption increased and tbis increase was accentuate-dwith an increase in duration of tbe intemtption. However, the signiiicauce of this increased survival decreases as tbe exposure period iucrea$es. The use of an exposure period, which produced over 99% mortality for tbe uninterrupted 1% oxygen exposure, meant that tkere was no increase in survival even after three 16-b ir@rruptions of 5% oxygen during such an exposure. Crown Copyright 0 1996 Published by Eisevier Science Ltd Key worris-stored
grain beetles, oxygen, nitrogen, mortality, time
*Author for correspondence. 187
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INTRODUCTION
Controlled atmosphere (CA) fumigation of bulk grain is a technology that has been available for over two decades (Banks et al., 1991) and is becoming a more attractive alternative to the traditional methods of grain storage. Although it costs more than the most widely used grain fumigant, phosphine (Annis, 1987), consumer demand has been moving towards pesticide-free produce. This market force may be the most important factor responsible for bringing CA storage to the forefront of the grain protection market. CA has an important role as an element in a new integrated control initiative (Banks et al., 1991) where it would be employed in conjunction with systems for drying and cooling the grain. In addition to effective control of arthropod pests, it also prevents mould growth, preserves grain quality and maintains the germination energy of the grain (Banks, 1981). The constraints on CA usage have always been the costs of gas provision and of the sealing of storage structures (Jay, 1984; Annis, 1987). The former has now been lessened with the development of systems for on-site generation, thus removing the need for transportation of gas. On-site generators, which include hydrocarbon burners, membrane separation units and pressure-swing adsorption systems, have a high initial cost. This can be offset against the low energy requirements for the gas production although some of these systems are still large consumers of energy and therefore costly to operate. It is important to find ways of lessening these costs particularly as insects require long exposures to the atmospheres to be controlled (Banks et al., 1991). This is especially true at low temperatures which prevail during the U.K. grain storage season. The main factor responsible for the loss of CAs from grain stores is the weather, especially on existing silo complexes which were not designed as far as sealing is concerned for use with CAs. High winds, rapid overnight drops in temperature and fluctuating atmospheric pressure may severely deplete the atmosphere in grain silos and thus raise the oxygen level. This may not be detected for some time and even with a higher gas application flow rate would take several hours to rectify. It is important to know whether it is necessary to compensate for this loss with an extension of exposure time. This project set out to determine the effect of interruptions of increased oxygen on the mortality of adult Cryptolestes ferrugineus, S. granarius and 0. surinamensis during exposures to 1% oxygen in a nitrogen atmosphere. These species are the major beetle pests found in grain in the U.K. (Muggleton et al., 1991). They all have the ability to overwinter in the British climate (Solomon and Adamson, 1956) without diapause (Kawamoto et al., 1989). C. ferrugineus, like 0. surinumensis, is classed as a secondary grain pest, being incapable of attacking whole grain (Freeman, 1952), but modern conveying methods result in sufficient grain damage for infestations to occur. These represent a serious threat to the maintenance of insect-free high quality grain (Kawamoto et al., 1989). Once established, C. ferrugineus in particular remains in grain stores on residues of grain and dust that accumulate in the fabric of the building (Cox and Parish, 1991).
MATERIALS
AND METHODS
Insects C. ferrugineus. Adults, from a laboratory phosphine-susceptible and a phosphine-resistant strain, were produced from separate cultures using a food mixture of 98 g of heat-sterilised rolled oats, 49 g of wheat flour and 8 g of yeast placed in a 1 kg glass jar. Two jars were prepared for each strain every two weeks. These were each set up with 150 adults and were kept in a culture room at 30°C and 70% r.h. The original adults were removed after 3 weeks. The start of emergence was noted and 3 weeks later the new adults were removed and combined from the two jars. They were then placed in a new jar with the same quantity of medium for a further 3 weeks before they were tested. Two days prior to testing, the adults were removed from the cultures. Fifty adults were placed in a crystallising dish (50 mm diameter x 30 mm), 30 dishes for each strain, containing a spoonful of culture food and topped by a filter paper identifying each dish. These were then covered with a piece of gauze which was held in place with elastic bands. They were then placed in a room at a temperature of 25°C and 70% r.h. for 24 h and finally moved to the exposure room at 20°C and 70% r.h., 24 h before the start of the exposures (4, 6 or 8 d).
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S. granarius. A laboratory malathion-susceptible and a malathion-resistant strain were used for this species. A similar culture regime was used as for C. ferrugineus. However, the food medium was 320 g of heat-sterilised whole wheat grains and the culture jars were kept at 25°C and 70% r.h. Each crystallising dish used for this species had fluon (polytetrafluoroethylene) wiped around the rim to prevent escape. The dishes were moved from the culturing conditions to the exposure room 24 h before the start of the exposures (4, 6 or 8 d). Similar strains and a similar culture regime was used for this species as for 0. surinamensis. S. granarius. The food used for this species was 150 g of heat-sterilised rolled oats and the exposures tested were 2, 4 and 6 d.
Test procedure
The adults were exposed to the atmosphere in glass desiccators (280 mm diameter x 240 mm height). The generated atmosphere of 1% oxygen was produced by passing nitrogen and compressed air through a gas blender (850 Series, Signal Instrument Co. Ltd, Camberley, Surrey, U.K.). The output was assessed for accuracy by using a paramagnetic oxygen analyser (Model no. 570A, Servomex Ltd, Crowborough, Sussex, U.K.). The humidity of the gas chosen for the experiments was 50% r.h., a level likely to increase the efficacy of low oxygen atmospheres. This was achieved by passing the gas stream on route to the desiccators over an aqueous solution of potassium hydroxide (Solomon, 1951) contained in a litre bottle. There was one bottle for each exposure chamber. The humidity was verified with the aid of a dew point meter (Model no. DP680, Protimeter Ltd, Marlow, Bucks, U.K.) after the gas left the exposure chamber. Insects were exposed for periods of 2, 4, 6 or 8 d. Running concurrently were experiments in which the insects were exposed for similar periods but, additionally, these were subjected to one, two or three periods of 8- or 16-h duration at a higher level of oxygen, either 3, 5 or 10%. The schedule for the regimes is shown in Fig. 1. These higher levels were achieved with the use of another gas blender (850 Series,
Fig. 1. Outline of the regime used to test the effect of increases in oxygen to 3, 5 or 10% for 8 or 16 h on mortality from exposure to a 1% oxygen atmosphere: (above) C. ferrugineus and S. grunurius; (below) 0. surinamensis. In both, black area indicates 1% oxygen; and grey area indicates 3, 5 or 10% oxygen for 8 or 16 h.
