Beta-cyfluthrin Resistance of Cigarette Beetle Lasioderma serricorne (F.) (Coleoptera: Anobiidae) and its Control Using Imidacloprid Space Sprays

INTRODUCTION

The cigarette beetles, Lasioderma serricorne (F.) (Coleoptera: Anobiidae), belong to a group of insects that infest stored products, and feed and live in many kinds of dried substances, such as cloth, paper, furniture, grain, coffee, and tobacco [1]. As the cosmopolitan and polyphagous pests, L. serricorne is generally considered to be one of the most destructive insects in tobacco processing and cigarette manufacturing, due to the capacity to develop in tobacco which is toxic for most other species [2-6]. In addition to weight losses of tobacco leaves caused by larvae feeding during tobacco processing and storage, if beetles get into final cigarette products, it can lead to customer complaints and even cause a cigarette brand to disappear from the market, resulting in huge economic losses [3, 7].

Like pest control in food processing and stored factories, management of L. serricorne in cigarette manufacturing areas has always depended heavily on sanitation, including promptly removing waste material and eliminating infected tobacco shreds [8]. However, merely cleaning in cigarette manufacturing area is not enough to control the pest completely, and the application of space aerosol insecticide is often used as a supplementary method [8, 9]. Application of residual and contact insecticide aerosols can effectively prevent pest infestation in food-processing facilities with several advantages, including low cost, short treatment time, and can be integrated with other management tactics such as sanitation [10, 11]. The application of insecticides as aerosols for managing stored-product pests is a well-established technique and was initially used for controlling L. serricorne in tobacco warehouses in the 1950s with dichlorvos (DDVP) aerosol [12]. It was reported that DDVP aerosol could effectively control stored-product pests, including black carpet beetles Attagenus megatoma (Fab.) (Coleoptera: Dermestidae), confused flour beetles Tribolium confusum (Jacquelin du Val) (Coleoptera: Pseudodontidae), Lasioderma serricorne, red flour beetles Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and almond moths Cadra cautella (Walker) (Lepidoptera: Pyralidae) in laboratory and field [10, 12-15]. However, DDVP aerosol has been discontinued because of the development of pest resistance and its high toxicity to mammals and the environment. At present, widely used pesticides for such applications mainly include pyrethroids and insect growth regulators. There have been quite a few reports on successful control of various insects, for example, prallethrin aerosol for Cadra cautella, Indian meal moths Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), saw-toothed grain beetles Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae), Trogoderma variabile Ballion (Coleoptera: Dermestidae) and Tribolium castaneum under laboratory conditions [16], cyfluthrin aerosol for Tribolium castaneum under laboratory conditions [17], esfenvalerate aerosol for Plodia interpunctella under laboratory conditions [18], and pyrethrin aerosol for Tribolium castaneum and Tribolium confusum in laboratory and in a commercial food-storage factory [11, 19-22].

It’s well known that beta-cyfluthrin is a synthetic pyrethroid with low environmental persistence, low toxicity to non-target organisms, and no bioaccumulation. In addition, this pesticide is also a broad-spectrum neurotoxin with a demonstrable effect in killing L. serricorne [20, 23]. In fact, a chemical insecticide that can be used in cigarette manufacturing factories is limited, and the insecticide resistance of L. serricorne is of great concern. Beta-cyfluthrin aerosol, as an assistant to sanitation, has been used for years to control L. serricorne in Hefei Cigarette Manufacturing Factory (Hefei City, Anhui province, China). Therefore, it is imperative to determine the sensitivity level of L. serricorne to beta-cyfluthrin for maintaining control efficiency and resistance management.

Herein, we assessed beta-cyfluthrin susceptibility level in the population of L. serricorne collected from Hefei Cigarette Manufacturing Factory. After multiple rounds of bioassays with different insecticides (not shown in this paper except imidacloprid), we finally selected imidacloprid as an alternative insecticide. Subsequent field trials were carried out in the cigarette manufacturing area of Hefei Cigarette Manufacturing Factory to evaluate the control effect of space spray with imidacloprid aerosol in practice. Our study is conducive to timely recognizing the development of insecticide resistance of L. serricorne in cigarette manufacturing factories and provides key suggestions for pest resistance management.

