Directory UMM :Journals:Journal of Stored Products Research:Vol 36.Issue3.Jul2000:
Journal of Stored Products Research 36 (2000) 297±308
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Resistance and some enzyme activities in Liposcelis
bostrychophila Badonnel (Psocoptera: Liposcelididae) in
relation to carbon dioxide enriched atmospheres
Jin-Jun Wang, Zhi-Mo Zhao, James H. Tsai*
Fort Lauderdale Research and Education Center, IFAS, University of Florida, 3205 College Avenue, Fort Lauderdale,
FL 33314-7799, USA
Accepted 3 November 1999
Abstract
Two populations (S-1 and S-2) of the psocid, Liposcelis bostrychophila Badonnel were exposed to
carbon dioxide enriched atmospheres. Carbon dioxide resistance developed at steady rates in these two
populations during this study period. Selection with 35 and 55% CO2 resulted in resistance development
as expressed by LT50. Resistance increased steadily under continuous selection to 4.6- and 5.3-fold by
generation F30 for S-1 and S-2, respectively. Throughout the selection process, the slopes of regression
lines were always lower than that of the control. The results of biochemical assays showed that the
activities of carboxyl esterase (CarE) and superoxide dismutase (SOD) in vitro increased in the selection
process. Exposure to higher CO2 content (HCC) resulted in a gradual decrease in CarE activity in both
selected and control populations. Although the induction eect of CO2 on SOD was brief, the induction
times for the S-1 and S-2 were greater than those of the control. The elevated catalase (CAT) activity in
association with resistance development was also evident, but no statistical correlation was found
between CAT activity and HCC resistance. No signi®cant dierences were found in acid phosphatase
and alkaline phosphatase activities in both selected and control populations during this study. This
study demonstrated that high CarE and SOD activities were positively correlated to CO2
resistance. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Liposcelis bostrychophila; Resistance; Controlled atmosphere; Enzyme activity
* Corresponding author. Tel: +1-954-475-8990; fax: +1-954-475-4125.
E-mail address: [email protected]¯.edu (J.H. Tsai).
0022-474X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 2 - 4 7 4 X ( 9 9 ) 0 0 0 5 1 - X
298
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
1. Introduction
The psocid, Liposcelis bostrychophila Badonnel, a member of the order Psocoptera, is usually
considered as a minor pest (Gorham, 1991) and of little economic importance (Mockford,
1993). In recent years, there has been a gradual worldwide recognition that psocids do pose a
series of distinct pest problems in the area of grain storage and stored products (Rees, 1994;
Turner, 1994). In the last decade, this insect has been reported as a pest in Europe (Turner,
1994), Canada (Sinha, 1988; Mills et al., 1992), Australia (Smither, 1984), New Zealand
(Somer®eld et al., 1980) and Asia (Leong and Ho, 1994) in commercial and domestic
situations. Routine fumigations of warehouses and storage facilities with methyl bromide have
failed to control this pest (Ho and Winks, 1995). The adverse eect of methyl bromide on the
atmospheric ozone layer and worker safety issues have prompted the enactment of the Clean
Air Act by the US Federal government which requires a phase-out of production and use by
January 1, 2005 (Anonymous, 1993).
Modi®ed temperatures and controlled atmospheres (CA) are alternative treatments to the use
of methyl bromide for post-harvest insect control (Soderstrom et al., 1990, 1996). Controlled
atmospheres with elevated carbon dioxide, depleted oxygen levels, or a combination of both
have been tested and showed promise for control of stored product pests (Annis, 1987; Bailey
and Banks, 1980; Jayas et al., 1991).
Development of resistance to the most commonly used fumigants such as phosphine, methyl
bromide and ethylene dibromide has been well documented (Bond, 1973; Champ and Dyte,
1976; Ho and Winks, 1995; Leong and Ho, 1994). Similarly, extensive use of CA in insect
control could lead to selection of insect populations resistant to hypercarbia and hypoxia
(Donahaye, 1990a±c).
In China, L. bostrychophila has posed an alarming threat to stored products, especially for
the storage facilities where CA treatment is commonly practiced. The dramatic increase of
psocid populations associated with CA treatment in storage was widely evident (Wang, 1997).
Several studies have demonstrated that stored product insects have the genetic potential to
develop resistance to CA (Bond and Buckland, 1979; Navarro et al., 1985; Donahaye, 1990a,b;
Zhao and Zhang, 1993; Wang, 1997). Little is known about the mechanism of resistance to CA
(Donahaye, 1992). Our purpose was to establish the eect of carbon dioxide alone on L.
bostrychophila and to determine some enzyme activities in relation to the development of
resistance.
2. Materials and methods
2.1. Controlled atmosphere apparatus
A controlled atmosphere unit capable of generating gaseous compositions of three gases
(CO2, O2 and N2) in the range of 0±100% was used in this study. The unit consisted of gas
cylinders and regulators, three ¯owmeters, a gas blender, and an insect chamber with 24
exposure units constructed of 100 ml Erlenmeyer ¯asks. All gas concentration measurements
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
299
were carried out with the same gas chromatography apparatus as previously described by
Wang et al. (1999). The apparatus was kept in a temperature controlled room at 28218C.
The mixed and humidi®ed gases were ¯ushed continuously into the exposure units of the
insect chamber in order to prevent changes of gas composition due to metabolic activities of
the test insects. The CO2 and O2 concentrations in the ¯asks were monitored by gas
chromatography at 2±3 h intervals to maintain the desired levels of gas during the exposure
periods.