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Signal Instrument Co. Ltd, Camberley, Surrey, U.K.). ‘T’ ball valves were used to switch the gas stream from one source to the other. At the start of the exposures, the desiccators were purged with a flow rate of 350 ml/min which was reduced to 100 ml/min for the duration of the exposure once the oxygen level had dropped to 1%. The same flow rates were used after alteration of the gas mixture to increase the oxygen level for the interruption in the 1% oxygen exposure, and again to restore the 1% level at the end of the period. The 8-h interruption periods began at 0900 and finished at 1700 on the day shown (Fig. 1) whereas the 16-h periods began at 1700 on that day and finished at 0900 on the next day. There were five replicate experiments (1500 insects used / experiment) which were run consecutively for each of the three elevated oxygen levels. A different culture of each strain of insect was used for each experiment. All of the seven exposure regimes within each experiment had three dishes from each strain which left nine from each strain to act as untreated controls. Once the exposures were completed, all the dishes were moved back to the original culture conditions. A count was made of the mortality after 2 weeks. A correction was made to the mortality figures from the exposures using the mortality found in the controls (Abbott, 1925). The corrected mortality figures were then transformed by angular sine to normalise their distribution (Parker, 1979). The computer program used radians and these figures were converted into degrees to give a broader distribution. The data for each replicate at the different oxygen levels were then compared to test for any significant differences which would prevent their grouping. The data were then combined and a general linear model (GLM) (Minitab Inc., State College, PA, U.S.A.) was used to test for significant differences between the two strains, the oxygen regimes and the exposure time periods. For this test the 8-d exposures with two and three periods of higher oxygen were grouped together to give a balanced model. If there was no difference between the two strains used for each species then their data were pooled for further analysis. The means were then tested separately using a one-way analysis of variance with the separation of the mean values for the 8-d exposures with two and three periods of higher oxygen. Any significant differences (P < 0.05) were assessed using Tukey’s HSD Test (Kirk, 1968) after the means had been converted back to percentages. There was a further GLM analysis carried out to assess the difference between the 8- and the 16-h periods used for C. ferrugineus.
RESULTS C. ferrugineus The results from the GLM analysis of the effects of the 8-h interruptions showed that there was no significant difference in a comparison between the two strains (F = 0.4, P > 0.05). However there were significant differences between the four oxygen regimes (F = 47.7, P < 0.05) and between the three exposure periods (F = 437.0, P < 0.05). The same responses were observed in the results from 16-h interruptions with no significant difference between the strains (F = 0.8, P > 0.05) but significant differences due to oxygen regime (F = 81.9, P < 0.05) and exposure length (F = 658.4, P < 0.05). These results alone give no indication of the extent to which each factor contributed to the differences observed. The subsequent one-way analysis of variance provided the treatment means for each strain (Table 1) and the pooled mean mortality for both strains showed the effect of the different exposures on the mortality of the adult beetle. For all the different oxygen regimes used, as expected, mortality increased as the exposure time increased. When looking at the results from the experiment on 8-h periods of interruption, the increase in mortality was only significant (P < 0.05) for each increase in exposure time for the continuous 1% oxygen exposure. For the other three interruption regimes at different oxygen levels, the increase in exposure time from 6 to 8 d was not significant (P > 0.05) and there was no significant difference (P > 0.05) between the 8-d exposures with two or three periods of increased oxygen. When the effect of the different oxygen levels was considered, there was a significant decrease in mortality (P < 0.05) with 3% oxygen interruptions for only the 4- and 6-d exposures. The increase to 5% oxygen gave another significant decrease (P < 0.05) for both 4 and 8 d. The increase from 5 to 10% oxygen did not achieve any further significant decrease (P > 0.05) in mortality. For the 16-h interruption period experiment, the results for the increase in exposure time were
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Table 1. Mean percentage mortalities ( f SE., n = 5) for a phosphine-resistant (R) and a laboratory-susceptible (S) strain of adult C. firrugineus after exposure to a 1% oxygen atmosphere and to increases in oxygen (to 3, 5 or 10%) for 8 or 16-h periods during exposure to 1% oxygen
8 Hour
Atmosphere 1% + 3%
I%+5%
1% + 10%
81.8(1.4) 80.6(1.4) 98.7(0.4) ’ ’ 97.8(0.5)
65.4(1.7) ’ 59.9(1.8) 97.5(0.6) 24 99.1(0.3)
64.0(1.8) ’ 57.9(1.8) 88.3tl.2) 4 87.6( 1.2)
99.9(0.1) 2 4 99.9(0.1) 98.9(0.4) * 4 92.1(1.0)
92.6(1.0) ’ 4 96.1(0.7) 94.0(0.9) ’ 4 94.1(0.9)
89.1(1.1) 3’ 91.1(1.0) 87.8(1.2) 3’ 92.7(1.0)
1%+3%
1% + 5%
1% + 10%
38.4( 1.8)2 35.0(1.7) 73.6(1.6) ’ 72.0( 1.6) 92.2(1.0) 2 3 4 88.2(1.2) __. 89.7(1.1) 1 ’ 4 91.6(1.0)
31.8(1.7) 31.9(1.7) 75.1(1.6) 70.1(1.7) 85.7(1.3) 88.6(1.2) 91.3(1.0) 91.1(1.0)
1%
Strain R S R S
Days 4 6
R S R S
8 8
68.3(1.0) 70.2( 1.O) 92.7(0.6) 92.9(0.6)
Periods’ 2 2
2
99.5tO.2) 2 99.5(0.2) 99.5(0.2) 2 99.5(0.2)
3
16 Hour
Atmosphere 1% Strain R S R S R S R S
Days 4 6 8 8
65.9(1.0) 65.8(1.0) 84.9(0.8) 84.9(0.8) 94.5(0.5) * 95.1(0.5) 94.5(0.5) * 95.1(0.5)
Periods ’ 2 2 2 3
2 * ’4 “ 4
35.3(1.8) 35.2(1.7) 77.5(1.5) 69.4(1.7) 88.1(1.2) 90.0(1.1) 84.1(1.3) 89.0(1.1)
2 ? ’* 34
‘Number of periods with increased oxygen.