MATERIALS AND METHODS

Insecticide, pheromone trap, atomizer, and spray tower

Insecticides used in this study were commercial formulations, as follows: beta-cyfluthrin (12.5% [a.i.]), suspension concentrate (SC), provided by Jiangsu Yangnong Chemical Co. LTD. (Jiangsu, China), imidacloprid (100g [a.i] L^-1, SC), provided by Zhejiang Haizheng Chemical co. LTD. (Zhejiang, China).

Pheromone traps were used to monitor the number of insects captured in the cigarette manufacturing area of Hefei Cigarette Manufacturing Factory, provided by Japan Fuji Flavor Co. LTD. (Japan). A lure permitting the controlled release of sex pheromones is mounted on the sticky board that catches beetles for counting.

Electric atomizer (Nanjing Minghai Health Technology Development Co. LTD., Nanjing, China) was used in field trials for delivering spray particles of 50 ± 10 μm in size, with spray efficiency of 500 mL/min, Hand-held ultra-low volume atomizer gun (Aima Instrument Factory, Guangdong, China) with calibration was used to produce mist in spray toxicity bioassay.

The cylindrical glass spray tower was made by Beijing Wuyi Glass Instrument Factory (Beijing, China) with spray settings at a volume of 10 L and, a height of 40 cm. The inner diameter of the tower bottom is 20 cm, and 3 cm for the tower mouth. The Spray tower has no upper and lower bases, but has a glass tower cap.

Insect strains and rearing conditions

Tobacco leaf fragments and tobacco shorts were carefully collected from processing machinery in the cigarette manufacturing area of Hefei Cigarette Manufacturing Factory and brought back to the laboratory. With the aid of a stereoscopic microscope, larvae of L. serricorne were searched and collected from fragments and tobacco shorts and reared into adults (LS_H). LS_H strain was cultured for more than three generations in the laboratory before bioassays. Reference susceptible strain (LS_LAB) was obtained from our laboratory with no pesticide exposure for several years. Both strains were maintained in dark incubators with a temperature of 28 ℃ and relative humidity of 65-75%. Wheat raw material, previously frozen for more than 7 days under -18 degrees, was crushed into small particles. L. serricorne was reared on wheat particles mixed with active yeast (10:1, w/w). Unsexed adults of 1-3 days were used for the following bioassays.

Spray toxicity bioassay

In this study, spray toxicity bioassays were conducted to study the toxicity of beta-cyfluthrin and imidacloprid against L. serricorne in the laboratory as suggested by relative literature [24, 25]. Preliminary trials were carried out to determine appropriate ranges of testing concentration, with 156.25 mg mL^-1, 83.64 mg mL^-1, 41.82 mg mL^-1, 13.94 mg mL^-1, 6.35 mg mL^-1, 3.17 mg mL^-1 for LS_H strain, and 57.11 mg mL^-1, 19.04 mg mL^-1, 6.35 mg mL^-1, 3.17 mg mL^-1, 0.63 mg mL^-1, 0.13 mg mL^-1, 0.03 mg mL^-1 for LS_LAB strain, and distilled water as the control in beta-cyfluthrin spray toxicity bioassay. In imidacloprid spray toxicity bioassay, testing concentrations of LS_H strain and LS_LAB strain were 1.75 mg mL^-1, 0.35 mg mL^-1, 3.50 ´ 10^-2 mg mL^-1, 3.50 ´ 10^-3 mg mL^-1, 3.50 ´ 10^-4 mg mL^-1 and 3.50 ´ 10^-5 mg mL^-1, and distilled water as the control. In the laboratory, at 27 °C, the spray tower was placed on a flat marble tabletop to ensure that the bottom of the spray tower fitted perfectly with the tabletop to prevent insects from escaping. After slowly introducing 20 adults to the tower bottom from the tower mouth, 0.5 mL liquid was sprayed into the spray chamber at the tower mouth with a hand-held ultra-low volume atomizer gun, at a distance of 35 cm. The tower mouth was covered with a glass cap immediately (not sealed). Dead individuals were documented 2 and 24 hours after treatment under dark conditions. An adult was considered dead if its appendages did not move when prodded with a hairbrush [26]. Three replicates were conducted for each dose.

Field trials

Field trials were carried out in the cigarette manufacturing area (shred-making section, storage section, blending section, cigarette-making section, and ripper section) of Hefei Cigarette Manufacturing Factory in Hefei, Anhui, China. Before the test, five sections worked normally and were cleaned normally following written sanitation procedures. Pheromone traps were used to monitor the number of insects captured [8, 11, 27-31]. The number and hanging height of the pheromone trap in each section were based on CORESTA [8]. Traps were placed on steel nails of the wall or column and marked for position. The numbers of L. serricorne caught by traps were recorded once a week, and traps were replaced every 30 days.

