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Weight loss and mortality of three life stages of Tribolium castaneum (Herbst) when exposed to four modified atmospheres
WEIGHT LOSS AND MORTALITY OF THREE LIFE STAGES OF ~~~~U~~~~ ~~~~~~~~~ (HERBST) WHEN EXPOSED TO FOUR MODIFIED ATMOSPHERES EDWARD
G. JAY’ and WILFRED
CUFF’
‘Stored-Product Insects Research and Development Laboratory, Agricultural Research Service, USDA, Savannah. Georgia. U.S.A. and “CSIRO. Division of Computing Research, Canberra, A.C.T., Australia
Abstract-Larvae. pupae, and adults of Triboliltm eastuneuin (Herbst) were exposed to atmospheres containing high nitrogen (NJ) or carbon dioxide (COz) con~ntrations at about 50:/i r.h. and 27 C for periods up to 72 hr. Overall, 99”,, Nz caused greater mortality in adults than did 58”, CO2 while 58:, CO* was more effective against pupae. The difference in mortality to larvae exposed to the two atmospheres was not significant, though 99”h N2 produced greater mortalities by 48 hr. Also, the differences in mortality for larvae and pupae exposed to atmospheres of 58% CO, and 97q, CO2 were not significant though 977” COz caused a significantly greater mortality in adults than did 58% COZ. Both mortality and weight loss of all life stages tested were small when the insects were exposed to an atmosphere of 91% N,, but weight toss was generally smalf and mortality was Iarge for those insects exposed to 977; CO,. When overall weight loss was compared for those insects exposed to 587; COz and 99”>;N2, larval weight loss was greater for those exposed to the COZ atmosphere; pupal weight loss was not significantly different between the two atmospheres; adult weight loss was greater for those exposed to the NZ atmosphere.
IN THE PAST few years,considerable effort has been placed on the development of control measures for stored-product insects that would replace or supplement conventional fumigants and insecticides. A portion of this effort has been directed toward controlling these insects by the modification of storage atmospheres through the introduction of carbon dioxide (CC+) or nitrogen (N,). Field tests have been conducted in the USA. by JAY et ul. (1970) and by JAY and FEARMAN (1973), in Australia by BANKSand ANNIS(1977), and in Italy by SHEJBALet al. (1973). Also, many investigators have conducted laboratory studies in this area, and YOSHIDA(1975), in a review paper, lists 78 references on the effects of N2 atmospheres on insects, the majority being stored-product pests. BAILEYand BANKS (1975) published a paper which reviews the effects of CO2 atmospheres on storedproduct insects. The relative humidity (r.h.) of an atmosphere was found by JAY et af. (1971) in laboratory studies to have an important effect on insect mortality. This mortality was attributed to water loss and the eventual dessication that occurs when insects open spiracles in response to either low oxygen (0,) when exposed to high N2 atmospheres or to high CO2 concentrations. Also, laboratory studies by NAVARRO and CALDERON (1973, 1974) on the effect of r.h. on weight loss and emergence of ~~~~s~~~~~~r~~~u(Walker) pupae showed that even in atmospheres of high r.h. adult emergence is inhibited when the pupae are exposed to high CO2 atmospheres. However, the effect of r-h. has not been verified under field conditions. For example, when JAY and PEARMAN(1973) purged the atmosphere in a large silo containing maize with 7-9% r.h. gaseous CO2 they found that the r.h. of interstitial areas of the grain did not change. The r.h. apparently adjusted to differences in interstitial r.h. by the corn giving up moisture. Thus, in the field, changes in r.h. probably play a smaller role in control than atmospheric con~ntration and temperature in actual situations where modified atmospheres are used to purge storage facilities for insect control.
118
EDWARD G. JAY and
WILFRED CUFF
The tests reported here were conducted at about 507; atmospheric r.h., the r.h. of wheat at about 11.5% moisture (GAY, 1946), and were designed to investigate the weight loss and mortality of three life stages of Tribolium castaneum (Herbst) when they were exposed to four concentrations of modified atmospheres for five time intervals. The gas concentrations used were essentially those that one would expect to attain and maintain in a purge of a relatively gas-tight commercial-type storage facility: 1. the concentration about 58% CO2 is near the 60% concentration recommended by JAY (1971) (the O2 and N2 concentrations approximate those found when purging grain with this concentration of CO,); 2. the 97% CO2 concentration is what one could hope to attain and maintain when the existing atmosphere in an upright concrete silo is purged with a loo”,, CO? concentration (JAY and PEARMAN,unpublished field studies); and 3. the 999; N, concentration was used in field tests by BANKSand ANNIS (1977). The 97% N, concentration was included for comparison purposes with the 99% N2 concentration. MATERIALS AND METHODS The test insects were the Savannah laboratory strain of T. castanrun~, which were reared at 26.7”C + 1 (range) and 60 + 5% (range) r.h. in a 1: 1 mixture of white wheat b flour and maize meal containing 5% brewer’s yeast. When exposed to modified atmospheres, the larvae were 16-20 days old, the pupae were 3-6 days old and the adults were 2-14 days old. Representatives from each of the three life stages of T. ca~taneum were exposed to a commercially prepared ternary mixture of COZ, 02, and Nz, to a binary mixture of O2 and Nz, to 1OO’YCOZ, to 100% Nz, and to breathing quality air. Prior to exposure, the insects were weighed in groups of 25 in a gelatin capsule. They were then placed in wire mesh cages and immediately transferred to the exposure chambers, which had previously been flushed with compressed air. After exposure for various periods of time, the caged insects were placed in a desiccator maintained at 50% r.h. through the use of a saturated salt solution. They were reweighed 30 min to 1 hr after removal from the atmospheres. All weighings were done on a Sartorius type 2642 analytical balance*. After the final weighing, the insects were transferred to petri dishes containing a small amount of rearing media and held at 26.7”C f 1 and 60 k 5”/dr.h. Seven days later they were examined for mortality. Those that did not move when subjected to vibration of the petri dishes were considered dead. The exposure chambers were similar to those described by HAREINand PRESS(1968) and consisted of 2.8 liter glass jars that were partly submerged in laboratory baths filled with water, which was maintained at 26.7”C + 1. The jars were closed with metal screwtop lids fitted with 23 and 2.5 cm lengths of 0.6 cm dia. copper tubing that were used as gas inlet and gas exit tubes, respectively. The lids were also fitted with a neoprene stopper that supported a calibrated dial thermometer and a second stopper for the insertion of a humidity sensor. During exposure the cages containing the insects were suspended in the chamber from a 5-cm length of steel wire hung from the underside of the neoprene stopper holding the thermometer. The gas mixtures were released from the cylinders through two-stage regulators and flowed through a micrometering valve and flowmeter into gas washing bottles that contained the glycerin-water mixture that adjusted the r.h. of the gases to c. SOS?;.Flow rates of 2OOcc/min were used for the first hour and 30 cc/min for the balance of the exposure periods. The r.h. was monitored with an electric hygrometer (model 15-2001 humidity indicator and narrow range humidity sensors, Hygrodynamics, Inc.), and the temperature was recorded daily by using the dial thermometers. A Fisher-Hamilton model 29 gas partitioner equipped with dual columns was used for daily analysis of ternary mixtures. A Vidar model 6300 digital integrator was used to measure the areas under the peaks. A Beckman model E-2 oxygen analyzer was used for daily analysis of binary mixtures. * Mention of a commercial product in this paper does not constitute an endorsement of this product by the USDA.