* and 3 Means with the same number in the same row which are not significantly different (P > 0.05). 4Means in the same column which are not significantly different (P > 0.05).
the same except that the increase in mortality from 6 to 8 d was significant for all treatments (P < 0.05). For the interruptions, the addition of 3% oxygen produced a significant decrease (P < 0.05) for the 4- and 6-d exposures, but the increases in mortality with further increases in oxygen to 5 and 10% were not significant (P > 0.05). A direct comparison between the mean mortalities due to these increased levels of oxygen for the 8- and 16-h periods (Table 2) shows a trend of decreasing difference (F = 223.2, P < 0.05) as exposure length increases. It becomes almost negligible and was not significant (P > 0.05) during the 8-d exposure with the three periods at 5 and 10% oxygen. S. granarius The results from the GLM analysis gave a significant difference for all factors being assessed: strain (F = 486.6, P < 0.05), oxygen regime (F = 128.2, P -c 0.05) and exposure length (F = 2143.0, P -c 0.05). The means are shown in Table 3 and the strain difference is significant (P < 0.05) for similar oxygen conditions for all 4- and 6-d exposure lengths, but only for the 10% oxygen interruption for both two and three periods for the 8 d. Taking the R strain alone it showed significant increases (P < 0.05) in mortality for each increase in exposure length, but there was no significant difference between the two and three periods of higher oxygen during the 8-d exposure. The trend with the increasing oxygen showed significant decreases (P < 0.05) between 1 and 3% interruptions of oxygen except for the 8-d with three periods. A further significant decrease (P < 0.05) occurred with the increase in oxygen from 3 to 5% oxygen was seen in the 4- and 6-d
Table 2. A comparison of the mean percentage mortalities from the 8- and 16-h periods of increased oxygen for C. ferrugineus (mean of the R and S strains) Exposure regime 4
Davs r
%02
Hours 8 16
1+3
1+5
81.2’ 36.7
62.7’ 31.8
6 1+10
1+3
1+5
98.3’ 72.8
98.4’ 72.6
8 1+10 87.9’ 73.6
1+3
1+5
99.9’ 94.5’ 90.3 87.2
1+10 90.2 89.1
1+3x3 96.2’ 90.7
1+5x3
1+10x3
94. I 91.2
90.4 86.6
‘Significant difference (P < 0.05) between mean mortalities due to difference in time period for increased oxygen.
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Table 3. Mean percentage mortalities ( + SE., n = 5) for a phosphine-resistant (R) and a laboratory-susceptible (S) strain of adult S. granarius and 0. surinamensis after exposure to a 1% oxygen atmosphere and to increases in oxygen (to 3, 5 or 10%) for 8- or 16-h periods during exposure to 1% oxygen S. granarius
Atmosphere 1%
Days 4
6 8 8
1% + 5% __. _.
1% + 10%
13.2(I.2)2 34.5(1.7) 72.2( 1.6)2 98.2(0.5) ’ 6 97.5(0.6) 3 5 100 ’ 6 95.4(0.8) 3 ’ 99.9(0.1) 3 6
2.5(0.6)’ ’ 15.7(1.3) 56.7( 1.8)3 ’ 98.2(0.5) 3 6 94.5(0.8) 3 4 5 99.8(0.1) 3 6 93.2(0.9)j 4 5 99.9(0.1) 3 b
3.9(0.2)3 9.7(1.1) 57.9(1.8)’ J. 94.9(0.8) ’ 6 t39&1” 4 J
1%+3%
1%+5%
1% + 10%
90.8(1.1)’ * 62.8(1.8) 3 99.9(0.1) 3 5 97.2(0.6) 4 3 ’ 100 J 3 100 36
87.4(1.2)3 * 61.2(1.8) 3 99.9(0.1)3 2 5 94.3(0.8) ‘ 100 ’ 5
59.0(1.8)2 27.6(1.6) 96.2(0.7)’ * ’ 83.1(1.4) 100 ’ 5 99.9(0.1) 3
Periods ’
Strain R S R S R S R S
1% + 3%
24.1(0.9)’ 59.5(1.0) 88.1(0.7)’ 99.8(0.1) 3 6 99.8(0.1)’ 100 ’ 6 99.8(0.1)3 100 3 6
2 2 2 3
0, surinamensis
Atmosphere 1%
Days 2 4 6
86.4( 1.3)’ 4 5 99.7(0.2) ’ 6
Periods ’
Strain R S R S R S
96.2(0.4)’ ’ 77.9(0.9) 100 3 ’ 36 99W
1 2 2
!OO’
‘Number of periods with increased oxygen. 2Pairs of R and S mean mortalities which are significantly different (P < 0.05). 3 and * Means with the same number in the same row which are not signi6cantly different (P > 0.05). IR means in the same column which are not significantly different (P > 0.05). 6S means in the same colutnn which are not significantly different (P > 0.05).
exposures but with the further increase in oxygen to lo%, any decrease in the mortality was not significant (P > 0.05). The S strain was less tolerant of these atmospheres. Therefore significant decreases (P < 0.05) in mortality due to exposure length were only seen in the increase from 4 to 6 d. Any significant decrease (P < 0.05) in mortality due to the increases in oxygen during the interruptions were noted only for the 4-d exposures and occurred for each increase in oxygen. 0. surinamensis The GLM analysis gave a significant difference for all factors being assessed: strain (F = 220.9, P < 0.05), oxygen regime (F = 140.6, P < 0.05) and exposure length (F = 952.0, P < 0.05). The means are shown in Table 3 and the strain difference was only significant (P -z 0.05) for each of the 2-d comparisons and for the 5 and 10% interruptions for the 4-d. In contrast to S. granaries, the S strain was more tolerant of these atmospheres. There was a significant increase in mortality of both strains due to increased exposure time between 2 and 4 d except for the R strain with the exposure in 1% oxygen. When the interruptions of higher oxygen are considered, there is a similar pattern for both strains for the 2-d exposure. There is a significant decrease (P < 0.05) in mortality with the addition of 3% oxygen and in the change from 5 to 10% oxygen. However after this there is no significant difference for any of the mortalities from the 4- and 6-d exposures for the R strain. The S strain only exhibited a significant decrease (P < 0.05) for the 4-d exposure with the addition of 10% oxygen. DISCUSSION
The efficacy of a 1% oxygen atmosphere was in general only slightly affected by fluctuations of up to 16 h duration in which oxygen levels rose to 10% during the exposure. From the results, complete mortahty of adult C. ferrugineus would be expected from an exposure slightly longer than 8 d in a 1% oxygen atmosphere at 20°C and 50% r.h. Krishnamurthy et al. (1986) obtained complete mortality after only 7 d at the same temperature but used a higher humidity. This could be seen as a discrepancy as the lower humidity of the present results should have increased the mortality due to its desiccant effect (Jay et al., 1971). However carbon dioxide ( > 10%) was
Insect mortality in interrupted low oxygen atmospheres
193
included in the gas mixture used by Krishnamurthy et al. (1986). This level of carbon dioxide has been shown to enhance the mortality in low oxygen atmospheres and may have a synergistic effect (Calderon and Navarro, 1980) rather than the direct toxic effect of higher carbon dioxide levels as shown by Rameshbabu ef al. (1991). The exact conditions of a test are very important as shown by the attainment of complete mortality of adults of this beetle in a 100% nitrogen atmosphere in less than 2 d at a higher temperature (Anon., 1983). It is important in a control situation to lower the oxygen content as far as possible. However, though this is desirable, the results from this experiment have shown that an exposure of 8 d with three periods of 16 h where the oxygen level reaches 5% will not give a significant decrease (P > 0.05) in mortality in this the most tolerant of the three species tested here, but that there is a significant decrease (P < 0.05) in mortality when 10% oxygen is used. This higher level of survival at the 10% level, but not the 5%, agrees with the tests of Bailey (1965) where adults of C. ferrugineus were not adversely affected by low oxygen atmospheres in air-tight storage until the level dropped to 5% and below. For S. granarius, Lindgren and Vincent (1970) produced 95% mortality in adults after 5.3 d with 100% nitrogen at 70°F and 60-70% r.h. In the present experiment 95% mortality for either the R or the S strain was attained between 6 and 8 d. The