The details of the cigarette manufacturing area in the Hefei Cigarette Manufacturing Factory and the locations of traps in each section can be seen in Figure 1. Shred shredding section has a construction area of 1600 m² (20 m ´ 80 m), a height of 10 m, and thus, a volume of 16000 m³. A total of 9 pheromone traps were placed evenly in this section, with 8 traps placed on 80-meter-long walls, at 1.5 m above the ground, and one trap placed on the center column at 5 m above the ground. The storage section has a construction area of 600 m² (20 m ´ 30 m), a height of 5 m, thus, a volume of 3000 m³. A total of 3 pheromone traps were placed evenly on three non-entrance walls, at 1.5m above the ground. The blending section has a construction area of 600 m² (20 m ´ 30 m), a height of 5m, thus, a volume of 3000 m³. A total of 3 pheromone traps were placed evenly on three non-entrance walls, at 1.5m above the ground. The cigarette-making section has a construction area of 1200 m² (20 m ´ 60 m), a height of 5 m, thus, a volume of 6000 m³. A total of 6 pheromone traps were placed evenly on 60-meter-long walls, at 1.5 m above the ground. The ripper section has a construction area of 200 m² (20 m ´ 10 m), and a height of 5m, thus, a volume of 1000 m³. Only one pheromone trap was placed on the center column, at 1.5 m above the ground. Temperature was at 26 ± 2℃ and relative humidity was 60 ± 5% in all, but the ripper section.

In field trials, the application concentration and dose of beta-cyfluthrin and imidacloprid were determined according to the guidelines for the use of commercial pesticides and pre-experiment. Beta-cyfluthrin aerosols were sprayed in the space of five sections with a dose of 10 L/3000 m³(1 L of beta-cyfluthrin commercial formulation in 9 L water). Seven weeks later, imidacloprid aerosols were sprayed in the space of five sections with a dose of 10 L/3000 m³ (0.5 L of imidacloprid commercial formulation in 9.5 L water). Both applications were conducted during the factory break time, and with doors and windows closed. The next morning, windows (equipped with insect-proof gauze net) were opened for ventilation, and sanitation, following standardized sanitation protocols. The people spraying and counting are skilled workers in the factory.

Data analyses

Probit analysis (IBM SPSS^® Statistics 25, IBM Corp., NY, USA) was used for analyzing the bioassay data to estimate the concentration of killing 50% (LC₅₀) of L. serricorne. The chi-square test was applied to ensure the goodness-of-fit of the model.

In field trials, monitoring data of L. serricorne in five sections were analyzed by graphing software (Origin 2018, OriginLab Corp., Northampton, Massachusetts, USA).

N_W0 is the number of detected L. serricorne in the week before application, Nwn is the number of detected L. serricorne in week n after application.

RESULTS AND DISCUSSION

Spray toxicity bioassay

Results of spray toxicity bioassay are shown in Table 1. At 2 h after beta-cyfluthrin application, the LC₅₀ value of the LS_H strain was 71.98 mg mL^-1, which was 7.48 folds that of the LS_LAB strain. At 24 h, the LC₅₀ value of the LS_H strain was 19.78 mg mL^-1, which was 15.10 folds that of the LS_LAB strain. In addition, LC₅₀ values of LS_H strain were 1.33-fold and 1.00-fold that of LS_LAB strain at 2 h and 24 h after imidacloprid application, respectively.

Table 1. Toxicity of insecticides to LS_LAB strain and LS_H strain of L. serricorne at 2 h and 24 h in bioassays (in the laboratory, at 27 ℃)

^aLS_LAB, reference susceptible strain; LS_H, L. serricorne collected from Hefei Cigarette Manufacturing Factory.

^bFiducial Limit.

^cDegree of freedom.

^dP ³ 0.05 indicates a significant fit between the observed data and the expected linear regression model in Probit analysis.

^eNumber of insects used totally

^fResistance Ratio: LC₅₀ of LS_H strain/LC₅₀ of LS_LAB strain.