2.2. Selection of test insect and rearing conditions
Stock colonies of L. bostrychophila were started with nymphs collected from a wheat
warehouse in Chongqing, The People's Republic of China in 1990. The colonies were
maintained on an arti®cial diet consisting of whole wheat ¯our, skim milk and yeast powder
(10:1:1) in an air conditioned room at 28218C, 70±80% r.h., and a scotoperiod of 24 h. Three
populations of L. bostrychophila were maintained. One population (S-1) was treated with 35%
CO2, 21% O2, balance N2; the other (S-2) was treated with 55% CO2, 21% O2, balance N2;
the third population was without blended gas treatments as a control. The selection pressure
maintained about 70% mortality per generation. Adults obtained from surviving populations
after each exposure were reared in 50 plastic oviposition vials (1 cm high, 2.4 cm diameter) for
3 wk. Eggs were collected through a sieve (1 by 1 mm mesh) at 5 d intervals. Approximately
5000 eggs were placed in 100 plastic vials. Newly emerged adults were transferred to new vials
for use in the subsequent generation. The study was made from September 1992 to May 1997
totaling 30 selected generations.
2.3. Bioassay
A group of 100 newly emerged adults was placed in each of six ¯asks. Five ¯asks were
exposed to CO2 enriched atmosphere for ®ve exposure times as determined by their LT50 (the
estimated exposure time to achieve 50% mortality) values, the 6th ¯ask served as a control.
Each experiment was replicated ®ve times totaling 30 ¯asks. At the end of each exposure time,
the ¯asks were removed from the apparatus, and held at 28 2 18C and 80% r.h. for 48 h. At
the end of 48 h, the mortality was counted and corrected mortality was calculated (Abbott,
1925). The base-line sensitivity of the control population was determined at generation F2 by
probit analysis (Donahaye, 1990a). The sensitivities of the treated populations (S-1 and S-2)
and control population were determined in 5-generation increments.
2.4. Enzyme preparation and assays
Fifty adults were ground in 3 ml of ice-cold 0.04 M sodium phosphate buer (pH 7.0) in a
tissue grinder. The crude homogenate was centrifuged at 10,000 g, for 5 min at 48C in a
ultracentrifuge (Beckman L5-50B, Schaumburg, IL). The pellet was discarded and the resulting
supernatant was immediately used for enzyme assays.
Six enzymes were assayed in vitro throughout the selection process. The assay methods for
carboxyl esterase (CarE) activity measurement have been previously outlined by van Asperen
300
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
(1962) using a-naphthyl acetate as substrate. Alkaline phosphatase (ALP) and acid
phosphatase (ACP) activities were measured with 4-nitrophenyl phosphate disodium salt as
substrate according to Bessy et al. (1946). Superoxide dismutase (SOD) activity was assayed
using nitroblue tetrazolium as a substrate as described by Li et al. (1994). Catalase (CAT)
activity assay was identical to that of Chance and Machly (1955) using hydrogen peroxide as a
substrate. Peroxidase (POD) activity was monitored with guaiacol as a substrate (Simon et al.,
1974).
2.5. Data analysis
Mortalities were transformed into probits to stabilize the variance. The regression lines of
the log-time against probit-mortality for the selected generations were calculated using the
General Linear Model (GLM) Procedure (SAS Institute, 1988). LT50s were estimated from the
models, and the resistance factors were obtained by comparing LT50s between the selected and
control populations. The relationship between LT50 values or enzyme activities and selected
generations was described using linear regression ( y=a+bx ) (REG Procedure, SAS Insitute,
1988). Separate analyses of variance (ANOVA) were run for each enzyme measurement.
Dierences between means were examined with least signi®cant dierence values (SAS
Institute, 1988). Correlations between enzyme activities and resistance factors were examined
with Pearson correlation coecients (Sokal and Rohlf, 1969).
3. Results
3.1. Resistance development
The exposure times required to obtain LT50 values for successive generations of L.
bostrychophila adults are summarized in Table 1. According to the regression analysis of LT50
values over selected times, the values of LT50 in the control populations remained unchanged
throughout the selection process based on the value of slope (b=ÿ0.0006; P > 0.05), and
coincidence of 95% CI of LT50 values (Table 1) whereas the selected populations (S-1 and S-2)
evolved a low but signi®cant resistance to CO2 (bs-1=1.3852; bs-2=1.5607; P < 0.05; Table 1).
The calculated resistance factors (RF) based on LT50 for L. bostrychophila exposed to 35 and
55% CO2 increased to 4.6- and 5.3-fold by generation F30, respectively. The steady increase in
RF values for the S-2 population was greater than those of the S-1 population. The resistance
to hypercarbia in both treated populations also increased steadily (Table 1).
3.2. Changes of hydrolase activity
We found that CarE activities were signi®cantly higher in both S-1 and S-2 populations than
in the control population (Table 2; P < 0.05). The CarE measurements for the control
population remained unchanged based on the value of the slope (b = 0.001) of CarE activities
over selected generations (P > 0.05). However, the CarE activities for S-1 and S-2 increased
from generation to generation throughout this study period (bs-1=0.9564; bs-2=1.1781; P <
S-1
S-2
Control population
Fx
Slope2SE
w2a
LT50(h)
95% CIb
RFc
Slope2SE
w2a
LT50(h)
95% CIb
RFc
Slope2SE
w2a
LT50(h)
95%CIb
F0
F5
F10
F15
F20
F25
F30
7.5820.47
4.3920.21
5.7920.05
7.0420.72
6.6020.33
6.4020.39
6.4620.57
0.87
1.04
2.01
0.96
1.32
1.04
2.59
11
21
27
35
44
49
51
10±12
20±22
24±29
33±37
40±49
45±53
50±52
1.0
1.86
2.36
3.15
3.95
4.35
4.56
7.5820.47
4.2520.32
4.8920.54
5.5020.69
6.5720.71
5.8320.21
5.4720.09
0.87
2.30
1.48
3.06
2.57
3.02
1.57
11
23
31
40
46
53
59
10±12
21±25
27±35
39±42
43±49
50±56
57±61
1.0
2.05
2.80
3.58
4.12
4.72
5.26
7.5820.47
7.5420.37
7.6120.38
7.4320.62
7.5020.71
7.4920.12
7.5120.40
0.87
0.97
1.10
1.23
1.43
0.82
0.98
11
11
11
11
11
11
11
10±12
11±12
10±12
10±12
10±13
11±12
10±12
a
Chi-square goodness-of-®t test.