absence of oxygen in the former study and the slightly higher temperature than the present experiment would be expected to shorten the time taken to reach 95% mortality though the higher humidity used would have had the opposite affect. Adler (1991) produced an average of 99% mortality from 10 strains in 6.6 + 1.6 d (20°C and 75%) with 99% nitrogen and 1% oxygen. This result is comparable to those obtained in the present study. However, with the difference in the tolerance of strains to modified atmospheres apparent in the present experiment it becomes difficult to make direct comparisons with other works involving single strains. This difference in the tolerance of different strains to modified atmospheres has already been noted by Adler (1991) who found significant differences in the responses of S. granarius from different countries. The results for 0. surinumensis agree with results from Krishnamurthy et al. (1986) (20°C and 70% r.h.) although, as for C. ferrugineus, this experiment did incorporate 10% carbon dioxide which may have increased the mortality. Ninety-seven per cent mortality was achieved by 4 d with complete control by 6 d. The biggest increase in mortality for this species from the present study was from 2 to 4 d and for the other two species from 4 to 6 d. This indicates that for each species there is a particular time interval after which irreparable damage has been done to their metabolism. The critical time period for irreversible physiological damage has been quoted as 3-5 d at 27°C (Storey, 1980), but, as the present study was carried out at 20°C there were individuals from all three species that were capable of recovering from a 4-d exposure. The periods of higher oxygen broke up the continuous exposures into shorter time periods. Environmental conditions are very important considerations when using controlled atmospheres. The temperature chosen for these experiments would be similar to the conditions at the beginning of the U.K. grain storage season. Temperature is the most important factor because as it decreases, there is a significant extension required to the exposure time (Navarro and Calderon, 1980; Storey, 1980). This is always going to be an important consideration for the usage of controlled atmospheres on grain in the U.K. as storage occurs during the winter. Any operation will be adversely affected by changes in weather conditions with the resultant losses in the generated atmosphere. These experiments were carried out with the existing U.K. grain silo complexes in mind and these were not constructed for use with CAs. However, from the data presented here, provided that the loss does not cause the oxygen level to rise above 5% for over 16 h, there is no need to extend the exposure required to ensure 100% mortality of adults of the commonest grain pests, though this may not be the case for all developmental stages or other insect species. Acknowledgements-This work was funded by MAFF Pesticide Safety Division and their support acknowledged. We also thank Miss S. Cooper for her technical assistance.
is gratefully
REFERENCES Abbott W. S. (1925) A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18, 265-267.
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Adler C. S. (1991) Efficacy of controlled atmospheres on ten strains of the granary weevil Sitophilus granarius (L.) from different places of origin. In Proceedings of the 5th International Working Conference on Stored-Product Protection (Edited by Fleurat-Lessard F. and Ducom P.). pp. 727-735. Bordeaux-Blanquefort, France. Anon. (1983) Control of storage pests using modijed atmosphere. Research and development report. Storage pests 1983. Reference book 251(83). HMSO, London. Annis P. C. (1987) Towards rational controlled atmosphere dosage schedules: a review of current knowledge. In Proceedings of the 4th International Working Conference on Stored-Product Protection (Edited by Donahaye E. and Navarro S.), pp. 128-148. Caspit, Jerusalem. Bailey S. W. (1965) Air-tight storage of grain; its effect on insect pests-IV. Rhyropertha dominica (F.) and some other Coleoptera that infest stored grain. Journal of Stored Products Research 1, 25-33. Banks H. J. (1981) Effects of controlled atmosphere storage on grain quality: a review. Food Technology in Australia 33, 335-340. Banks H. J., Annis P. C. and Rigby G. R. (1991) Controlled atmosphere storage techniques for grain: the known and the future. In Proceedings of the 5th International Working Conference on Stored-Product Protection (Edited by Fleurat-Lessard F. and Ducom P.), pp. 695-708. Bordeaux-Blanquefort, France. Calderon M. and Navarro S. (1980) Synergistic effect of carbon dioxide and oxygen mixtures on two grain insect pests. In Controlled Atmosphere Storage of Grains (Edited by Shejbal J.), pp. 79-84. Elsevier, Amsterdam. Cox P. D. and Parish W. E. (1991) Effects of refuge content and food availability on the refuge-seeking behaviour in Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae). Journal of Stored Products Research 27, 135-139. Freeman J. A. (1952) Laemophloeus spp. as major pests of stored grain. Plant Pathology 1, 6976. Jay E. G. (1984) Imperfections in our current knowledge of insect biology as related to their response to controlled atmospheres. In Controlled and Fumigation in Grain Storages. Proceedings of an international Symposium on Practical Aspects of Controlled and Fumigation in Grain Storages, Perth, Australia (Edited by Ripp B. E. et al.), pp. 493-508. Elsevier, Amsterdam. Jay E. G., Arbogast R. T. and Pearman G. C. Jr. (1971) Relative humidity: its importance in the control of stored product insects with modified atmospheric gas concentrations. Journal of Stored Products Research 6, 325-329. Kawamoto H., Sinha R. N. and Muir W. E. (1989) Effect of temperature on adult survival and potential fecundity of the Rusty Grain Beetle Cryptolestes ferrugineus. Applied Entomology and Zoology 24, 418-423. Kirk R. E. (1968) Experimental Design: Procedures for the Behaoioural Scientist. Wadsworth Publishing Co. Inc., Belmont, CA.. Krishnamurthy T. S., Spratt E. C. and Bell C. H. (1986) The toxicity of carbon dioxide to adult beetles in low oxygen atmospheres. Journal of Stored Products Research 22, 145-151. Lindgren D. L. and Vincent L. E. (1970) Effect of atmospheric gases alone or in combination on the mortality of granary and rice weevils. Journal of Economic Entomology 63, 19261929. Muggleton J., Llewellin J. A. and Prickett A. J. (1991) Insecticide resistance in populations of Gryzaephilus surinamensis and Cryptolestes ferrugineus from grain stores in the United Kingdom. In Proceedings of the 5th International Working Conference on Stored-Product Protection (Edited by Fleurat-Lessard F. and Ducom P.), pp. 695-708. Bordeaux-Blanquefort, France. Parker R. E. (1979) Introductory Statistics for Biology. Edward Arnold, London. Rameshbabu M., Jayas D. S. and White N. D. G. (1991) Mortality of Cryptolestes ferrugineus (Stephens) adults and eggs in elevated carbon dioxide and depleted oxygen atmospheres. Journal of Stored Products Research 27, 163-170. Solomon M. E. (1951) The control of humidity with potassium hydroxide, sulphuric acid, and other solutions. Bulletin of Entomological Research 42, 543-554. Solomon M. E. and Adamson B. E. (1956) The powers of survival of storage and domestic pests under winter conditions in Britain. Bulletin of Entomological Research 46, 31 l-355. Storey C. L. (1980) Mortality of various stored product insects in low oxygen atmospheres produced by an exothermic inert atmosphere generator. In Controlled Atmosphere Storage of Grains (Edited by Shejbal J.), pp. 85-92. Elsevier, Amsterdam.