Triholium
in modified
atmospheres
119
The intended atmospheric concentrations in the chambers are given in Table 1. Small leaks in the system reduced the concentrations of lOOo/, N2 and 100% CO2 to the indicated levels. These changes were expected to occur prior to the initiation of the test. Nine cages containing 25 insects of each life stage were exposed to each of these gas concentrations for 6, 16, 24, 48 or 72 hr. The data were analyzed-using Tektronix software on a 4051 terminal-as a series of two-way Analysis of Variance (AOV) tests, over five levels of each of an ‘atmosphere’ and ‘time’ factor and with nine observations per cell. The data for larvae, pupae and adults were analyzed separately for each of two variables: weight, loss and number of dead insects. Within cell replication was equal in all larval and pupal tests (9), but in the 97% N2 level of each of the two adult tests only six replicates were observed in the 16, 24, 48, and 72 hr levels of the time factor. The analyses were thus conducted as non-orthogonal two-way AOV’s (STEELand TORRIE,1960). .4 priori ‘linear comparisons’ (SOKALand ROHLF, 1969) were set out as follows: air vs other concentrations or levels, 99% N2 vs 587” CO*, 99”:; Nz vs 97% COZ, 99”; N2 vs 97”,, NJ. and 58”;) CO2 vs 979~ COz. RESULTS The actual atmospheric concentrations in the exposure chambers are given in Table 1. These concentrations varied marginally from the desired concentrations due to variations in the concentrations in the mixed gases or to small leaks around the lids of the exposure chamber. Actual r.h. (+ SD) in the chambers averaged 49.0 f 2.6, 51.8 + 3.8 and 53.0 f 2.5”,, for the larval, pupal and adult experiments, respectively. The results from the series of AOV’s are summarized in Table 2. Both atmospheric concentrations and time exerted statistically significant effects on both weight loss and deaths for all life stages. As evidenced by the composition of the atmosphere and time mean squares (MSS), time had a larger effect on weight loss than atmosphere for larvae and pupae but with adults the two factors had about the same effect. Conversely, atmosphere had a larger effect on mortality than did time for all life stages. The interaction MSS are much smaller than either the atmosphere or time MSS, with significant interactions being declared only for larval weight loss and mortality. The analyses in Table 3 were conducted on log transformed data. This transformation was suggested by an apparent positive correlation between mean and variance (see Twl_k- I. I\TFNDED AND MEASURED ATMOSPHERIC CONCENTRATIONS THREE LIFE STAGES OF 7: CUStUnt'Ultl WERE EXPOSED
Stage
Actual
Intended atmospheric concentration (“,>I
atmospheric
concentration
TO WHICH
(“,)
@angel CO?
02
N*
Larvae
Air* 99 Nz 97 N, 97 co2 58 CO,
0.03 0.0 0.0 96.497.6 57.658.2
20.8-20.9 0% I.2 2.9-3.1 0.2- 0.5 9.4 9.6
78.1-78.2 98.8-99.2 96.9-97.1 2.2 ~3.1 32.4 32.8
Pupae
Air* 99 N, 97 N2 97 co2 5x co2
0.03 0.0 0.0 97C97.3 59.3-59.5
20.9. 21.1 0.7-0.9 2.8-3.1 O.hO.6 8.4 8.8
77.9-78.1 99.1-99.3 96.9.-97.2 2.1 -2.5 31.9-32.1
Adults
Air* 99 N, 97 NZ 97 co2 58 co,
0.03 0.0 0.0 97.5-98.4 60.4-60.9
21.0 21.1 0.6 0.7 2.3 3.1 0.3 -0.5 83 8.4
77.9-78.0 99.3-99.4 96.9-97.7 [email protected] 30.8-3 1.2
* Balance
of composition
is argon
and rare gases.
120
TABLE~.MEAN
G. JAY and WILFRED CUFF
EDWARD
SQUARESFROMTWO-WAYANALYSESOFVARIANCE(LOGGED THE 5 TIME EXPOSURES
Weight
DATA)
loss
Source
Larvae
Pupae
Atmosphere Time Interaction
5.44 19.18 0.11
10.07 28.13 0.18NS
OF THE FIVE ATMOSPHERES OVER
Mortality Adults 24.52 24.22 o.OQ5NS
Larvae
Pupae
503.81 225.33 37.69
50.40 24.14 5.03NS
Adults* 732.56 484.75 8.88NS
All atmosphere and time MSS are significant. but only the larval weight loss @ = 0.035) and mortality (P < 0.01) interactions are significant. * This non-orthogonal Analysis of Variance was done by the method of weighed squares of means (STEEL and TORRIE, 1960).This method gives a better testfor the main effects when there is significant interaction than given by the method of fitting constants. By the latter method the atmosphere MS was 699.14 while the time MS was 504.19 and the Interaction MS was 77.65 (P < 0.01).
Tables 4-6). A plotting of the untransformed data showed the interactions to be a result of relatively slow rates of increase over time in mortality and weight loss for the air and 97% N2 relative to the other concentrations. Besides stabilizing the variance estimates, this transformation made subsequent analyses less complex to interpret by nullifying many of the significant interactions found on analyses of all sets of untransformed data. A lack of significant interactions for many of the tests allowed a priori linear comparisons among the atmosphere factor that apply across all times. Where the comparisons make misleading declarations due to significant interactions, mention will be made to this effect. The general nature of the statistical differences among the atmospheric factors is examined in Table 3; the dynamic aspects of these differences can be clarified by a study of the mean values ( f SE) for larvae (Table 4), pupae (Table 5) and adults (Table 6). Consistently large differences were found between air and the remaining concentrations over all life stages for both variables (Table 3). Weight loss and number dead were lower under an air atmosphere than under any other atmosphere, except for pupal mortality at 6 and 24 hr (Table 5) and adult mortality at 6 and 72 hr (Table 6). In these cases, however, the size of the SE’s suggest that none of the pairs were significantly different.