95% CI means con®dence interval.
c
Resistance factors (RF)=LT50 selected population6LT50 control population.
b
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
Table 1
Responses of two selected populations of Liposcelis bostrychophila adults to CO2 exposure
301
302
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
Table 2
Changes of CarE activity (mmol/l/30 min/psocid) during the selection processa
Fx
Control population
S-1
S-2
F0
F5
F10
F15
F20
F25
F30
12.1220.02a
12.0920.21a
12.3320.72a
12.3020.06a
12.1920.18a
12.0620.11a
12.1920.20a
12.1220.02a
12.3120.13a
14.0120.11b
19.8520.11c
26.1620.10d
33.2520.35e
38.7420.26f
12.1220.02a
14.0120.21b
20.1320.08c
25.1320.53d
34.1120.31e
40.0220.18f
45.1020.20g
Each value represents the mean (x- 2 SE) of three replicates; each replicate consists of 50 adults. Means within a
column followed by same letters are not signi®cantly dierent (P > 0.05). ANOVA statistics were: control population, F = 0.31; df=6, 14; P = 0.512; S-1, F = 12.31; df=6, 14; P < 0.0001; S-2, F = 302.9; df=6, 14; P < 0.0001.
a
0.05). By generation F30, the CarE activities in S-1 and S-2 were 3.18- and 3.70-fold higher
than that of the control population, respectively. Furthermore, the CarE actvities were
positively correlated to RF values (rs-1=0.9680; rs-2=0.9906; P < 0.01).
The mean values of ACP and ALP (OD400) were 0.0028 and 0.0022 per insect respectively
for the control population. The ACP and ALP activities for S-1 and S-2 increased slightly with
the resistance development. By generation F30, the ACP and ALP activities in S-1 were 0.0038
and 0.0028, and in S-2 were 0.0038 and 0.0031, respectively. However, the ACP and ALP
activities did not dier signi®cantly among the three populations tested (P > 0.05).
3.3. Changes of SOD, CAT and POD activities
The SOD activities for the control population remained stable throughout this study based
on the value of slope (b=ÿ0.002) of SOD activities over selected generations (P > 0.05) and
the mean value was 0.1307 enzyme unit per 15 min (Table 3). The activities for selected
Table 3
Changes of SOD activity (unit/15 min/10 psocids) during the selection processa
Fx
Control population
S-1
S-2
F0
F5
F10
F15
F20
F25
F30
1.3020.15a
1.3020.21a
1.3220.10a
1.3220.09a
1.3020.02a
1.3120.01a
1.2920.01a
1.3020.15a
2.3820.05b
2.7920.02c
3.8120.02d
4.1420.10e
4.8220.16f
5.1320.06g
1.3020.15a
2.5120.15b
3.3820.01c
4.2220.04d
4.8620.04e
5.3620.05f
5.8420.05g
Each value represents the mean (x- 2 SE) of three replicates; each replicate consists of 50 adults. Means within a
column followed by same letters are not signi®cantly dierent (P > 0.05). ANOVA statistics were: control population, F = 0.44; df=6, 14; P = 0.487; S-1, F = 62.74; df=6, 14; P < 0.001; S-2, F = 65.31; df=6, 14; P < 0.001.
a
303
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
populations increased steadily up to 3.97- and 4.52-fold by generation F30 for S-1 and S-2,
respectively, (bs-1=0.1266; bs-2=0.1486). The SOD activities were positively correlated with
resistance factors (rs-1=0.9893; rs-2=0.9986; P < 0.01).
The CAT activity for the control population was 3.3797 mg H2O2/min/insect (Table 4). The
respective CAT activities for S-1 and S-2 populations increased slightly up to 1.31- and 1.39fold by generation F30. There was no signi®cant correlation between resistance factor values
and CAT activities (P > 0.05).
The POD activities could not be detected for any population throughout the study.
3.4. Eects of exposure to CO2 on CarE and SOD
Figs. 1 and 2 showed the inhibition of CarE and SOD activities in all three populations
tested at generation F30. The percentage reduction of CarE activity for the control population
was signi®cantly greater than those of selected populations (P < 0.05; Fig. 1). After 8 h
exposure, the inhibition percentages for the control, S-1, and S-2 were 67.2, 30.1, and 20.3%,
respectively. The SOD activity for S-1 and S-2 increased during the ®rst 4±6 h, and then
decreased signi®cantly (P < 0.05; Fig. 2), whereas the respective SOD activity in the control
population increased only for the ®rst 2 h. After 8 h exposure, the respective SOD activities for
S-1 and S-2 were 6.40- and 8.97-fold higher than that of the control population.
4. Discussion
Controlled atmospheres have been used to kill a wide range of quarantine and storage pests
eectively, including members of such families as Tephritidae, Tortricidae, Curculionidae,
Miridae and Liposcelididae (Carpenter and Potter, 1994; Ke and Kader, 1992; Leong and Ho,
1995b; Soderstrom et al., 1990; Whiting et al., 1992). The CA treatment involves manipulation
of the concentration of CO2, O2, and N2 in the atmosphere surrounding the stored product
Table 4
Changes of CAT (mg H2O2/min/psocid) during the selection processa
Fx
Control population
S-1
S-2
F0
F5
F10
F15
F20
F25
F30
3.3520.45a
3.3720.43a
3.4020.48a
3.3720.05a
3.3620.12a
3.4020.03a
3.3720.02a
3.3520.45a
3.5020.05ab
3.6120.21ab
3.7220.14ab
3.7920.40b
3.9520.31bc
4.4220.48c
3.3520.45a
3.6720.10a
3.7120.11ab
4.0520.24b
4.2120.30bc
4.3720.58c
4.6920.10c
Each value represents the mean (x- 2 SE) of three replicates; each replicate consists of 50 adults. Means within a
column followed by same letters are not signi®cantly dierent (P > 0.05). ANOVA statistics were: control population, F = 0.24; df=6, 14; P = 0.588; S-1, F = 18.97; df=6, 14; P < 0.001; S-2, F = 16.37; df=6, 14; P < 0.001.
a
304
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
Fig. 1. Percentage inhibition of CarE activities by CO2 exposure for control population (solid dot), S-1 (circle) and
S-2 (triangle) at generation F30.
and the pest. Carbon dioxide is an important factor aecting the ecacy of CA treatments for
pest mortality. Generally the combination of low O2 and high CO2 leads to higher mortality
than either gas alone because of the combined eects of anoxia and hypercarbia (FleuratLessard, 1990).