J. storedProd. Res. Vol. 32, No. 3, pp. 187-194. 1996 Crown Copyright 0 1996 Published by Elsevier Science Ltd Printed in Great Britain 0022-474X/96 $15.00 + 0.00 SOO22-474X(%)OOO29-X
The Effect on the Mortality of Adult Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae), Sitophilus granarius (L.) (Coleoptera: Curculionidae) and Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) of Interrupting Low Oxygen Exposures with Periods of Elevated Oxygen SIMON T. CONYERS* and CHRISTOPHER
H. BELL
Central Science Laboratory, Ministry of Agriculture, Fisheries and Food, London Road, Slough, Berks SL3 7HJ, U.K. (Accepted 1 July 1996)
Abstract-A pbospbine-resistant (R) and a laboratory-susceptible (S) strain of Cryptolestes ferrugineus (Stephens) and malathion-resistant (R) and a laboratory-susceptible (S) strain of Sitopkilus granarius (L.) and Oryzaephilus surinumensis (L.) were exposed as adults to a controlled atmosphere generated from mixing nitrogen and compressed air to give an oxygen content of 1%. These exposures all took place at 20°C and 50% r.b. They lasted for 4, 6 and 8 d for C. ferrugineus and S. granarius, and for 2,4 and 6 d for 0. surinamensis. Simultaneously, similar exposures were carried out wbicb included up to three periods at bigber oxygen levels (3, 5 or 10%) during the course of tbe exposure to 1% oxygen. These periods with increased oxygen concentratious lasted for 8 (only C. ferrugineus) or 16 b (all tbree species). There was no significant difference (P > 0.05) in the response of the two strains of C. ferrugineus to the atmosphere so furtber analysis was carried out using pooled data of both straius. However, there was a significant difference (Z’ < 0.05) between tbe strains of the other two species. Tbis ditierence was not tbe same for botb species as tbe R strain was more tolerant than the S strain of S. granarius whereas the reverse was true of 0. surinamensis. Adult C. ferrugikeus of either strain appeared the most tolerant to the 1% oxygen atmosphere, followed by the R strain of S. granarius, tben its S straiu and 6nally tbe S and tben tbe R strain of 0. surinamensis. Mortality of all species increased as tbe exposure period increased. Comporisous of mortalities between the continuous 1% exposures and interruptions witb bigber oxygen levels produced similar patterns of response for the three species, even when tbeir tolerances to tke atmosphere differed. Tbere was increased survival as tke level of oxygen during the interruption increased and tbis increase was accentuate-dwith an increase in duration of tbe intemtption. However, the signiiicauce of this increased survival decreases as tbe exposure period iucrea$es. The use of an exposure period, which produced over 99% mortality for tbe uninterrupted 1% oxygen exposure, meant that tkere was no increase in survival even after three 16-b ir@rruptions of 5% oxygen during such an exposure. Crown Copyright 0 1996 Published by Eisevier Science Ltd Key worris-stored
grain beetles, oxygen, nitrogen, mortality, time
*Author for correspondence. 187
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INTRODUCTION
Controlled atmosphere (CA) fumigation of bulk grain is a technology that has been available for over two decades (Banks et al., 1991) and is becoming a more attractive alternative to the traditional methods of grain storage. Although it costs more than the most widely used grain fumigant, phosphine (Annis, 1987), consumer demand has been moving towards pesticide-free produce. This market force may be the most important factor responsible for bringing CA storage to the forefront of the grain protection market. CA has an important role as an element in a new integrated control initiative (Banks et al., 1991) where it would be employed in conjunction with systems for drying and cooling the grain. In addition to effective control of arthropod pests, it also prevents mould growth, preserves grain quality and maintains the germination energy of the grain (Banks, 1981). The constraints on CA usage have always been the costs of gas provision and of the sealing of storage structures (Jay, 1984; Annis, 1987). The former has now been lessened with the development of systems for on-site generation, thus removing the need for transportation of gas. On-site generators, which include hydrocarbon burners, membrane separation units and pressure-swing adsorption systems, have a high initial cost. This can be offset against the low energy requirements for the gas production although some of these systems are still large consumers of energy and therefore costly to operate. It is important to find ways of lessening these costs particularly as insects require long exposures to the atmospheres to be controlled (Banks et al., 1991). This is especially true at low temperatures which prevail during the U.K. grain storage season. The main factor responsible for the loss of CAs from grain stores is the weather, especially on existing silo complexes which were not designed as far as sealing is concerned for use with CAs. High winds, rapid overnight drops in temperature and fluctuating atmospheric pressure may severely deplete the atmosphere in grain silos and thus raise the oxygen level. This may not be detected for some time and even with a higher gas application flow rate would take several hours to rectify. It is important to know whether it is necessary to compensate for this loss with an extension of exposure time. This project set out to determine the effect of interruptions of increased oxygen on the mortality of adult Cryptolestes ferrugineus, S. granarius and 0. surinamensis during exposures to 1% oxygen in a nitrogen atmosphere. These species are the major beetle pests found in grain in the U.K. (Muggleton et al., 1991). They all have the ability to overwinter in the British climate (Solomon and Adamson, 1956) without diapause (Kawamoto et al., 1989). C. ferrugineus, like 0. surinumensis, is classed as a secondary grain pest, being incapable of attacking whole grain (Freeman, 1952), but modern conveying methods result in sufficient grain damage for infestations to occur. These represent a serious threat to the maintenance of insect-free high quality grain (Kawamoto et al., 1989). Once established, C. ferrugineus in particular remains in grain stores on residues of grain and dust that accumulate in the fabric of the building (Cox and Parish, 1991).
