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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% CO2resulted 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 CO2content (HCC) resulted in a gradual decrease in CarE activity in both

selected and control populations. Although the induction eect of CO2on 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

Keywords: Liposcelis bostrychophila; Resistance; Controlled atmosphere; Enzyme activity Journal of Stored Products Research 36 (2000) 297±308

www.elsevier.com/locate/jspr

* Corresponding author. Tel: +1-954-475-8990; fax: +1-954-475-4125.

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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

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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. bostrychophilawere 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 28218C 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.

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(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

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Table 1

Responses of two selected populations ofLiposcelis bostrychophilaadults to CO2exposure

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 7.5820.47 0.87 11 10±12 1.0 7.5820.47 0.87 11 10±12 1.0 7.5820.47 0.87 11 10±12

F5 4.3920.21 1.04 21 20±22 1.86 4.2520.32 2.30 23 21±25 2.05 7.5420.37 0.97 11 11±12

F10 5.7920.05 2.01 27 24±29 2.36 4.8920.54 1.48 31 27±35 2.80 7.6120.38 1.10 11 10±12

F15 7.0420.72 0.96 35 33±37 3.15 5.5020.69 3.06 40 39±42 3.58 7.4320.62 1.23 11 10±12

F20 6.6020.33 1.32 44 40±49 3.95 6.5720.71 2.57 46 43±49 4.12 7.5020.71 1.43 11 10±13

F25 6.4020.39 1.04 49 45±53 4.35 5.8320.21 3.02 53 50±56 4.72 7.4920.12 0.82 11 11±12

F30 6.4620.57 2.59 51 50±52 4.56 5.4720.09 1.57 59 57±61 5.26 7.5120.40 0.98 11 10±12

a

Chi-square goodness-of-®t test.

b_{95% CI means con®dence interval.} c

Resistance factors (RF)=LT50selected population6LT50control population.

J.-J.

Wang

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297±308

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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 2

Changes of CarE activity (mmol/l/30 min/psocid) during the selection processa

Fx Control population S-1 S-2

F0 12.1220.02a 12.1220.02a 12.1220.02a

F5 12.0920.21a 12.3120.13a 14.0120.21b

F10 12.3320.72a 14.0120.11b 20.1320.08c

F15 12.3020.06a 19.8520.11c 25.1320.53d

F20 12.1920.18a 26.1620.10d 34.1120.31e

F25 12.0620.11a 33.2520.35e 40.0220.18f

F30 12.1920.20a 38.7420.26f 45.1020.20g

a

Each value represents the mean (x-2SE) 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 popu-lation,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.

Table 3

Changes of SOD activity (unit/15 min/10 psocids) during the selection processa

F0 1.3020.15a 1.3020.15a 1.3020.15a

F5 1.3020.21a 2.3820.05b 2.5120.15b

F10 1.3220.10a 2.7920.02c 3.3820.01c

F15 1.3220.09a 3.8120.02d 4.2220.04d

F20 1.3020.02a 4.1420.10e 4.8620.04e

F25 1.3120.01a 4.8220.16f 5.3620.05f

F30 1.2920.01a 5.1320.06g 5.8420.05g

a

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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 1.39-fold 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 CO2on 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

F0 3.3520.45a 3.3520.45a 3.3520.45a

F5 3.3720.43a 3.5020.05ab 3.6720.10a

F10 3.4020.48a 3.6120.21ab 3.7120.11ab

F15 3.3720.05a 3.7220.14ab 4.0520.24b

F20 3.3620.12a 3.7920.40b 4.2120.30bc

F25 3.4020.03a 3.9520.31bc 4.3720.58c

F30 3.3720.02a 4.4220.48c 4.6920.10c

a

Each value represents the mean (x-2SE) 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 popu-lation,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.

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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 (Fleurat-Lessard, 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. 1. Percentage inhibition of CarE activities by CO2exposure for control population (solid dot), S-1 (circle) and

S-2 (triangle) at generation F30.

Fig. 2. Percentage induction of SOD activities by CO2 exposure for control population (solid dot), S-1 (circle) and

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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 CO2resistance. 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 postornataHampson. 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.

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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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