We compared the eects of two gas combinations, 35% CO2 in 21% O2+44% N2 and 55%
CO2 in 21% O2+24% N2 and showed that L. bostrychophila is capable of developing
resistance to CO2 enriched atmospheres. The resistance to CO2 appears to be related to the
Fig. 2. Percentage induction of SOD activities by CO2 exposure for control population (solid dot), S-1 (circle) and
S-2 (triangle) at generation F30.
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
305
increase of CarE and SOD activities. The increase in resistance was greater in the S-2
population than in the S-1 population after 30 generations. The calculated resistance factors
based on LT95 values at the 30th generation were similar to resistance factors based on LT50
values. This is dierent from the report by Navarro et al. (1985). Their study showed that the
increase of resistance in rice weevil, Sitophilus oryzae L. treated with 40% CO2 was greater
than those treated with 75% CO2. In experiments with the granary weevil, S. granarius (L.),
Bond and Buckland (1979) found that resistance increased to 3.3- and 1.8-fold by generation
F7 and F4 for the populations treated with 42 and 75% CO2, respectively. Dierences between
our results and those of others may be due to dierences in the CO2 concentrations used or to
dierent species of insects exhibiting dierent adaptabilities.
Our study indicated that the slopes of regression lines remained essentially unchanged
throughout 30 generations indicating that the high heterogeneity of response continued in the
selection process (Table 1). We showed that the development of CO2 resistance in L.
bostrychophila was closely related to the increased CarE activity. This ®nding was further
supported by an inhibition study. Exposure of L. bostrychophila to CO2 enriched atmospheres
resulted in a decrease in CarE activities, but the inhibition range for the untreated population
was signi®cantly greater than those of the selected populations (P < 0.05; Fig. 1). The results
strongly suggest that CarE plays an important role in CO2 resistance. However, no relationship
between ACP or ALP activity and CO2 resistance in L. bostrychophila was observed,
suggesting that ACP and ALP were not related to CO2 resistance.
The endogenous enzymes of protective systems (EEPS) including SOD, CAT, and POD were
found in a number of organisms (Fridovich, 1977). SOD functions as an oxygen carrier with
antioxidant properties. SOD±CAT could decrease the formation of oxygen radicals (D'Agnillo
and Chang, 1998). It has been shown that the elevated EEPS activities are closely related to
the resistance of organisms to unfavorable environments (Packer, 1984). Li et al. (1994) found
that SOD and CAT activities were related to organophosphate insecticide resistance in Pieris
rapae L., Parasa consocia Walker and Thosea postornata Hampson. Grubor-Lajsic et al. (1997)
reported that cold exposure signi®cantly increased activities of the antioxidant enzymes of two
larval Lepidoptera, Ostrinia nubilalis Hubn. and Sesamia cretica Led. Aucoin et al. (1991)
reported that CATs may be inducible defenses against phototoxins by insects. We have also
observed the correlation between the SOD activity and CO2 resistance (Table 3). Carbon
dioxide could induce SOD activity after a short time exposure, while long term exposure would
inhibit SOD activity (Fig. 2) suggesting that elevated SOD activity positively aected CO2
resistance. Although CAT activity also increased with resistance development, there was no
close relationship between CAT activity and resistance level suggesting that CAT was not the
main factor aecting CO2 resistance (Table 4). In this study, we have not detected any POD
activity throughout the selection process.
Currently, not all aspects of the mode of action of CO2 on insects are elucidated (Donahaye,
1990c). Investigations with Ephestia cautella Walker pupae have shown that CO2 aected
several metabolic sites which might facilitate resistance development in the insect (Friedlander
and Navarro, 1979; Friedlander et al., 1983). These studies indicated that the resistance to CA
not only involved changes of physiological aspects, but also involved changes of biochemical
aspects. The possible mechanisms of CarE, SOD and CAT in relation to CO2 resistance
were under the stress of carbon dioxide, the toxic products and the formation of free
306
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
oxygen radicals. The enhanced CarE and anti-oxidation enzymes (SOD and CAT) activities
could reduce the eects of these toxic products on insects resulting in the insects' resistance to
CO2 increasing. Therefore, grain storage and warehouse operators should be aware that CA
treatments can induce the esterase enzymes which in turn can promote selection of pest strains
resistant to CA treatments (Bond and Buckland, 1979) and the rates of development of CA
resistance are similar to those recorded for laboratory induced resistance to fumigants
(Donahaye, 1990c). Many similarities exist between CA resistance and resistance to methyl
bromide (Monro, 1964; Upitis et al., 1973). Whether CA treatments could increase the
tolerance of pests to certain insecticides containing an ester linkage such as organophosphates,
carbamates, and pyrethroids (Ho and Winks, 1995; Leong and Ho, 1995a; Yu and Valles,
1997) will require unequivocal experimental proof.
Acknowledgements
Appreciation is extended to Dr. B. Turner, King's College London, UK for his
encouragement. This research was funded in part by the National Natural Sciences Foundation
(39800017) and Doctoral Research Grant of P.R. China to J. J. Wang. J. J. Wang is a
postdoctoral research associate and Z. M. Zhao is professor of the Department of Plant
Protection, Southwest Agricultural University, Chongqing, China. This is Florida Agricultural
Experiment Station Journal Series No. R-07113.