MATERIALS
AND METHODS
Insects C. ferrugineus. Adults, from a laboratory phosphine-susceptible and a phosphine-resistant strain, were produced from separate cultures using a food mixture of 98 g of heat-sterilised rolled oats, 49 g of wheat flour and 8 g of yeast placed in a 1 kg glass jar. Two jars were prepared for each strain every two weeks. These were each set up with 150 adults and were kept in a culture room at 30°C and 70% r.h. The original adults were removed after 3 weeks. The start of emergence was noted and 3 weeks later the new adults were removed and combined from the two jars. They were then placed in a new jar with the same quantity of medium for a further 3 weeks before they were tested. Two days prior to testing, the adults were removed from the cultures. Fifty adults were placed in a crystallising dish (50 mm diameter x 30 mm), 30 dishes for each strain, containing a spoonful of culture food and topped by a filter paper identifying each dish. These were then covered with a piece of gauze which was held in place with elastic bands. They were then placed in a room at a temperature of 25°C and 70% r.h. for 24 h and finally moved to the exposure room at 20°C and 70% r.h., 24 h before the start of the exposures (4, 6 or 8 d).
Insect mortality in interrupted low oxygen atmospheres
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S. granarius. A laboratory malathion-susceptible and a malathion-resistant strain were used for this species. A similar culture regime was used as for C. ferrugineus. However, the food medium was 320 g of heat-sterilised whole wheat grains and the culture jars were kept at 25°C and 70% r.h. Each crystallising dish used for this species had fluon (polytetrafluoroethylene) wiped around the rim to prevent escape. The dishes were moved from the culturing conditions to the exposure room 24 h before the start of the exposures (4, 6 or 8 d). Similar strains and a similar culture regime was used for this species as for 0. surinamensis. S. granarius. The food used for this species was 150 g of heat-sterilised rolled oats and the exposures tested were 2, 4 and 6 d.
Test procedure
The adults were exposed to the atmosphere in glass desiccators (280 mm diameter x 240 mm height). The generated atmosphere of 1% oxygen was produced by passing nitrogen and compressed air through a gas blender (850 Series, Signal Instrument Co. Ltd, Camberley, Surrey, U.K.). The output was assessed for accuracy by using a paramagnetic oxygen analyser (Model no. 570A, Servomex Ltd, Crowborough, Sussex, U.K.). The humidity of the gas chosen for the experiments was 50% r.h., a level likely to increase the efficacy of low oxygen atmospheres. This was achieved by passing the gas stream on route to the desiccators over an aqueous solution of potassium hydroxide (Solomon, 1951) contained in a litre bottle. There was one bottle for each exposure chamber. The humidity was verified with the aid of a dew point meter (Model no. DP680, Protimeter Ltd, Marlow, Bucks, U.K.) after the gas left the exposure chamber. Insects were exposed for periods of 2, 4, 6 or 8 d. Running concurrently were experiments in which the insects were exposed for similar periods but, additionally, these were subjected to one, two or three periods of 8- or 16-h duration at a higher level of oxygen, either 3, 5 or 10%. The schedule for the regimes is shown in Fig. 1. These higher levels were achieved with the use of another gas blender (850 Series,
Fig. 1. Outline of the regime used to test the effect of increases in oxygen to 3, 5 or 10% for 8 or 16 h on mortality from exposure to a 1% oxygen atmosphere: (above) C. ferrugineus and S. grunurius; (below) 0. surinamensis. In both, black area indicates 1% oxygen; and grey area indicates 3, 5 or 10% oxygen for 8 or 16 h.
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S. T. Conyers and C. H. Bell
Signal Instrument Co. Ltd, Camberley, Surrey, U.K.). ‘T’ ball valves were used to switch the gas stream from one source to the other. At the start of the exposures, the desiccators were purged with a flow rate of 350 ml/min which was reduced to 100 ml/min for the duration of the exposure once the oxygen level had dropped to 1%. The same flow rates were used after alteration of the gas mixture to increase the oxygen level for the interruption in the 1% oxygen exposure, and again to restore the 1% level at the end of the period. The 8-h interruption periods began at 0900 and finished at 1700 on the day shown (Fig. 1) whereas the 16-h periods began at 1700 on that day and finished at 0900 on the next day. There were five replicate experiments (1500 insects used / experiment) which were run consecutively for each of the three elevated oxygen levels. A different culture of each strain of insect was used for each experiment. All of the seven exposure regimes within each experiment had three dishes from each strain which left nine from each strain to act as untreated controls. Once the exposures were completed, all the dishes were moved back to the original culture conditions. A count was made of the mortality after 2 weeks. A correction was made to the mortality figures from the exposures using the mortality found in the controls (Abbott, 1925). The corrected mortality figures were then transformed by angular sine to normalise their distribution (Parker, 1979). The computer program used radians and these figures were converted into degrees to give a broader distribution. The data for each replicate at the different oxygen levels were then compared to test for any significant differences which would prevent their grouping. The data were then combined and a general linear model (GLM) (Minitab Inc., State College, PA, U.S.A.) was used to test for significant differences between the two strains, the oxygen regimes and the exposure time periods. For this test the 8-d exposures with two and three periods of higher oxygen were grouped together to give a balanced model. If there was no difference between the two strains used for each species then their data were pooled for further analysis. The means were then tested separately using a one-way analysis of variance with the separation of the mean values for the 8-d exposures with two and three periods of higher oxygen. Any significant differences (P < 0.05) were assessed using Tukey’s HSD Test (Kirk, 1968) after the means had been converted back to percentages. There was a further GLM analysis carried out to assess the difference between the 8- and the 16-h periods used for C. ferrugineus.