TABLE 3. F VALUESFOR a
COMPARISONS AMONG DIFFERENT MODIFIED ATMOSPHERES FOR WEIGHT LOSS AND MORTALITY OF THREE LIFE STAGES OF 7: CUStUneUWI*
priori
Weight Comparison
description
Air vs other levels
Mortality
loss
Larvae
Pupae
Adults
Larvae
Pupae
Adults
243’
220H
7818
137H
34”
171R
6’
9A
997; N, vs 58’/ 0 CO z
6’
llA
< INS’
997; N2 vs 97% CO1
29A
26A
20*
4”SZ
99% N, vs 97% N,
39A
56A
528A
7A’
58% CO2 vs 97% CO2
62A
14*
2NS
1”s
p
2’S 3NS’
INS
’ The means of Table 4 suggest that a significant difference develops by 48 hr. with 99”,, N, showing the greatest effect. * P = 0.06. ’ A strong interaction exists between 997; N2 and 97”,, N2. Table 4 shows that at 6 hr 97”” Nz z 99”” N2 but by 48 hr 997; N2 p 977: N2. a The means of Table 5 suggest that a significant difference develops by 48 hr, with 99”; N2 showing the greatest effect. * Where both FO.OS [1200] for orthogonal analyses and F,,,, [1187] for non-orthogonal analyses equal 3.9. An A (first of the two comparisons) or B (second of the two comparisons) beside a value indicates which of the two contrasting treatments caused the greatest weight loss or highest mortality unless the contrast was nonsignificant (NS).
in modified
7”riholium
TABLE 4. LARVAE: MEAN
WEIGHT
LOSS AND
MEAN
atmospheres
NUMBER
121
OF DEATHS FOR EACH GROUP
OF 25 7: ~u~tun4unl
EXPOSED TO MODIFIED ATMOSPHERES (+SE)
Time (hr) Atmosphere t”,,)
16
6
Weight Au 99 Nz 97 N, 97 CO2 5x CO*
27 44 30 32 49
* f & i *
5 2 3 2 4
46 94 71 77 122
f 6 + 4 k6 i 4 f 5 Mortality
Air 99 Nz 97 N2 91 CO, 58 co2
0.0 0.4 2.0 1.9 1.9
&- 0
0.4 1.9 1.9 2.9 1.8
* + f It *
* + f *
0.2 0.8 1.0 1.0
72
48
24 loss (IO-“g) 54 * 4 117 c 7 99 1 7 91 *4 149 & 4 (number out of 25)
0.2 0.5 0.4 0.6 0.5
0.2 2.8 1.7 22.7 3.9
* + f f *
0.1 0.6 0.6 0.8 0.5
66 190 118 135 201
f f + f &
8 13 8 I 11
157 258 184 185 247
k f * i: i_
30 24 13 IO IO
0.1 22.0 1.9 22.3 12.0
f 0.1 If- 1.7 + 0.6 + 1.4 + 2.0
1.3 25.0 3.2 ‘4.7 20.1
* & f f f
0.7 0 I.5 0.2 1.5
The relationships among the modified atmospheres are most easily understood by examination of the means (Tables 4-6). On an average, the two largest weight losses resulted from 99% Nz (first for pupae and adults) and 587; CO* (first for larvae). The 97% CO2 caused the third largest weight loss of all life stages, but this loss was inconsistent for larvae (Table 4). Except for air, the 97% N2 level caused the lowest weight losses overall except for the inconsistent loss for larvae at 24 hr. These same tendencies are not shown by the mortality data. Reversals (Table 4) in rank order occurred between the 58% CO2 and the 97J?/,CO2 levels for larvae, with 589, CO2 producing on an average the largest weight loss and the third largest mortality. A similar reversal occurred between 99% Nz and 58”,/, CO2 for pupae, with 99”/1 Nz producing the largest weight loss and the second largest mortality. When the trends discussed in the two previous paragraphs were interpreted in terms of the statistical comparisons of Table 3, it was found that the 58:; CO2 level had the greatest effect on larval weight loss, followed (significantly) by 99% N, and (significantly) by 97’:; COz. In terms of larval mortality, the 97% C02, 99% Nz, and 58% CO2 all produced statistically equivalent rates of mortality, although the data suggest that by 48 hr 99% N2 produced significantly greater mortality than 58% COz. Also, 99’?/, N, produced a significantly larger weight loss and mortality rate than 97% NZ. For pupae, 997; N2 and 58% COz produced the largest, but statistically equivalent, effects on weight loss followed significantly by 97Pg COz. The 99% Nz produced a TABLE 5. PUPAE:MEAN
WEIGHT LOSSAND
MEAN NUMBER OF DEATHSFOR EACH GROUP OF 25 7: castancum TO MODIFIED ATMOSPHERES(+ SE)
EXPOSED
Time (hr) Atmosphere (“J
6
16
24
Weight Air 99 Nz 97 N2 97 co, 58 CO2
8+1 16 k lo+ 13 + 17 f
3 1 2 2
23 53 35 36 48
_+ 4 i 4 * 2 f 3 + 3 Mortality
Air 99 N2 97 NZ 97 co* 58 CO1
3.2 3.1 4.1 6.1 8.9
k f * k k
0.9 1.0 1.3 1.4 1.0
3.4 5.3 3.9 7.1 11.3
* f k + *
0.9 0.4 0.8 0.6 1.7
loss (lo-“ 17 84 34 45 57 (number 3.8 6.5 3.5 7.8 17.3
* k i + +
48
72
g) 3 22 2 5 5
27 112 52 70 96
+ f f * +
3 4 5 3 4
2.6 21.8 4.4 14.2 22.4
& + + f +
0.7 1.6 1.1 1.3 0.6
41 155 86 100 128
+4 i 13 * 9 k 6 i_ 4
out of 25) & + + + +
0.9 1.2 0.9 1.0 1.0
4.4 25.0 6.3 25.0 24.7
+ + k k +
0.8 0 0.7 0 0.2
122
G.JAY and WILFRED CUFF
EDWARD
TABLE 6. ADULTS: MEAN WEIGHT LOSS AND MEAN NUMBER OF DEATHS FOR EACH GROUP OF 25 7: castunrum EXPOSED TO MODIFIED ATMOSPHERES(f SE) Time (hr) Atmosphere ("0)
6
24
16
12
48
Weight loss(IO-“g) Air 99 NZ 91 N, 97 co1 58 COZ
15 56 16 44 48
* f + f +
3 2 2 1 2
Air 99 N2 97 NZ 91 co1 58 CO2
0.1 0.0 0.1 0.0 0.2
+ f f * +
0.1 0 0.1 0 0.2
21 142 38 102 109
* f f + f
3 3 5 2 5
29 168 49 130 131
* & * + *
2 2 4 4 3
55 312 80 229 249
& k i k f
3 4 6 4 2
0.2 25.0 0.5 25.0 23.9
i f * + It
0.1 0 0.3 0 0.4
73 352 102 315 340
f * + f &
6 3 3 2 5
0.5 25.0 0.2 25.0 24.7
+ t f f *
0.3 0 0.2 0 0.2
Mortality (number out of 25) 0.0 24.8 0.1 21.0 1.1
* + + k f
0 0.1 0.1 0.8 0.6
0.2 * 0.1 24.8 _+ 0.1 1.3 * 1.3 25.0 + 0 7 1 f 0.6
significantly larger weight loss than 977; Nz. On the other hand, 587; CO* produced the largest mortality, followed (significantly) by 99% Nz. The 97% CO2 level was next but was not significantly different from either 58% CO2 or 99% N1. The 97% N2 level was next; it was not declared significantly different from 99% Nz, though a difference appears to arise at 48 hr (Table 5). For adults, the largest effect on weight loss resulted from 99% NZ, followed significantly by 58% CO2 and 97% CO*, which are statistically equivalent. The 99% N, produced a significantly larger weight loss than 97% NZ. The largest adult mortality was caused equivalently by 99% N2 and 977: COz, followed (significantly) by 58’4 COz and (significantly) by 97’0 Nz. DISCUSSION The mortality data presented in Table 3 show that of the three atmospheres previously proved practical in laboratory and field tests (99% N2, 97% COz, and 58% COJ, 999,; N2 was more effective than 58% CO2 against adults of 7: castaneum, and 58’4 CO2 was more effective against the pupae. The differences between the effects of these two atmospheres on larvae were not significant, though 99”; N2 produced larger mortalities by 48 hr. When 58% CO1 was compared with 97% CO*, the difference in mortality of the two atmospheres to larvae and pupae was not significant, but 97% CO* caused a greater mortality in the adults than did the 58:/i COz. The tests were terminated at 72 hr. It is likely from Tables 4-6 that all three atmospheres would have caused