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www.elsevier.com/locate/jspr
Resistance and some enzyme activities in Liposcelis
bostrychophila Badonnel (Psocoptera: Liposcelididae) in
relation to carbon dioxide enriched atmospheres
Jin-Jun Wang, Zhi-Mo Zhao, James H. Tsai*
Fort Lauderdale Research and Education Center, IFAS, University of Florida, 3205 College Avenue, Fort Lauderdale,
FL 33314-7799, USA
Accepted 3 November 1999
Abstract
Two populations (S-1 and S-2) of the psocid, Liposcelis bostrychophila Badonnel were exposed to
carbon dioxide enriched atmospheres. Carbon dioxide resistance developed at steady rates in these two
populations during this study period. Selection with 35 and 55% CO2 resulted in resistance development
as expressed by LT50. Resistance increased steadily under continuous selection to 4.6- and 5.3-fold by
generation F30 for S-1 and S-2, respectively. Throughout the selection process, the slopes of regression
lines were always lower than that of the control. The results of biochemical assays showed that the
activities of carboxyl esterase (CarE) and superoxide dismutase (SOD) in vitro increased in the selection
process. Exposure to higher CO2 content (HCC) resulted in a gradual decrease in CarE activity in both
selected and control populations. Although the induction eect of CO2 on SOD was brief, the induction
times for the S-1 and S-2 were greater than those of the control. The elevated catalase (CAT) activity in
association with resistance development was also evident, but no statistical correlation was found
between CAT activity and HCC resistance. No signi®cant dierences were found in acid phosphatase
and alkaline phosphatase activities in both selected and control populations during this study. This
study demonstrated that high CarE and SOD activities were positively correlated to CO2
resistance. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Liposcelis bostrychophila; Resistance; Controlled atmosphere; Enzyme activity
* Corresponding author. Tel: +1-954-475-8990; fax: +1-954-475-4125.
E-mail address: [email protected]¯.edu (J.H. Tsai).
0022-474X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 2 - 4 7 4 X ( 9 9 ) 0 0 0 5 1 - X
298
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
1. Introduction
The psocid, Liposcelis bostrychophila Badonnel, a member of the order Psocoptera, is usually
considered as a minor pest (Gorham, 1991) and of little economic importance (Mockford,
1993). In recent years, there has been a gradual worldwide recognition that psocids do pose a
series of distinct pest problems in the area of grain storage and stored products (Rees, 1994;
Turner, 1994). In the last decade, this insect has been reported as a pest in Europe (Turner,
1994), Canada (Sinha, 1988; Mills et al., 1992), Australia (Smither, 1984), New Zealand
(Somer®eld et al., 1980) and Asia (Leong and Ho, 1994) in commercial and domestic
situations. Routine fumigations of warehouses and storage facilities with methyl bromide have
failed to control this pest (Ho and Winks, 1995). The adverse eect of methyl bromide on the
atmospheric ozone layer and worker safety issues have prompted the enactment of the Clean
Air Act by the US Federal government which requires a phase-out of production and use by
January 1, 2005 (Anonymous, 1993).
Modi®ed temperatures and controlled atmospheres (CA) are alternative treatments to the use
of methyl bromide for post-harvest insect control (Soderstrom et al., 1990, 1996). Controlled
atmospheres with elevated carbon dioxide, depleted oxygen levels, or a combination of both
have been tested and showed promise for control of stored product pests (Annis, 1987; Bailey
and Banks, 1980; Jayas et al., 1991).
Development of resistance to the most commonly used fumigants such as phosphine, methyl
bromide and ethylene dibromide has been well documented (Bond, 1973; Champ and Dyte,
1976; Ho and Winks, 1995; Leong and Ho, 1994). Similarly, extensive use of CA in insect
control could lead to selection of insect populations resistant to hypercarbia and hypoxia
(Donahaye, 1990a±c).
In China, L. bostrychophila has posed an alarming threat to stored products, especially for
the storage facilities where CA treatment is commonly practiced. The dramatic increase of
psocid populations associated with CA treatment in storage was widely evident (Wang, 1997).
Several studies have demonstrated that stored product insects have the genetic potential to
develop resistance to CA (Bond and Buckland, 1979; Navarro et al., 1985; Donahaye, 1990a,b;
Zhao and Zhang, 1993; Wang, 1997). Little is known about the mechanism of resistance to CA
(Donahaye, 1992). Our purpose was to establish the eect of carbon dioxide alone on L.
bostrychophila and to determine some enzyme activities in relation to the development of
resistance.
2. Materials and methods
2.1. Controlled atmosphere apparatus
A controlled atmosphere unit capable of generating gaseous compositions of three gases
(CO2, O2 and N2) in the range of 0±100% was used in this study. The unit consisted of gas
cylinders and regulators, three ¯owmeters, a gas blender, and an insect chamber with 24
exposure units constructed of 100 ml Erlenmeyer ¯asks. All gas concentration measurements
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
299
were carried out with the same gas chromatography apparatus as previously described by
Wang et al. (1999). The apparatus was kept in a temperature controlled room at 28218C.
The mixed and humidi®ed gases were ¯ushed continuously into the exposure units of the
insect chamber in order to prevent changes of gas composition due to metabolic activities of
the test insects. The CO2 and O2 concentrations in the ¯asks were monitored by gas
chromatography at 2±3 h intervals to maintain the desired levels of gas during the exposure
periods.
