- Email: [email protected]
Activity of the German Cockroach, Blattella germanica (L.) (Orthoptera: Blattellidae), at Different Microhabitats in Semi-natural Conditions when Treated with Sublethal Doses of Chlorpyrifos and Permethrin*
J. Asia-Pacific Entomol. 1(1): 99-107 (1998)
Activity of the German Cockroach, Blattella germanica (L.) (Orthoptera: Blattellidae), at Different Microhabitats in Semi- natural Conditions when Treated with Sublethal Doses of Chlorpyrifos and Permethrin* Chon, Tae-Soo*, Young Seuk Park and Mary H. Ross' Abstract - Male adults of a mutant, or (orange body), and KNIH (Korea National Institute of Health) strains of the German cockroach, Blattella germanica (L.), were individually treated with chlorpyrifos and permethrin in sublethal doses. Local activity at different microhabitats for harborage, food, water, and other neighbor cockroaches was measured by observing visiting frequency for 4-5 days continuously after the chemical treatment. Also long-range activity was observed by counting the number of crossing imaginary center-lines within the observation cage during the observation period. The local activity was generally decreased after the treatment of chemicals at most microhabitats. Trends of the activity observed in the control also appeared in response to the chemical treatments; local activity was the highest at harborage, and was low at the other microhabitats. The dieI difference observed in the local activity, however, disappeared in the or strain by the treatment of both insecticides. Especially high values in local and long-range activity at scotophase were decreased greatly. In the KNIH strain, the local activity was less affected with chlorpyrifos. Diel difference persisted in the chlorpyrifos treatment, but disappeared in the permethrin treatment. The long-range activity was increased in the chlorpyrifos treatment in the KNIH strain. Key Words - Blattella germanica
(L.), Activity, Semi-natural conditions, Chlorpyrifos, Perme-
thrin
Introduction Although there have been numerous studies on the application of insecticides to the German cockroach, Blattella germanica (L.), few studies have been directly related to the responding behavior after the treatment of chemicals, especially in the long-term observation. Most studies were mainly aimed to determine the dose-mortality relationship, but the long-term behavior after the chemical treatment has been seldom observed, except for some short-term studies on immediate movement (Bret and Ross, 1986), population dispersal (Wooster and
Ross, 1989) and avoidance (e.g., Ross, 1993) after the chemical treatment. Recently Chon et al. (1997) continuously observed resting behavior of adult males of the German cockroach in harborage in two strains, or (a mutant having an orange body) and KNIH. In overall terms the within-harborage time, quiescent phase of the resting behavior, showed diel difference in both strains. The active phase of the resting behavior, represented by visiting frequency at harborage, however, responded differently between the two strains; while no clear diurnal difference was observed in the KNIH strain, diurnal difference appeared in the or strain. This suggested that the quie-
*To whom correspondence should be addressed. Department of Biology, Pusan National University, Pusan, 609-735, Korea. E-mail: [email protected] I Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0319, USA
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scent and active phase could separately represent the time pattern of the resting behavior of the German cockroach and this resting behavior could be differentiated in the systematic study of behavioral study, with the possibility of genetic difference. Chon et al. (Unpublished observation) subsequently treated these two strains with chlorpyrifos and permethrin in sublethal doses individually, and investigated how these two phases in resting behavior would be extrapolated under the effect of toxic chemicals in a relatively long period. Although individual variation was high in these observations, the response showed some consistencies and differences. Visiting frequency and within-harborage time were generally decreased in both strains with the chlorpyrifos treatment. With the permethrin treatment, however, the two strains showed different responses. Those were decreased in the KNIH strain, while within-harborage time was not changed in the or strain. Also visiting frequency in scotophase was selectively decreased in a large amount by the chemical treatment. This study indicated that the chemical treatment would impose a wide spectrum of resting behavior, and could reveal the different mechanism of activity in the German cockroach. The systematic measurements in the continuous observation could produce indicators cha-
Adult males about 7 days after emergence were treated with the insecticides and observed individually for 4-5 days in a rectangle-shape cage (200 mm x 200 mm x 7 mm), made of transparent acrylic board (2 mm in thickness) where microhabitats for harborage (H), feeding (F), drinking (W) and other individuals (0), were provided at each comer of the cage (see Chon et al., 1997). An organophosphate, chlorpyrifos, and a pyrethroid, permethrin, were treated to each insect in sublethal doses equivalent to LD lO • Based on Scott et al. (1986), permethrin was applied with 118 ng per male in this test, while chlorpyrifos was treated with 92 ng per male according to Shim and Lee (1979). With this amount of sublethal doses, 4 individuals were killed out of 24 individuals in 48 hours after the chlorpyrifos treatment for both strains, while two and three individuals were dead out of 20 individuals, respectively, for the or and KNIH strain in the permethrin treatment. The applied dose of LD lO based on other sources still showed the similar level of mortality response in this study also. The chemicals, dissolved in acetone (2111), were applied to the ventral part of the insect abdomen for testing. Sixteen to twenty eight individuals were tested for each treatment of the chemicals and for the control. The other detailed method for the rearing and observation could be
racterizing these response behavior.
referred to Chon et al. (1997).
In the present study we tried to investigate further how these response patterns in the local activity at harborage could be extended to other microhabitats in the observation cage such as feeding, drinking and neighbor individuals in the chemical treatment. We also measured the relationship of their local activity with the long-range activity.