complete mortality in an additional exposure of about 24 hr. For example, Table 4 shows that one atmosphere gave complete larval mortality in 72 hr while another gave 24.7 mean larval deaths in that time. Table 5 shows that two of the three atmospheres gave complete pupal mortality in 72 hr. Table 6 shows that two of the three atmospheres gave complete mortality after only 48 hr. These data compare favorably with those of JAY and PEARMAN (1971) who observed 1000/dmortality of ?: castaneum in 2 days when adults were exposed to a gas mixture containing 99.77; N2 and 0.37; O2 at 26.7”C. Also, since ALINIAZEE and LINDGREN (1970) showed that T. castaneum eggs did not hatch after a 5 day exposure to an atmosphere containing 20% CO2 and 16.504 O2 (balance N2), it is likely that a 3 day exposure to the higher CO1 concentration and at the temperature used in the tests would also prevent egg hatch. Plainly, all of these atmospheres tested here could be considered usable in field situations against the three life stages of T. castaneum tested here. The mortality that resulted from the 97% N, atmosphere was generally similar to that described in other papers (i.e., low). However, in our tests, weight loss was much lower in the 97:/, N2 concentration than in the 99% N2 concentration, which suggest that water
7‘riholium in modified
atmospheres
I!3
loss is a major cause of death in high Nz atmospheres. When the insects were exposed to 97”~ COz, the weight loss was lower but the mortality was higher (except for pupae) than when the insects were exposed to 58% COz. This suggests that high COz concentrations had an effect similar to that described by NAVARROand CALDERON(1974) for E. c’autella pupae treated at high r.h.‘s. Weight loss in 7: castaneum adults increased after 24 hr exposures to 979; CO2 even though all of the insects were dead (Table 6), which is attributed to dessication continuing after death. The factors influencing weight loss vs mortality that operated for 97”,, CO, and 97O,, N, seem to eliminate them from direct comparison with each other or with the other two modified atmospheres. When weight loss is compared with mortality for 58”,, CO* and 99”,, N2 (Table 3), larval weight loss was greater for those exposed to 58”;; COz, but mortality was about the same for the two atmospheres. On the other hand, pupal weight loss was not significantly different for the two atmospheres, but mortality was higher for those exposed to 58s; COz. Adult weight loss and mortality were significantly higher for the group exposed to 990,:,N,. This comparison between these two concentrations seem to show that even within the same species the effects on weight loss and mortality vary for the different life stages. The data also demonstrate that research should be conducted to establish the effects of modified atmospheres on different life stages of each species of economically important stored-grain insects before we can determine what concentration of what modified atmosphere will be effective against insects actually infesting stored commodities. The final decision about the use of CO1 or N, in practical field situations will probably depend on economics, which will in turn depend on cost per unit (tons or m3) at the distribution point plus the costs of transportation and application. For example. it may be more reasonable, cost-wise, to provide a 6- to 8-day treatment with one atmosphere than a 4-day treatment with another. Careful consideration of all these interrelated factors will allow for the determination of the least costly treatment.
.l~,~,lo~l,/~,~yc,rt~~~~~ -The authors wish to acknowledge the assistance of G. C. PEARMAN. Jr.. of USDA ARS. Stored-Product Insects Research and Development Laboratory. Savannah. GA. U.S.A.. in the actual conduct of the laboratory phases of this research.
REFERENCES ALINIAZEE. M. T. Tenebrlonidae) BAIIXY. S. W. and iny.\ ,$the Fir.st pp. 362 374. BANKS, H. J. and CSIRO
Division
and LINDGREN, D. L. (1970) Egg hatch of Tribolium confusum and 7: c’usfuneurn (Cnleoptera: in different carbon dioxide and nitrogen atmospheres. J. econ. Em. 63, 101~1012. BANKS, H. J. (1975) The use of controlled atmospheres for the storage of grain. In ProcrrdIntcrntrtionul
Working
Co@zrmce
on Storrd-Product
Entomolo~g~. Strrunncd~. G.4. (‘.S. A. 1914.
ANNIS, P. C. (1977) Suggested oj Entomology
procedures for controlled atmosphere storage of dry grain. 13. 23 pp. on the moisture content-relative humidity equilibria of wheat
Tech. Puper No.
GAY. F. J. (1946) The effect of temperature Commun. Sci. Ind. Rr.\. J. 19, 187-189. HAREIN. P. K. and PRESS. A. F. (1968) Mortality of stored-peanut insects exposed to mixtures of atmospheric gases at various temperatures. J. .\toret/ Prod. Rrs. 4. 77-82. JAY. E. G. (1971) Suggested conditions and procedures for using carbon dioxide to control insects m grain storage facilities. USDA .4RS 51-46, September 1971. 6 pp. JA\. E. G. and PEARMAN. G. C.. Jr (1971) Susceptibility of two species of Triboliwtl (Coleoptera: Tenebrionidae) to alterations of atmospheric gas concentrations. J. srorrtl Prod. Res. 7, 181 -186. .I). E. G. and PEARMAN. G. C., Jr. (1973) Carbon dioxide for control of an insect infestation in stored corn (maize). J. \torrd Prod. Rrs. 9, 25-29. JOY. E. G., ARRIXAST, R. T. and PEARMAN, G. C., Jr. (1971) Relative humidity: Its importance in the control of stored-product insects with modified atmospheric gas concentrations. J. stored Prod. Rrs. 6, 325-329. JAY. E. G., REDI.INGER, L. M. and LAUDANI, H. (1970) The application and distribution of carbon dioxide in a peanut (groundnut) silo for insect control. J. stored Prod. Rex 6, 247-254. NAVARRO. S. and CALDERON, M. (1973) Carbon dioxide and relative humidity: Interrelated factors affecting the 10~s of water and mortality of Ephrstiu caurella (Wlk.) (Lepidoptera: Phycitidae). Israel J. Ent. 8, 143- 152. NAVARKO.S. and CALDERON, M. (1974) Exposure of Ephestia cuutella (Wlk.) pupae to carbon dioxide concentrations at dimerent relative humidities: The etlect on adult emergence and loss of weight. J. storrd Prod. Rr\. IO, 237 241.
124
EDWARDG. JAY and WILFREDCUFF
SHWBAL,J., TONOLO,A. and CARERI,G. (1973) Conservation of wheat in silos and under nitrogen. Ann. Technol. agric. 22, 773-785. SOKAL,R. R. and ROHLF.F. J. (1969) Biometry. Freeman, San Francisco, U.S.A. 776 pp. STEEL,R. G. D. and TORRIE,J. H. (1960) Principles und Procedures o~Sruristic.s. McGraw Hill. New York, 481 PP. YOSHIDA,T. (1975) Nitrogen atmosphere and pest insects. J. Fd Hry. Sot., Jupon 16, l-l 1.