2.2. Selection of test insect and rearing conditions
Stock colonies of L. bostrychophila were started with nymphs collected from a wheat
warehouse in Chongqing, The People's Republic of China in 1990. The colonies were
maintained on an arti®cial diet consisting of whole wheat ¯our, skim milk and yeast powder
(10:1:1) in an air conditioned room at 28218C, 70±80% r.h., and a scotoperiod of 24 h. Three
populations of L. bostrychophila were maintained. One population (S-1) was treated with 35%
CO2, 21% O2, balance N2; the other (S-2) was treated with 55% CO2, 21% O2, balance N2;
the third population was without blended gas treatments as a control. The selection pressure
maintained about 70% mortality per generation. Adults obtained from surviving populations
after each exposure were reared in 50 plastic oviposition vials (1 cm high, 2.4 cm diameter) for
3 wk. Eggs were collected through a sieve (1 by 1 mm mesh) at 5 d intervals. Approximately
5000 eggs were placed in 100 plastic vials. Newly emerged adults were transferred to new vials
for use in the subsequent generation. The study was made from September 1992 to May 1997
totaling 30 selected generations.
2.3. Bioassay
A group of 100 newly emerged adults was placed in each of six ¯asks. Five ¯asks were
exposed to CO2 enriched atmosphere for ®ve exposure times as determined by their LT50 (the
estimated exposure time to achieve 50% mortality) values, the 6th ¯ask served as a control.
Each experiment was replicated ®ve times totaling 30 ¯asks. At the end of each exposure time,
the ¯asks were removed from the apparatus, and held at 28 2 18C and 80% r.h. for 48 h. At
the end of 48 h, the mortality was counted and corrected mortality was calculated (Abbott,
1925). The base-line sensitivity of the control population was determined at generation F2 by
probit analysis (Donahaye, 1990a). The sensitivities of the treated populations (S-1 and S-2)
and control population were determined in 5-generation increments.
2.4. Enzyme preparation and assays
Fifty adults were ground in 3 ml of ice-cold 0.04 M sodium phosphate buer (pH 7.0) in a
tissue grinder. The crude homogenate was centrifuged at 10,000 g, for 5 min at 48C in a
ultracentrifuge (Beckman L5-50B, Schaumburg, IL). The pellet was discarded and the resulting
supernatant was immediately used for enzyme assays.
Six enzymes were assayed in vitro throughout the selection process. The assay methods for
carboxyl esterase (CarE) activity measurement have been previously outlined by van Asperen
300
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
(1962) using a-naphthyl acetate as substrate. Alkaline phosphatase (ALP) and acid
phosphatase (ACP) activities were measured with 4-nitrophenyl phosphate disodium salt as
substrate according to Bessy et al. (1946). Superoxide dismutase (SOD) activity was assayed
using nitroblue tetrazolium as a substrate as described by Li et al. (1994). Catalase (CAT)
activity assay was identical to that of Chance and Machly (1955) using hydrogen peroxide as a
substrate. Peroxidase (POD) activity was monitored with guaiacol as a substrate (Simon et al.,
1974).
2.5. Data analysis
Mortalities were transformed into probits to stabilize the variance. The regression lines of
the log-time against probit-mortality for the selected generations were calculated using the
General Linear Model (GLM) Procedure (SAS Institute, 1988). LT50s were estimated from the
models, and the resistance factors were obtained by comparing LT50s between the selected and
control populations. The relationship between LT50 values or enzyme activities and selected
generations was described using linear regression ( y=a+bx ) (REG Procedure, SAS Insitute,
1988). Separate analyses of variance (ANOVA) were run for each enzyme measurement.
Dierences between means were examined with least signi®cant dierence values (SAS
Institute, 1988). Correlations between enzyme activities and resistance factors were examined
with Pearson correlation coecients (Sokal and Rohlf, 1969).
3. Results
3.1. Resistance development
The exposure times required to obtain LT50 values for successive generations of L.
bostrychophila adults are summarized in Table 1. According to the regression analysis of LT50
values over selected times, the values of LT50 in the control populations remained unchanged
throughout the selection process based on the value of slope (b=ÿ0.0006; P > 0.05), and
coincidence of 95% CI of LT50 values (Table 1) whereas the selected populations (S-1 and S-2)
evolved a low but signi®cant resistance to CO2 (bs-1=1.3852; bs-2=1.5607; P < 0.05; Table 1).
The calculated resistance factors (RF) based on LT50 for L. bostrychophila exposed to 35 and
55% CO2 increased to 4.6- and 5.3-fold by generation F30, respectively. The steady increase in
RF values for the S-2 population was greater than those of the S-1 population. The resistance
to hypercarbia in both treated populations also increased steadily (Table 1).
3.2. Changes of hydrolase activity
We found that CarE activities were signi®cantly higher in both S-1 and S-2 populations than
in the control population (Table 2; P < 0.05). The CarE measurements for the control
population remained unchanged based on the value of the slope (b = 0.001) of CarE activities
over selected generations (P > 0.05). However, the CarE activities for S-1 and S-2 increased
from generation to generation throughout this study period (bs-1=0.9564; bs-2=1.1781; P <
S-1
S-2
Control population
Fx
Slope2SE
w2a
LT50(h)
95% CIb
RFc
Slope2SE
w2a
LT50(h)
95% CIb
RFc
Slope2SE
w2a
LT50(h)
95%CIb
F0
F5
F10
F15
F20
F25
F30
7.5820.47
4.3920.21
5.7920.05
7.0420.72
6.6020.33
6.4020.39
6.4620.57
0.87
1.04
2.01
0.96
1.32
1.04
2.59
11
21
27
35
44
49
51
10±12
20±22
24±29
33±37
40±49
45±53
50±52
1.0
1.86
2.36
3.15
3.95
4.35
4.56
7.5820.47
4.2520.32
4.8920.54
5.5020.69
6.5720.71
5.8320.21
5.4720.09
0.87
2.30
1.48
3.06
2.57
3.02
1.57
11
23
31
40
46
53
59
10±12
21±25
27±35
39±42
43±49
50±56
57±61
1.0
2.05
2.80
3.58
4.12
4.72
5.26
7.5820.47
7.5420.37
7.6120.38
7.4320.62
7.5020.71
7.4920.12
7.5120.40
0.87
0.97
1.10
1.23
1.43
0.82
0.98
11
11
11
11
11
11
11
10±12
11±12
10±12
10±12
10±13
11±12
10±12
a
Chi-square goodness-of-®t test.