RESULTS C. ferrugineus The results from the GLM analysis of the effects of the 8-h interruptions showed that there was no significant difference in a comparison between the two strains (F = 0.4, P > 0.05). However there were significant differences between the four oxygen regimes (F = 47.7, P < 0.05) and between the three exposure periods (F = 437.0, P < 0.05). The same responses were observed in the results from 16-h interruptions with no significant difference between the strains (F = 0.8, P > 0.05) but significant differences due to oxygen regime (F = 81.9, P < 0.05) and exposure length (F = 658.4, P < 0.05). These results alone give no indication of the extent to which each factor contributed to the differences observed. The subsequent one-way analysis of variance provided the treatment means for each strain (Table 1) and the pooled mean mortality for both strains showed the effect of the different exposures on the mortality of the adult beetle. For all the different oxygen regimes used, as expected, mortality increased as the exposure time increased. When looking at the results from the experiment on 8-h periods of interruption, the increase in mortality was only significant (P < 0.05) for each increase in exposure time for the continuous 1% oxygen exposure. For the other three interruption regimes at different oxygen levels, the increase in exposure time from 6 to 8 d was not significant (P > 0.05) and there was no significant difference (P > 0.05) between the 8-d exposures with two or three periods of increased oxygen. When the effect of the different oxygen levels was considered, there was a significant decrease in mortality (P < 0.05) with 3% oxygen interruptions for only the 4- and 6-d exposures. The increase to 5% oxygen gave another significant decrease (P < 0.05) for both 4 and 8 d. The increase from 5 to 10% oxygen did not achieve any further significant decrease (P > 0.05) in mortality. For the 16-h interruption period experiment, the results for the increase in exposure time were
Insect mortality in interrupted low oxygen atmospheres
191
Table 1. Mean percentage mortalities ( f SE., n = 5) for a phosphine-resistant (R) and a laboratory-susceptible (S) strain of adult C. firrugineus after exposure to a 1% oxygen atmosphere and to increases in oxygen (to 3, 5 or 10%) for 8 or 16-h periods during exposure to 1% oxygen
8 Hour
Atmosphere 1% + 3%
I%+5%
1% + 10%
81.8(1.4) 80.6(1.4) 98.7(0.4) ’ ’ 97.8(0.5)
65.4(1.7) ’ 59.9(1.8) 97.5(0.6) 24 99.1(0.3)
64.0(1.8) ’ 57.9(1.8) 88.3tl.2) 4 87.6( 1.2)
99.9(0.1) 2 4 99.9(0.1) 98.9(0.4) * 4 92.1(1.0)
92.6(1.0) ’ 4 96.1(0.7) 94.0(0.9) ’ 4 94.1(0.9)
89.1(1.1) 3’ 91.1(1.0) 87.8(1.2) 3’ 92.7(1.0)
1%+3%
1% + 5%
1% + 10%
38.4( 1.8)2 35.0(1.7) 73.6(1.6) ’ 72.0( 1.6) 92.2(1.0) 2 3 4 88.2(1.2) __. 89.7(1.1) 1 ’ 4 91.6(1.0)
31.8(1.7) 31.9(1.7) 75.1(1.6) 70.1(1.7) 85.7(1.3) 88.6(1.2) 91.3(1.0) 91.1(1.0)
1%
Strain R S R S
Days 4 6
R S R S
8 8
68.3(1.0) 70.2( 1.O) 92.7(0.6) 92.9(0.6)
Periods’ 2 2
2
99.5tO.2) 2 99.5(0.2) 99.5(0.2) 2 99.5(0.2)
3
16 Hour
Atmosphere 1% Strain R S R S R S R S
Days 4 6 8 8
65.9(1.0) 65.8(1.0) 84.9(0.8) 84.9(0.8) 94.5(0.5) * 95.1(0.5) 94.5(0.5) * 95.1(0.5)
Periods ’ 2 2 2 3
2 * ’4 “ 4
35.3(1.8) 35.2(1.7) 77.5(1.5) 69.4(1.7) 88.1(1.2) 90.0(1.1) 84.1(1.3) 89.0(1.1)
2 ? ’* 34
‘Number of periods with increased oxygen.
* and 3 Means with the same number in the same row which are not significantly different (P > 0.05). 4Means in the same column which are not significantly different (P > 0.05).
the same except that the increase in mortality from 6 to 8 d was significant for all treatments (P < 0.05). For the interruptions, the addition of 3% oxygen produced a significant decrease (P < 0.05) for the 4- and 6-d exposures, but the increases in mortality with further increases in oxygen to 5 and 10% were not significant (P > 0.05). A direct comparison between the mean mortalities due to these increased levels of oxygen for the 8- and 16-h periods (Table 2) shows a trend of decreasing difference (F = 223.2, P < 0.05) as exposure length increases. It becomes almost negligible and was not significant (P > 0.05) during the 8-d exposure with the three periods at 5 and 10% oxygen. S. granarius The results from the GLM analysis gave a significant difference for all factors being assessed: strain (F = 486.6, P < 0.05), oxygen regime (F = 128.2, P -c 0.05) and exposure length (F = 2143.0, P -c 0.05). The means are shown in Table 3 and the strain difference is significant (P < 0.05) for similar oxygen conditions for all 4- and 6-d exposure lengths, but only for the 10% oxygen interruption for both two and three periods for the 8 d. Taking the R strain alone it showed significant increases (P < 0.05) in mortality for each increase in exposure length, but there was no significant difference between the two and three periods of higher oxygen during the 8-d exposure. The trend with the increasing oxygen showed significant decreases (P < 0.05) between 1 and 3% interruptions of oxygen except for the 8-d with three periods. A further significant decrease (P < 0.05) occurred with the increase in oxygen from 3 to 5% oxygen was seen in the 4- and 6-d
Table 2. A comparison of the mean percentage mortalities from the 8- and 16-h periods of increased oxygen for C. ferrugineus (mean of the R and S strains) Exposure regime 4
Davs r
%02
Hours 8 16
1+3
1+5
81.2’ 36.7
62.7’ 31.8
6 1+10
1+3
1+5
98.3’ 72.8
98.4’ 72.6
8 1+10 87.9’ 73.6
1+3
1+5
99.9’ 94.5’ 90.3 87.2
1+10 90.2 89.1
1+3x3 96.2’ 90.7
1+5x3
1+10x3
94. I 91.2
90.4 86.6
‘Significant difference (P < 0.05) between mean mortalities due to difference in time period for increased oxygen.
S. T. Conyers and C. H. Bell
192
Table 3. Mean percentage mortalities ( + SE., n = 5) for a phosphine-resistant (R) and a laboratory-susceptible (S) strain of adult S. granarius and 0. surinamensis after exposure to a 1% oxygen atmosphere and to increases in oxygen (to 3, 5 or 10%) for 8- or 16-h periods during exposure to 1% oxygen S. granarius
Atmosphere 1%
Days 4
6 8 8
1% + 5% __. _.
1% + 10%
13.2(I.2)2 34.5(1.7) 72.2( 1.6)2 98.2(0.5) ’ 6 97.5(0.6) 3 5 100 ’ 6 95.4(0.8) 3 ’ 99.9(0.1) 3 6
2.5(0.6)’ ’ 15.7(1.3) 56.7( 1.8)3 ’ 98.2(0.5) 3 6 94.5(0.8) 3 4 5 99.8(0.1) 3 6 93.2(0.9)j 4 5 99.9(0.1) 3 b
3.9(0.2)3 9.7(1.1) 57.9(1.8)’ J. 94.9(0.8) ’ 6 t39&1” 4 J
1%+3%
1%+5%
1% + 10%
90.8(1.1)’ * 62.8(1.8) 3 99.9(0.1) 3 5 97.2(0.6) 4 3 ’ 100 J 3 100 36
87.4(1.2)3 * 61.2(1.8) 3 99.9(0.1)3 2 5 94.3(0.8) ‘ 100 ’ 5
59.0(1.8)2 27.6(1.6) 96.2(0.7)’ * ’ 83.1(1.4) 100 ’ 5 99.9(0.1) 3
Periods ’
Strain R S R S R S R S
1% + 3%
24.1(0.9)’ 59.5(1.0) 88.1(0.7)’ 99.8(0.1) 3 6 99.8(0.1)’ 100 ’ 6 99.8(0.1)3 100 3 6
2 2 2 3
0, surinamensis
Atmosphere 1%
Days 2 4 6
86.4( 1.3)’ 4 5 99.7(0.2) ’ 6
Periods ’
Strain R S R S R S
96.2(0.4)’ ’ 77.9(0.9) 100 3 ’ 36 99W
1 2 2
!OO’
‘Number of periods with increased oxygen. 2Pairs of R and S mean mortalities which are significantly different (P < 0.05). 3 and * Means with the same number in the same row which are not signi6cantly different (P > 0.05). IR means in the same column which are not significantly different (P > 0.05). 6S means in the same colutnn which are not significantly different (P > 0.05).