G. JAY’ and WILFRED
CUFF’
‘Stored-Product Insects Research and Development Laboratory, Agricultural Research Service, USDA, Savannah. Georgia. U.S.A. and “CSIRO. Division of Computing Research, Canberra, A.C.T., Australia
Abstract-Larvae. pupae, and adults of Triboliltm eastuneuin (Herbst) were exposed to atmospheres containing high nitrogen (NJ) or carbon dioxide (COz) con~ntrations at about 50:/i r.h. and 27 C for periods up to 72 hr. Overall, 99”,, Nz caused greater mortality in adults than did 58”, CO2 while 58:, CO* was more effective against pupae. The difference in mortality to larvae exposed to the two atmospheres was not significant, though 99”h N2 produced greater mortalities by 48 hr. Also, the differences in mortality for larvae and pupae exposed to atmospheres of 58% CO, and 97q, CO2 were not significant though 977” COz caused a significantly greater mortality in adults than did 58% COZ. Both mortality and weight loss of all life stages tested were small when the insects were exposed to an atmosphere of 91% N,, but weight toss was generally smalf and mortality was Iarge for those insects exposed to 977; CO,. When overall weight loss was compared for those insects exposed to 587; COz and 99”>;N2, larval weight loss was greater for those exposed to the COZ atmosphere; pupal weight loss was not significantly different between the two atmospheres; adult weight loss was greater for those exposed to the NZ atmosphere.
IN THE PAST few years,considerable effort has been placed on the development of control measures for stored-product insects that would replace or supplement conventional fumigants and insecticides. A portion of this effort has been directed toward controlling these insects by the modification of storage atmospheres through the introduction of carbon dioxide (CC+) or nitrogen (N,). Field tests have been conducted in the USA. by JAY et ul. (1970) and by JAY and FEARMAN (1973), in Australia by BANKSand ANNIS(1977), and in Italy by SHEJBALet al. (1973). Also, many investigators have conducted laboratory studies in this area, and YOSHIDA(1975), in a review paper, lists 78 references on the effects of N2 atmospheres on insects, the majority being stored-product pests. BAILEYand BANKS (1975) published a paper which reviews the effects of CO2 atmospheres on storedproduct insects. The relative humidity (r.h.) of an atmosphere was found by JAY et af. (1971) in laboratory studies to have an important effect on insect mortality. This mortality was attributed to water loss and the eventual dessication that occurs when insects open spiracles in response to either low oxygen (0,) when exposed to high N2 atmospheres or to high CO2 concentrations. Also, laboratory studies by NAVARRO and CALDERON (1973, 1974) on the effect of r.h. on weight loss and emergence of ~~~~s~~~~~~r~~~u(Walker) pupae showed that even in atmospheres of high r.h. adult emergence is inhibited when the pupae are exposed to high CO2 atmospheres. However, the effect of r-h. has not been verified under field conditions. For example, when JAY and PEARMAN(1973) purged the atmosphere in a large silo containing maize with 7-9% r.h. gaseous CO2 they found that the r.h. of interstitial areas of the grain did not change. The r.h. apparently adjusted to differences in interstitial r.h. by the corn giving up moisture. Thus, in the field, changes in r.h. probably play a smaller role in control than atmospheric con~ntration and temperature in actual situations where modified atmospheres are used to purge storage facilities for insect control.
118
EDWARD G. JAY and
WILFRED CUFF
The tests reported here were conducted at about 507; atmospheric r.h., the r.h. of wheat at about 11.5% moisture (GAY, 1946), and were designed to investigate the weight loss and mortality of three life stages of Tribolium castaneum (Herbst) when they were exposed to four concentrations of modified atmospheres for five time intervals. The gas concentrations used were essentially those that one would expect to attain and maintain in a purge of a relatively gas-tight commercial-type storage facility: 1. the concentration about 58% CO2 is near the 60% concentration recommended by JAY (1971) (the O2 and N2 concentrations approximate those found when purging grain with this concentration of CO,); 2. the 97% CO2 concentration is what one could hope to attain and maintain when the existing atmosphere in an upright concrete silo is purged with a loo”,, CO? concentration (JAY and PEARMAN,unpublished field studies); and 3. the 999; N, concentration was used in field tests by BANKSand ANNIS (1977). The 97% N, concentration was included for comparison purposes with the 99% N2 concentration. MATERIALS AND METHODS The test insects were the Savannah laboratory strain of T. castanrun~, which were reared at 26.7”C + 1 (range) and 60 + 5% (range) r.h. in a 1: 1 mixture of white wheat b flour and maize meal containing 5% brewer’s yeast. When exposed to modified atmospheres, the larvae were 16-20 days old, the pupae were 3-6 days old and the adults were 2-14 days old. Representatives from each of the three life stages of T. ca~taneum were exposed to a commercially prepared ternary mixture of COZ, 02, and Nz, to a binary mixture of O2 and Nz, to 1OO’YCOZ, to 100% Nz, and to breathing quality air. Prior to exposure, the insects were weighed in groups of 25 in a gelatin capsule. They were then placed in wire mesh cages and immediately transferred to the exposure chambers, which had previously been flushed with compressed air. After exposure for various periods of time, the caged insects were placed in a desiccator maintained at 50% r.h. through the use of a saturated salt solution. They were reweighed 30 min to 1 hr after removal from the atmospheres. All weighings were done on a Sartorius type 2642 analytical balance*. After the final weighing, the insects were transferred to petri dishes containing a small amount of rearing media and held at 26.7”C f 1 and 60 k 5”/dr.h. Seven days later they were examined for mortality. Those that did not move when subjected to vibration of the petri dishes were considered dead. The exposure chambers were similar to those described by HAREINand PRESS(1968) and consisted of 2.8 liter glass jars that were partly submerged in laboratory baths filled with water, which was maintained at 26.7”C + 1. The jars were closed with metal screwtop lids fitted with 23 and 2.5 cm lengths of 0.6 cm dia. copper tubing that were used as gas inlet and gas exit tubes, respectively. The lids were also fitted with a neoprene stopper that supported a calibrated dial thermometer and a second stopper for the insertion of a humidity sensor. During exposure the cages containing the insects were suspended in the chamber from a 5-cm length of steel wire hung from the underside of the neoprene stopper holding the thermometer. The gas mixtures were released from the cylinders through two-stage regulators and flowed through a micrometering valve and flowmeter into gas washing bottles that contained the glycerin-water mixture that adjusted the r.h. of the gases to c. SOS?;.Flow rates of 2OOcc/min were used for the first hour and 30 cc/min for the balance of the exposure periods. The r.h. was monitored with an electric hygrometer (model 15-2001 humidity indicator and narrow range humidity sensors, Hygrodynamics, Inc.), and the temperature was recorded daily by using the dial thermometers. A Fisher-Hamilton model 29 gas partitioner equipped with dual columns was used for daily analysis of ternary mixtures. A Vidar model 6300 digital integrator was used to measure the areas under the peaks. A Beckman model E-2 oxygen analyzer was used for daily analysis of binary mixtures. * Mention of a commercial product in this paper does not constitute an endorsement of this product by the USDA.