95% CI means con®dence interval.
c
Resistance factors (RF)=LT50 selected population6LT50 control population.
b
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
Table 1
Responses of two selected populations of Liposcelis bostrychophila adults to CO2 exposure
301
302
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
Table 2
Changes of CarE activity (mmol/l/30 min/psocid) during the selection processa
Fx
Control population
S-1
S-2
F0
F5
F10
F15
F20
F25
F30
12.1220.02a
12.0920.21a
12.3320.72a
12.3020.06a
12.1920.18a
12.0620.11a
12.1920.20a
12.1220.02a
12.3120.13a
14.0120.11b
19.8520.11c
26.1620.10d
33.2520.35e
38.7420.26f
12.1220.02a
14.0120.21b
20.1320.08c
25.1320.53d
34.1120.31e
40.0220.18f
45.1020.20g
Each value represents the mean (x- 2 SE) of three replicates; each replicate consists of 50 adults. Means within a
column followed by same letters are not signi®cantly dierent (P > 0.05). ANOVA statistics were: control population, F = 0.31; df=6, 14; P = 0.512; S-1, F = 12.31; df=6, 14; P < 0.0001; S-2, F = 302.9; df=6, 14; P < 0.0001.
a
0.05). By generation F30, the CarE activities in S-1 and S-2 were 3.18- and 3.70-fold higher
than that of the control population, respectively. Furthermore, the CarE actvities were
positively correlated to RF values (rs-1=0.9680; rs-2=0.9906; P < 0.01).
The mean values of ACP and ALP (OD400) were 0.0028 and 0.0022 per insect respectively
for the control population. The ACP and ALP activities for S-1 and S-2 increased slightly with
the resistance development. By generation F30, the ACP and ALP activities in S-1 were 0.0038
and 0.0028, and in S-2 were 0.0038 and 0.0031, respectively. However, the ACP and ALP
activities did not dier signi®cantly among the three populations tested (P > 0.05).
3.3. Changes of SOD, CAT and POD activities
The SOD activities for the control population remained stable throughout this study based
on the value of slope (b=ÿ0.002) of SOD activities over selected generations (P > 0.05) and
the mean value was 0.1307 enzyme unit per 15 min (Table 3). The activities for selected
Table 3
Changes of SOD activity (unit/15 min/10 psocids) during the selection processa
Fx
Control population
S-1
S-2
F0
F5
F10
F15
F20
F25
F30
1.3020.15a
1.3020.21a
1.3220.10a
1.3220.09a
1.3020.02a
1.3120.01a
1.2920.01a
1.3020.15a
2.3820.05b
2.7920.02c
3.8120.02d
4.1420.10e
4.8220.16f
5.1320.06g
1.3020.15a
2.5120.15b
3.3820.01c
4.2220.04d
4.8620.04e
5.3620.05f
5.8420.05g
Each value represents the mean (x- 2 SE) of three replicates; each replicate consists of 50 adults. Means within a
column followed by same letters are not signi®cantly dierent (P > 0.05). ANOVA statistics were: control population, F = 0.44; df=6, 14; P = 0.487; S-1, F = 62.74; df=6, 14; P < 0.001; S-2, F = 65.31; df=6, 14; P < 0.001.
a
303
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
populations increased steadily up to 3.97- and 4.52-fold by generation F30 for S-1 and S-2,
respectively, (bs-1=0.1266; bs-2=0.1486). The SOD activities were positively correlated with
resistance factors (rs-1=0.9893; rs-2=0.9986; P < 0.01).
The CAT activity for the control population was 3.3797 mg H2O2/min/insect (Table 4). The
respective CAT activities for S-1 and S-2 populations increased slightly up to 1.31- and 1.39fold by generation F30. There was no signi®cant correlation between resistance factor values
and CAT activities (P > 0.05).
The POD activities could not be detected for any population throughout the study.
3.4. Eects of exposure to CO2 on CarE and SOD
Figs. 1 and 2 showed the inhibition of CarE and SOD activities in all three populations
tested at generation F30. The percentage reduction of CarE activity for the control population
was signi®cantly greater than those of selected populations (P < 0.05; Fig. 1). After 8 h
exposure, the inhibition percentages for the control, S-1, and S-2 were 67.2, 30.1, and 20.3%,
respectively. The SOD activity for S-1 and S-2 increased during the ®rst 4±6 h, and then
decreased signi®cantly (P < 0.05; Fig. 2), whereas the respective SOD activity in the control
population increased only for the ®rst 2 h. After 8 h exposure, the respective SOD activities for
S-1 and S-2 were 6.40- and 8.97-fold higher than that of the control population.
4. Discussion
Controlled atmospheres have been used to kill a wide range of quarantine and storage pests
eectively, including members of such families as Tephritidae, Tortricidae, Curculionidae,
Miridae and Liposcelididae (Carpenter and Potter, 1994; Ke and Kader, 1992; Leong and Ho,
1995b; Soderstrom et al., 1990; Whiting et al., 1992). The CA treatment involves manipulation
of the concentration of CO2, O2, and N2 in the atmosphere surrounding the stored product
Table 4
Changes of CAT (mg H2O2/min/psocid) during the selection processa
Fx
Control population
S-1
S-2
F0
F5
F10
F15
F20
F25
F30
3.3520.45a
3.3720.43a
3.4020.48a
3.3720.05a
3.3620.12a
3.4020.03a
3.3720.02a
3.3520.45a
3.5020.05ab
3.6120.21ab
3.7220.14ab
3.7920.40b
3.9520.31bc
4.4220.48c
3.3520.45a
3.6720.10a
3.7120.11ab
4.0520.24b
4.2120.30bc
4.3720.58c
4.6920.10c
Each value represents the mean (x- 2 SE) of three replicates; each replicate consists of 50 adults. Means within a
column followed by same letters are not signi®cantly dierent (P > 0.05). ANOVA statistics were: control population, F = 0.24; df=6, 14; P = 0.588; S-1, F = 18.97; df=6, 14; P < 0.001; S-2, F = 16.37; df=6, 14; P < 0.001.
a
304
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
Fig. 1. Percentage inhibition of CarE activities by CO2 exposure for control population (solid dot), S-1 (circle) and
S-2 (triangle) at generation F30.
and the pest. Carbon dioxide is an important factor aecting the ecacy of CA treatments for
pest mortality. Generally the combination of low O2 and high CO2 leads to higher mortality
than either gas alone because of the combined eects of anoxia and hypercarbia (FleuratLessard, 1990).