Field trials

Quantitative change of detected L. serricorne in the shred-making section

As shown in A1 of Figure 2, the number of monitored L. serricorne increased in the first five weeks after beta-cyfluthrin application from 17 in week 0 to 116 in week 5 and then decreased in week 6 and week 7. Compared to the beetle count in week 0, the numbers increased by 0%, 41.18%, 82.35%, -282.35%, -582.35%, -341.18%, and -300.0% for weeks 1-7 after application, respectively. As shown in A2 of Figure 2, the number of monitored L. serricorne decreased rapidly after application from 68 in week 0 to 7 in week 1, a decrease rate of 89.71%. In the following weeks, the decrease rate was 95.58%, 94.12%, 95.58%, 91.18%, 83.82%, and 69.12%, respectively.

Quantitative change of detected L. serricorne in the storage section

Results of B1 (Figure 3) showed that the decrease rate of detected L. serricorne in Week 1-7 was -15.91%, -11.36%, 11.36%, 68.18%, -50.0%, -20.45%, and -9.1%, respectively. It could be seen from B2 (Figure 3) that the number of monitored L. serricorne decreased obviously after imidacloprid application. The decrease rate of detected L. serricorne in weeks 1-7 were 95.83%, 95.83%, 95.83%, 100.0%, 97.92%, 100.0%, and 97.92%, respectively. The storage section has a construction area of 600 m² and a volume of 3000 m³. After imidacloprid treatment, only at most two L. serricorne were detected every week. This section is for storing shreds, as a Hazard Analysis Critical Control Point, with the greatest risk of L. serricorne invading cigarette products. Therefore, the fewer L. serricorne in this plant, the less risk of L. serricorne infecting products. In addition, none of the complicated machines and fewer shelters for L. serricorne in this plant were also the factors for the high control effect.

Quantitative change of detected L. serricorne in the blending section

After the application of beta-cyfluthrin, except in week 4 with decrease rate of detected L. serricorne of 89.47%, decrease rate in other weeks was maintained at 26.32%-47.37% (Figure 4). As shown in C2 (Figure 4), within 7 weeks after imidacloprid application, decrease rate of detected L. serricorne was maintained at 70.0%-100.0%. Only at most three L. serricorne were captured per week.

Quantitative change of detected L. serricorne in the cigarette-making section

This section has a complex structure, large space, and a high degree of machine integration, which is the last risk point for L. serricorne to invade cigarette products. The tobacco industry has strict requirements for pest control and sanitation. D1 in Figure 5a showed a weekly quantitative change of detected L. serricorne before and after the application of beta-cyfluthrin. As a whole, number of captured L. serricorne did not change obviously, and decrease rate of detected L. serricorne in week 1-7 was -15.79%, 57.89%, 63.16%, 10.53%, 36.84%, -31.58%, 31.58%, respectively. It could be seen from D2 (Figure 5a) that, the decrease rate of detected L. serricorne in week 1 was 84.62%, but only 46.15% and 15.38% in week 2 and week 3. We realized that the treatment effect differed significantly with other sections and adults were only found in a pheromone trap, and thus carried out an inspection immediately. After careful examination, it was found that most beetles were in obsolete equipment covered with plastic film that pesticide could not reach. Only those beetles that came out from the not-well sealing of the plastic cover were attracted and landed in the trap. Therefore, the obsolete equipment was removed and imidacloprid was applied again. Decreased rate of detected L. serricorne in weeks 4-7 was maintained at 84.62-100.0%, which reached the threshold index of pest control in this section (£ one L. serricorne/week/trap).

Quantitative change of detected L. serricorne in ripper section

In the cigarette manufacturing area, this section is relatively independent, and isolated from other plants in architecture and space. E1 (Figure 5b) showed that the number of L. serricorne in this section was few, but did not decrease obviously after application. E2 (Figure 5b) showed that after the application of imidacloprid, none of L. serricorne was detected in week 1-5, and only two were detected in week 6 and week 7.

Decrease rate of detected L. serricorne in cigarette manufacturing area after application with different insecticides

The decreased rate of detected L. serricorne weekly in cigarette manufacturing areas after application with different insecticides is shown in Table 2. After space spraying with beta-cyhalothrin aerosol, the decreasing rate of detected L. serricorne in the cigarette manufacturing area was maintained at -109% and 9%, with an average decreasing rate of -29%. After application of imidacloprid, the decreasing rate was maintained at 81.29-97.84%, with an average rate of 89.93%.

Table 2. Decrease rate of detected L. serricorne weekly in cigarette manufacturing area (five sections overall) after application