Triholium
in modified
atmospheres
119
The intended atmospheric concentrations in the chambers are given in Table 1. Small leaks in the system reduced the concentrations of lOOo/, N2 and 100% CO2 to the indicated levels. These changes were expected to occur prior to the initiation of the test. Nine cages containing 25 insects of each life stage were exposed to each of these gas concentrations for 6, 16, 24, 48 or 72 hr. The data were analyzed-using Tektronix software on a 4051 terminal-as a series of two-way Analysis of Variance (AOV) tests, over five levels of each of an ‘atmosphere’ and ‘time’ factor and with nine observations per cell. The data for larvae, pupae and adults were analyzed separately for each of two variables: weight, loss and number of dead insects. Within cell replication was equal in all larval and pupal tests (9), but in the 97% N2 level of each of the two adult tests only six replicates were observed in the 16, 24, 48, and 72 hr levels of the time factor. The analyses were thus conducted as non-orthogonal two-way AOV’s (STEELand TORRIE,1960). .4 priori ‘linear comparisons’ (SOKALand ROHLF, 1969) were set out as follows: air vs other concentrations or levels, 99% N2 vs 587” CO*, 99”:; Nz vs 97% COZ, 99”; N2 vs 97”,, NJ. and 58”;) CO2 vs 979~ COz. RESULTS The actual atmospheric concentrations in the exposure chambers are given in Table 1. These concentrations varied marginally from the desired concentrations due to variations in the concentrations in the mixed gases or to small leaks around the lids of the exposure chamber. Actual r.h. (+ SD) in the chambers averaged 49.0 f 2.6, 51.8 + 3.8 and 53.0 f 2.5”,, for the larval, pupal and adult experiments, respectively. The results from the series of AOV’s are summarized in Table 2. Both atmospheric concentrations and time exerted statistically significant effects on both weight loss and deaths for all life stages. As evidenced by the composition of the atmosphere and time mean squares (MSS), time had a larger effect on weight loss than atmosphere for larvae and pupae but with adults the two factors had about the same effect. Conversely, atmosphere had a larger effect on mortality than did time for all life stages. The interaction MSS are much smaller than either the atmosphere or time MSS, with significant interactions being declared only for larval weight loss and mortality. The analyses in Table 3 were conducted on log transformed data. This transformation was suggested by an apparent positive correlation between mean and variance (see Twl_k- I. I\TFNDED AND MEASURED ATMOSPHERIC CONCENTRATIONS THREE LIFE STAGES OF 7: CUStUnt'Ultl WERE EXPOSED
Stage
Actual
Intended atmospheric concentration (“,>I
atmospheric
concentration
TO WHICH
(“,)
@angel CO?
02
N*
Larvae
Air* 99 Nz 97 N, 97 co2 58 CO,
0.03 0.0 0.0 96.497.6 57.658.2
20.8-20.9 0% I.2 2.9-3.1 0.2- 0.5 9.4 9.6
78.1-78.2 98.8-99.2 96.9-97.1 2.2 ~3.1 32.4 32.8
Pupae
Air* 99 N, 97 N2 97 co2 5x co2
0.03 0.0 0.0 97C97.3 59.3-59.5
20.9. 21.1 0.7-0.9 2.8-3.1 O.hO.6 8.4 8.8
77.9-78.1 99.1-99.3 96.9.-97.2 2.1 -2.5 31.9-32.1
Adults
Air* 99 N, 97 NZ 97 co2 58 co,
0.03 0.0 0.0 97.5-98.4 60.4-60.9
21.0 21.1 0.6 0.7 2.3 3.1 0.3 -0.5 83 8.4
77.9-78.0 99.3-99.4 96.9-97.7 [email protected] 30.8-3 1.2
* Balance
of composition
is argon
and rare gases.
120
TABLE~.MEAN
G. JAY and WILFRED CUFF
EDWARD
SQUARESFROMTWO-WAYANALYSESOFVARIANCE(LOGGED THE 5 TIME EXPOSURES
Weight
DATA)
loss
Source
Larvae
Pupae
Atmosphere Time Interaction
5.44 19.18 0.11
10.07 28.13 0.18NS
OF THE FIVE ATMOSPHERES OVER
Mortality Adults 24.52 24.22 o.OQ5NS
Larvae
Pupae
503.81 225.33 37.69
50.40 24.14 5.03NS
Adults* 732.56 484.75 8.88NS
All atmosphere and time MSS are significant. but only the larval weight loss @ = 0.035) and mortality (P < 0.01) interactions are significant. * This non-orthogonal Analysis of Variance was done by the method of weighed squares of means (STEEL and TORRIE, 1960).This method gives a better testfor the main effects when there is significant interaction than given by the method of fitting constants. By the latter method the atmosphere MS was 699.14 while the time MS was 504.19 and the Interaction MS was 77.65 (P < 0.01).
Tables 4-6). A plotting of the untransformed data showed the interactions to be a result of relatively slow rates of increase over time in mortality and weight loss for the air and 97% N2 relative to the other concentrations. Besides stabilizing the variance estimates, this transformation made subsequent analyses less complex to interpret by nullifying many of the significant interactions found on analyses of all sets of untransformed data. A lack of significant interactions for many of the tests allowed a priori linear comparisons among the atmosphere factor that apply across all times. Where the comparisons make misleading declarations due to significant interactions, mention will be made to this effect. The general nature of the statistical differences among the atmospheric factors is examined in Table 3; the dynamic aspects of these differences can be clarified by a study of the mean values ( f SE) for larvae (Table 4), pupae (Table 5) and adults (Table 6). Consistently large differences were found between air and the remaining concentrations over all life stages for both variables (Table 3). Weight loss and number dead were lower under an air atmosphere than under any other atmosphere, except for pupal mortality at 6 and 24 hr (Table 5) and adult mortality at 6 and 72 hr (Table 6). In these cases, however, the size of the SE’s suggest that none of the pairs were significantly different.
TABLE 3. F VALUESFOR a
COMPARISONS AMONG DIFFERENT MODIFIED ATMOSPHERES FOR WEIGHT LOSS AND MORTALITY OF THREE LIFE STAGES OF 7: CUStUneUWI*
priori
Weight Comparison
description
Air vs other levels
Mortality
loss
Larvae
Pupae
Adults
Larvae
Pupae
Adults
243’
220H
7818
137H
34”
171R
6’
9A
997; N, vs 58’/ 0 CO z
6’
llA
< INS’
997; N2 vs 97% CO1
29A
26A
20*
4”SZ
99% N, vs 97% N,
39A
56A
528A
7A’
58% CO2 vs 97% CO2
62A
14*
2NS
1”s
p
2’S 3NS’
INS
’ The means of Table 4 suggest that a significant difference develops by 48 hr. with 99”,, N, showing the greatest effect. * P = 0.06. ’ A strong interaction exists between 997; N2 and 97”,, N2. Table 4 shows that at 6 hr 97”” Nz z 99”” N2 but by 48 hr 997; N2 p 977: N2. a The means of Table 5 suggest that a significant difference develops by 48 hr, with 99”; N2 showing the greatest effect. * Where both FO.OS [1200] for orthogonal analyses and F,,,, [1187] for non-orthogonal analyses equal 3.9. An A (first of the two comparisons) or B (second of the two comparisons) beside a value indicates which of the two contrasting treatments caused the greatest weight loss or highest mortality unless the contrast was nonsignificant (NS).