We compared the eects of two gas combinations, 35% CO2 in 21% O2+44% N2 and 55%
CO2 in 21% O2+24% N2 and showed that L. bostrychophila is capable of developing
resistance to CO2 enriched atmospheres. The resistance to CO2 appears to be related to the
Fig. 2. Percentage induction of SOD activities by CO2 exposure for control population (solid dot), S-1 (circle) and
S-2 (triangle) at generation F30.
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
305
increase of CarE and SOD activities. The increase in resistance was greater in the S-2
population than in the S-1 population after 30 generations. The calculated resistance factors
based on LT95 values at the 30th generation were similar to resistance factors based on LT50
values. This is dierent from the report by Navarro et al. (1985). Their study showed that the
increase of resistance in rice weevil, Sitophilus oryzae L. treated with 40% CO2 was greater
than those treated with 75% CO2. In experiments with the granary weevil, S. granarius (L.),
Bond and Buckland (1979) found that resistance increased to 3.3- and 1.8-fold by generation
F7 and F4 for the populations treated with 42 and 75% CO2, respectively. Dierences between
our results and those of others may be due to dierences in the CO2 concentrations used or to
dierent species of insects exhibiting dierent adaptabilities.
Our study indicated that the slopes of regression lines remained essentially unchanged
throughout 30 generations indicating that the high heterogeneity of response continued in the
selection process (Table 1). We showed that the development of CO2 resistance in L.
bostrychophila was closely related to the increased CarE activity. This ®nding was further
supported by an inhibition study. Exposure of L. bostrychophila to CO2 enriched atmospheres
resulted in a decrease in CarE activities, but the inhibition range for the untreated population
was signi®cantly greater than those of the selected populations (P < 0.05; Fig. 1). The results
strongly suggest that CarE plays an important role in CO2 resistance. However, no relationship
between ACP or ALP activity and CO2 resistance in L. bostrychophila was observed,
suggesting that ACP and ALP were not related to CO2 resistance.
The endogenous enzymes of protective systems (EEPS) including SOD, CAT, and POD were
found in a number of organisms (Fridovich, 1977). SOD functions as an oxygen carrier with
antioxidant properties. SOD±CAT could decrease the formation of oxygen radicals (D'Agnillo
and Chang, 1998). It has been shown that the elevated EEPS activities are closely related to
the resistance of organisms to unfavorable environments (Packer, 1984). Li et al. (1994) found
that SOD and CAT activities were related to organophosphate insecticide resistance in Pieris
rapae L., Parasa consocia Walker and Thosea postornata Hampson. Grubor-Lajsic et al. (1997)
reported that cold exposure signi®cantly increased activities of the antioxidant enzymes of two
larval Lepidoptera, Ostrinia nubilalis Hubn. and Sesamia cretica Led. Aucoin et al. (1991)
reported that CATs may be inducible defenses against phototoxins by insects. We have also
observed the correlation between the SOD activity and CO2 resistance (Table 3). Carbon
dioxide could induce SOD activity after a short time exposure, while long term exposure would
inhibit SOD activity (Fig. 2) suggesting that elevated SOD activity positively aected CO2
resistance. Although CAT activity also increased with resistance development, there was no
close relationship between CAT activity and resistance level suggesting that CAT was not the
main factor aecting CO2 resistance (Table 4). In this study, we have not detected any POD
activity throughout the selection process.
Currently, not all aspects of the mode of action of CO2 on insects are elucidated (Donahaye,
1990c). Investigations with Ephestia cautella Walker pupae have shown that CO2 aected
several metabolic sites which might facilitate resistance development in the insect (Friedlander
and Navarro, 1979; Friedlander et al., 1983). These studies indicated that the resistance to CA
not only involved changes of physiological aspects, but also involved changes of biochemical
aspects. The possible mechanisms of CarE, SOD and CAT in relation to CO2 resistance
were under the stress of carbon dioxide, the toxic products and the formation of free
306
J.-J. Wang et al. / Journal of Stored Products Research 36 (2000) 297±308
oxygen radicals. The enhanced CarE and anti-oxidation enzymes (SOD and CAT) activities
could reduce the eects of these toxic products on insects resulting in the insects' resistance to
CO2 increasing. Therefore, grain storage and warehouse operators should be aware that CA
treatments can induce the esterase enzymes which in turn can promote selection of pest strains
resistant to CA treatments (Bond and Buckland, 1979) and the rates of development of CA
resistance are similar to those recorded for laboratory induced resistance to fumigants
(Donahaye, 1990c). Many similarities exist between CA resistance and resistance to methyl
bromide (Monro, 1964; Upitis et al., 1973). Whether CA treatments could increase the
tolerance of pests to certain insecticides containing an ester linkage such as organophosphates,
carbamates, and pyrethroids (Ho and Winks, 1995; Leong and Ho, 1995a; Yu and Valles,
1997) will require unequivocal experimental proof.
Acknowledgements
Appreciation is extended to Dr. B. Turner, King's College London, UK for his
encouragement. This research was funded in part by the National Natural Sciences Foundation
(39800017) and Doctoral Research Grant of P.R. China to J. J. Wang. J. J. Wang is a
postdoctoral research associate and Z. M. Zhao is professor of the Department of Plant
Protection, Southwest Agricultural University, Chongqing, China. This is Florida Agricultural
Experiment Station Journal Series No. R-07113.
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