exposures but with the further increase in oxygen to lo%, any decrease in the mortality was not significant (P > 0.05). The S strain was less tolerant of these atmospheres. Therefore significant decreases (P < 0.05) in mortality due to exposure length were only seen in the increase from 4 to 6 d. Any significant decrease (P < 0.05) in mortality due to the increases in oxygen during the interruptions were noted only for the 4-d exposures and occurred for each increase in oxygen. 0. surinamensis The GLM analysis gave a significant difference for all factors being assessed: strain (F = 220.9, P < 0.05), oxygen regime (F = 140.6, P < 0.05) and exposure length (F = 952.0, P < 0.05). The means are shown in Table 3 and the strain difference was only significant (P -z 0.05) for each of the 2-d comparisons and for the 5 and 10% interruptions for the 4-d. In contrast to S. granaries, the S strain was more tolerant of these atmospheres. There was a significant increase in mortality of both strains due to increased exposure time between 2 and 4 d except for the R strain with the exposure in 1% oxygen. When the interruptions of higher oxygen are considered, there is a similar pattern for both strains for the 2-d exposure. There is a significant decrease (P < 0.05) in mortality with the addition of 3% oxygen and in the change from 5 to 10% oxygen. However after this there is no significant difference for any of the mortalities from the 4- and 6-d exposures for the R strain. The S strain only exhibited a significant decrease (P < 0.05) for the 4-d exposure with the addition of 10% oxygen. DISCUSSION
The efficacy of a 1% oxygen atmosphere was in general only slightly affected by fluctuations of up to 16 h duration in which oxygen levels rose to 10% during the exposure. From the results, complete mortahty of adult C. ferrugineus would be expected from an exposure slightly longer than 8 d in a 1% oxygen atmosphere at 20°C and 50% r.h. Krishnamurthy et al. (1986) obtained complete mortality after only 7 d at the same temperature but used a higher humidity. This could be seen as a discrepancy as the lower humidity of the present results should have increased the mortality due to its desiccant effect (Jay et al., 1971). However carbon dioxide ( > 10%) was
Insect mortality in interrupted low oxygen atmospheres
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included in the gas mixture used by Krishnamurthy et al. (1986). This level of carbon dioxide has been shown to enhance the mortality in low oxygen atmospheres and may have a synergistic effect (Calderon and Navarro, 1980) rather than the direct toxic effect of higher carbon dioxide levels as shown by Rameshbabu ef al. (1991). The exact conditions of a test are very important as shown by the attainment of complete mortality of adults of this beetle in a 100% nitrogen atmosphere in less than 2 d at a higher temperature (Anon., 1983). It is important in a control situation to lower the oxygen content as far as possible. However, though this is desirable, the results from this experiment have shown that an exposure of 8 d with three periods of 16 h where the oxygen level reaches 5% will not give a significant decrease (P > 0.05) in mortality in this the most tolerant of the three species tested here, but that there is a significant decrease (P < 0.05) in mortality when 10% oxygen is used. This higher level of survival at the 10% level, but not the 5%, agrees with the tests of Bailey (1965) where adults of C. ferrugineus were not adversely affected by low oxygen atmospheres in air-tight storage until the level dropped to 5% and below. For S. granarius, Lindgren and Vincent (1970) produced 95% mortality in adults after 5.3 d with 100% nitrogen at 70°F and 60-70% r.h. In the present experiment 95% mortality for either the R or the S strain was attained between 6 and 8 d. The absence of oxygen in the former study and the slightly higher temperature than the present experiment would be expected to shorten the time taken to reach 95% mortality though the higher humidity used would have had the opposite affect. Adler (1991) produced an average of 99% mortality from 10 strains in 6.6 + 1.6 d (20°C and 75%) with 99% nitrogen and 1% oxygen. This result is comparable to those obtained in the present study. However, with the difference in the tolerance of strains to modified atmospheres apparent in the present experiment it becomes difficult to make direct comparisons with other works involving single strains. This difference in the tolerance of different strains to modified atmospheres has already been noted by Adler (1991) who found significant differences in the responses of S. granarius from different countries. The results for 0. surinumensis agree with results from Krishnamurthy et al. (1986) (20°C and 70% r.h.) although, as for C. ferrugineus, this experiment did incorporate 10% carbon dioxide which may have increased the mortality. Ninety-seven per cent mortality was achieved by 4 d with complete control by 6 d. The biggest increase in mortality for this species from the present study was from 2 to 4 d and for the other two species from 4 to 6 d. This indicates that for each species there is a particular time interval after which irreparable damage has been done to their metabolism. The critical time period for irreversible physiological damage has been quoted as 3-5 d at 27°C (Storey, 1980), but, as the present study was carried out at 20°C there were individuals from all three species that were capable of recovering from a 4-d exposure. The periods of higher oxygen broke up the continuous exposures into shorter time periods. Environmental conditions are very important considerations when using controlled atmospheres. The temperature chosen for these experiments would be similar to the conditions at the beginning of the U.K. grain storage season. Temperature is the most important factor because as it decreases, there is a significant extension required to the exposure time (Navarro and Calderon, 1980; Storey, 1980). This is always going to be an important consideration for the usage of controlled atmospheres on grain in the U.K. as storage occurs during the winter. Any operation will be adversely affected by changes in weather conditions with the resultant losses in the generated atmosphere. These experiments were carried out with the existing U.K. grain silo complexes in mind and these were not constructed for use with CAs. However, from the data presented here, provided that the loss does not cause the oxygen level to rise above 5% for over 16 h, there is no need to extend the exposure required to ensure 100% mortality of adults of the commonest grain pests, though this may not be the case for all developmental stages or other insect species. Acknowledgements-This work was funded by MAFF Pesticide Safety Division and their support acknowledged. We also thank Miss S. Cooper for her technical assistance.
is gratefully
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