in modified
7”riholium
TABLE 4. LARVAE: MEAN
WEIGHT
LOSS AND
MEAN
atmospheres
NUMBER
121
OF DEATHS FOR EACH GROUP
OF 25 7: ~u~tun4unl
EXPOSED TO MODIFIED ATMOSPHERES (+SE)
Time (hr) Atmosphere t”,,)
16
6
Weight Au 99 Nz 97 N, 97 CO2 5x CO*
27 44 30 32 49
* f & i *
5 2 3 2 4
46 94 71 77 122
f 6 + 4 k6 i 4 f 5 Mortality
Air 99 Nz 97 N2 91 CO, 58 co2
0.0 0.4 2.0 1.9 1.9
&- 0
0.4 1.9 1.9 2.9 1.8
* + f It *
* + f *
0.2 0.8 1.0 1.0
72
48
24 loss (IO-“g) 54 * 4 117 c 7 99 1 7 91 *4 149 & 4 (number out of 25)
0.2 0.5 0.4 0.6 0.5
0.2 2.8 1.7 22.7 3.9
* + f f *
0.1 0.6 0.6 0.8 0.5
66 190 118 135 201
f f + f &
8 13 8 I 11
157 258 184 185 247
k f * i: i_
30 24 13 IO IO
0.1 22.0 1.9 22.3 12.0
f 0.1 If- 1.7 + 0.6 + 1.4 + 2.0
1.3 25.0 3.2 ‘4.7 20.1
* & f f f
0.7 0 I.5 0.2 1.5
The relationships among the modified atmospheres are most easily understood by examination of the means (Tables 4-6). On an average, the two largest weight losses resulted from 99% Nz (first for pupae and adults) and 587; CO* (first for larvae). The 97% CO2 caused the third largest weight loss of all life stages, but this loss was inconsistent for larvae (Table 4). Except for air, the 97% N2 level caused the lowest weight losses overall except for the inconsistent loss for larvae at 24 hr. These same tendencies are not shown by the mortality data. Reversals (Table 4) in rank order occurred between the 58% CO2 and the 97J?/,CO2 levels for larvae, with 589, CO2 producing on an average the largest weight loss and the third largest mortality. A similar reversal occurred between 99% Nz and 58”,/, CO2 for pupae, with 99”/1 Nz producing the largest weight loss and the second largest mortality. When the trends discussed in the two previous paragraphs were interpreted in terms of the statistical comparisons of Table 3, it was found that the 58:; CO2 level had the greatest effect on larval weight loss, followed (significantly) by 99% N, and (significantly) by 97’:; COz. In terms of larval mortality, the 97% C02, 99% Nz, and 58% CO2 all produced statistically equivalent rates of mortality, although the data suggest that by 48 hr 99% N2 produced significantly greater mortality than 58% COz. Also, 99’?/, N, produced a significantly larger weight loss and mortality rate than 97% NZ. For pupae, 997; N2 and 58% COz produced the largest, but statistically equivalent, effects on weight loss followed significantly by 97Pg COz. The 99% Nz produced a TABLE 5. PUPAE:MEAN
WEIGHT LOSSAND
MEAN NUMBER OF DEATHSFOR EACH GROUP OF 25 7: castancum TO MODIFIED ATMOSPHERES(+ SE)
EXPOSED
Time (hr) Atmosphere (“J
6
16
24
Weight Air 99 Nz 97 N2 97 co, 58 CO2
8+1 16 k lo+ 13 + 17 f
3 1 2 2
23 53 35 36 48
_+ 4 i 4 * 2 f 3 + 3 Mortality
Air 99 N2 97 NZ 97 co* 58 CO1
3.2 3.1 4.1 6.1 8.9
k f * k k
0.9 1.0 1.3 1.4 1.0
3.4 5.3 3.9 7.1 11.3
* f k + *
0.9 0.4 0.8 0.6 1.7
loss (lo-“ 17 84 34 45 57 (number 3.8 6.5 3.5 7.8 17.3
* k i + +
48
72
g) 3 22 2 5 5
27 112 52 70 96
+ f f * +
3 4 5 3 4
2.6 21.8 4.4 14.2 22.4
& + + f +
0.7 1.6 1.1 1.3 0.6
41 155 86 100 128
+4 i 13 * 9 k 6 i_ 4
out of 25) & + + + +
0.9 1.2 0.9 1.0 1.0
4.4 25.0 6.3 25.0 24.7
+ + k k +
0.8 0 0.7 0 0.2
122
G.JAY and WILFRED CUFF
EDWARD
TABLE 6. ADULTS: MEAN WEIGHT LOSS AND MEAN NUMBER OF DEATHS FOR EACH GROUP OF 25 7: castunrum EXPOSED TO MODIFIED ATMOSPHERES(f SE) Time (hr) Atmosphere ("0)
6
24
16
12
48
Weight loss(IO-“g) Air 99 NZ 91 N, 97 co1 58 COZ
15 56 16 44 48
* f + f +
3 2 2 1 2
Air 99 N2 97 NZ 91 co1 58 CO2
0.1 0.0 0.1 0.0 0.2
+ f f * +
0.1 0 0.1 0 0.2
21 142 38 102 109
* f f + f
3 3 5 2 5
29 168 49 130 131
* & * + *
2 2 4 4 3
55 312 80 229 249
& k i k f
3 4 6 4 2
0.2 25.0 0.5 25.0 23.9
i f * + It
0.1 0 0.3 0 0.4
73 352 102 315 340
f * + f &
6 3 3 2 5
0.5 25.0 0.2 25.0 24.7
+ t f f *
0.3 0 0.2 0 0.2
Mortality (number out of 25) 0.0 24.8 0.1 21.0 1.1
* + + k f
0 0.1 0.1 0.8 0.6
0.2 * 0.1 24.8 _+ 0.1 1.3 * 1.3 25.0 + 0 7 1 f 0.6
significantly larger weight loss than 977; Nz. On the other hand, 587; CO* produced the largest mortality, followed (significantly) by 99% Nz. The 97% CO2 level was next but was not significantly different from either 58% CO2 or 99% N1. The 97% N2 level was next; it was not declared significantly different from 99% Nz, though a difference appears to arise at 48 hr (Table 5). For adults, the largest effect on weight loss resulted from 99% NZ, followed significantly by 58% CO2 and 97% CO*, which are statistically equivalent. The 99% N, produced a significantly larger weight loss than 97% NZ. The largest adult mortality was caused equivalently by 99% N2 and 977: COz, followed (significantly) by 58’4 COz and (significantly) by 97’0 Nz. DISCUSSION The mortality data presented in Table 3 show that of the three atmospheres previously proved practical in laboratory and field tests (99% N2, 97% COz, and 58% COJ, 999,; N2 was more effective than 58% CO2 against adults of 7: castaneum, and 58’4 CO2 was more effective against the pupae. The differences between the effects of these two atmospheres on larvae were not significant, though 99”; N2 produced larger mortalities by 48 hr. When 58% CO1 was compared with 97% CO*, the difference in mortality of the two atmospheres to larvae and pupae was not significant, but 97% CO* caused a greater mortality in the adults than did the 58:/i COz. The tests were terminated at 72 hr. It is likely from Tables 4-6 that all three atmospheres would have caused complete mortality in an additional exposure of about 24 hr. For example, Table 4 shows that one atmosphere gave complete larval mortality in 72 hr while another gave 24.7 mean larval deaths in that time. Table 5 shows that two of the three atmospheres gave complete pupal mortality in 72 hr. Table 6 shows that two of the three atmospheres gave complete mortality after only 48 hr. These data compare favorably with those of JAY and PEARMAN (1971) who observed 1000/dmortality of ?: castaneum in 2 days when adults were exposed to a gas mixture containing 99.77; N2 and 0.37; O2 at 26.7”C. Also, since ALINIAZEE and LINDGREN (1970) showed that T. castaneum eggs did not hatch after a 5 day exposure to an atmosphere containing 20% CO2 and 16.504 O2 (balance N2), it is likely that a 3 day exposure to the higher CO1 concentration and at the temperature used in the tests would also prevent egg hatch. Plainly, all of these atmospheres tested here could be considered usable in field situations against the three life stages of T. castaneum tested here. The mortality that resulted from the 97% N, atmosphere was generally similar to that described in other papers (i.e., low). However, in our tests, weight loss was much lower in the 97:/, N2 concentration than in the 99% N2 concentration, which suggest that water
7‘riholium in modified
atmospheres
I!3
loss is a major cause of death in high Nz atmospheres. When the insects were exposed to 97”~ COz, the weight loss was lower but the mortality was higher (except for pupae) than when the insects were exposed to 58% COz. This suggests that high COz concentrations had an effect similar to that described by NAVARROand CALDERON(1974) for E. c’autella pupae treated at high r.h.‘s. Weight loss in 7: castaneum adults increased after 24 hr exposures to 979; CO2 even though all of the insects were dead (Table 6), which is attributed to dessication continuing after death. The factors influencing weight loss vs mortality that operated for 97”,, CO, and 97O,, N, seem to eliminate them from direct comparison with each other or with the other two modified atmospheres. When weight loss is compared with mortality for 58”,, CO* and 99”,, N2 (Table 3), larval weight loss was greater for those exposed to 58”;; COz, but mortality was about the same for the two atmospheres. On the other hand, pupal weight loss was not significantly different for the two atmospheres, but mortality was higher for those exposed to 58s; COz. Adult weight loss and mortality were significantly higher for the group exposed to 990,:,N,. This comparison between these two concentrations seem to show that even within the same species the effects on weight loss and mortality vary for the different life stages. The data also demonstrate that research should be conducted to establish the effects of modified atmospheres on different life stages of each species of economically important stored-grain insects before we can determine what concentration of what modified atmosphere will be effective against insects actually infesting stored commodities. The final decision about the use of CO1 or N, in practical field situations will probably depend on economics, which will in turn depend on cost per unit (tons or m3) at the distribution point plus the costs of transportation and application. For example. it may be more reasonable, cost-wise, to provide a 6- to 8-day treatment with one atmosphere than a 4-day treatment with another. Careful consideration of all these interrelated factors will allow for the determination of the least costly treatment.
.l~,~,lo~l,/~,~yc,rt~~~~~ -The authors wish to acknowledge the assistance of G. C. PEARMAN. Jr.. of USDA ARS. Stored-Product Insects Research and Development Laboratory. Savannah. GA. U.S.A.. in the actual conduct of the laboratory phases of this research.
REFERENCES ALINIAZEE. M. T. Tenebrlonidae) BAIIXY. S. W. and iny.\ ,$the Fir.st pp. 362 374. BANKS, H. J. and CSIRO
Division
and LINDGREN, D. L. (1970) Egg hatch of Tribolium confusum and 7: c’usfuneurn (Cnleoptera: in different carbon dioxide and nitrogen atmospheres. J. econ. Em. 63, 101~1012. BANKS, H. J. (1975) The use of controlled atmospheres for the storage of grain. In ProcrrdIntcrntrtionul
Working
Co@zrmce
on Storrd-Product
Entomolo~g~. Strrunncd~. G.4. (‘.S. A. 1914.
ANNIS, P. C. (1977) Suggested oj Entomology
procedures for controlled atmosphere storage of dry grain. 13. 23 pp. on the moisture content-relative humidity equilibria of wheat
Tech. Puper No.
GAY. F. J. (1946) The effect of temperature Commun. Sci. Ind. Rr.\. J. 19, 187-189. HAREIN. P. K. and PRESS. A. F. (1968) Mortality of stored-peanut insects exposed to mixtures of atmospheric gases at various temperatures. J. .\toret/ Prod. Rrs. 4. 77-82. JAY. E. G. (1971) Suggested conditions and procedures for using carbon dioxide to control insects m grain storage facilities. USDA .4RS 51-46, September 1971. 6 pp. JA\. E. G. and PEARMAN. G. C.. Jr (1971) Susceptibility of two species of Triboliwtl (Coleoptera: Tenebrionidae) to alterations of atmospheric gas concentrations. J. srorrtl Prod. Res. 7, 181 -186. .I). E. G. and PEARMAN. G. C., Jr. (1973) Carbon dioxide for control of an insect infestation in stored corn (maize). J. \torrd Prod. Rrs. 9, 25-29. JOY. E. G., ARRIXAST, R. T. and PEARMAN, G. C., Jr. (1971) Relative humidity: Its importance in the control of stored-product insects with modified atmospheric gas concentrations. J. stored Prod. Rrs. 6, 325-329. JAY. E. G., REDI.INGER, L. M. and LAUDANI, H. (1970) The application and distribution of carbon dioxide in a peanut (groundnut) silo for insect control. J. stored Prod. Rex 6, 247-254. NAVARRO. S. and CALDERON, M. (1973) Carbon dioxide and relative humidity: Interrelated factors affecting the 10~s of water and mortality of Ephrstiu caurella (Wlk.) (Lepidoptera: Phycitidae). Israel J. Ent. 8, 143- 152. NAVARKO.S. and CALDERON, M. (1974) Exposure of Ephestia cuutella (Wlk.) pupae to carbon dioxide concentrations at dimerent relative humidities: The etlect on adult emergence and loss of weight. J. storrd Prod. Rr\. IO, 237 241.
124
EDWARDG. JAY and WILFREDCUFF
SHWBAL,J., TONOLO,A. and CARERI,G. (1973) Conservation of wheat in silos and under nitrogen. Ann. Technol. agric. 22, 773-785. SOKAL,R. R. and ROHLF.F. J. (1969) Biometry. Freeman, San Francisco, U.S.A. 776 pp. STEEL,R. G. D. and TORRIE,J. H. (1960) Principles und Procedures o~Sruristic.s. McGraw Hill. New York, 481 PP. YOSHIDA,T. (1975) Nitrogen atmosphere and pest insects. J. Fd Hry. Sot., Jupon 16, l-l 1.