The local and long-range activities were observed through CCTV and analyged with computeraided data analysis system. The long-range activity, representing a large-scale movement of the test insect inside the observation cage, was measured by counting the number of the cockroaches crossing the imaginary center-lines (horizontal and vertical) in the cage every 3 hrs under 12L112D regime. The local activity was represented by a small-scale movement in and out of microhabitats for harborage, feeding, drinking and other neighbor individuals. Visiting frequency was defined as the number of entering the area of the harborage during each 3 hr time block. Each measurement representing activity was first averaged for each individual during the observation period, and subsequently averaged again for all the observed individuals for each 3 hr time block. The detailed measurement process was reported in Chon et al. (1997). For quantitative analysis of the observed data, ANOVA
Materials and Methods A mutant, insecticide susceptible strain, orange body (or) (Ross and Cochran, 1962), and the KNIH strain (Bang et al., 1993), have been reared at temperature of 27 ± 1°C and relative humidity of 60 ± 10 %, under the light regime of 12L/12D (at 1,500 lux of fluorescence in photophase and less than 10 lux of red light at scotophase). The KNIH strain had been reared in the Korea National Institute of Health since 1978 without exposure to insecticides.
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(Norusis, 1986) was conducted. Probability for significance in ANOVA was expressed up to three significant digits after the decimal point in this report (e.g., p= 0.000).
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Local Activity In general, local activity was decreased after the treatment of the chemicals. In the or strain nontreated, the highest freguency of visiting was observed at harborage (H), followed by the microhabitats of drinking (W) and then other individuals (0) (Fig. 1). Although visiting frequencies were consistently decreased after the treatment of the insecticides, pattern of the activity observed in the control persisted. Visiting frequency at harborage was the highest among all the microhabitats, as observed in the control. Local activity at the other microhabitats was generally low. Visiting frequencies at drinking place and other individuals were decreased in higher degrees after the treatment of the insecticides. With the treatment of chlorpyrifos, visiting frequencies were generally decreased at all microhabitats (Fig. 1). There was no apparent activity difference between drinking and feeding places after the treatment of the insecticides on the average. The response in activity to the permethrin treatment was
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generally similar to that of the treatment of chlorpyrifos with visiting frequency consistently decreased at all microhabitats (Fig. 1). In the KNIH strain the local activity tended to be lower when compared with the or strain in the control, and the general trend of local activity was also similar to that of the or strain (Fig. 2). It was the highest at H, and lower at other microhabitats. At the microhabitat W, however, the visiting frequency was not as high as in the or strain, and the local activity at Wand F were similar in average values in contrast to the or strain. Local activity at 0 appeared generally high, as shown in the case of the or strain. The effect of insecticide treatment to the KNIH strain was in general also similar to that of the or strain; local activity was decreased at most microhabitats with few exceptions. When treated with chlorpyrifos, visiting frequency at H, for example, was 10.0, while it was 17.1 for the control (Fig. 2). At 0, however, the visiting frequency appeared to be slightly increased after the treatment. At the other microhabitats, visiting frequencies were lower. The impact of the treatment of permethrine was similar to that of the chlorpyrifos treatment, being decreased in a larger amount consistently at all microhabitats.Visiting frequency at H was highest, and was low at the other microhabitats.
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Diel Difference The or strain showed diel difference in their local activity; higher during scotophase at all microhabitats (Fig. 3). At H, for example, visiting frequency was higher at scotophase with 22.1, and lower at photophase 16.0 (Fig. 3a). This local activity in scotophase, however, was abruptly decreased after the treatment of chemicals at all microhabitats, both by
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chlorpyrifos and permethrin. In most cases, after the decrease in visiting frequency in scotophase, local activity showed similar levels in the two phases. At W, visiting frequency was decreased in photophase after the treatment of insecticides. The diel difference in visiting frequency was not clear in the KNIH strain in the control at harborage, H (Fig. 4a), while it appeared at all other micro-
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Fig. 3. Daily temporal patterns of local activity (a-d) (number of visits during 3 hrs) at different microhabitats (a; harborage, b; other individuals, c; drinking and d; feeding) and long-range activity (e) when the or strain was treated with ch1orpyrifos and permethrin in sublethal doses. The activity was measured every 3 hrs during the photophase (P) and the scotophase (5).
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Time block Fig. 4. Daily temporal pattern of local activity (a-d) (number of visit during 3 hrs) at different microhabitats and longrange activity (e) when the KNIH strain was treated with chlorpyrifos and permethrin in sublethal doses. (a; harborage, b; other individuals, c; drinking and d; feeding).
habitats of W, F and a (Fig. 4b-d), as shown in the or strain (Fig. 3); lower at photophase and higher at scotophase. The KNIH strain responded differently to the treatment of different insecticides. The visiting frequency was decreased consistently in the case of the permethrin treatment, similar to the case
of the or strain. In the chlorpyrifos treatment, however, the impact was relatively low and the diel difference tended to persist, similar to that of the control except H. At H, the visiting frequency was also decreased by chlorpyrifos.
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Long-range Activity The long-range activity appeared to be higher in the or strain than in the KNIH strain in the control (Figs. 3e and 4e). The total number of crossing lines per 3 hrs in the or strain was 37.3 while that of the KNIH strain was 25.4. Diurnal difference appeared clearly in the long range activity in both strains, higher at scotophase and lower at photophase. The insecticide treatment also caused substantial difference in the response of the long-range activity. With the permethrin treatment the activity was decreased in both strains in total; from 37.3 to 17.9 in the or strain (Fig. 3e), and from 25.4 to 16.7 in the KNIH strain (Fig. 4e). With the chloropyrifos treatment, this activity actually appeared to be increased from 25.4 to 45.8 in the KNIH strain. In the or strain, the response was about the same level to the control, both showing 37.2-38.8 in average values. The insecticide treatment also elicited different responses in different light phases. The or strain showed no clear difference in the long-range activity during the photophase, when treated with insecticides. During the scotophase, however, there was a great difference in the long-range activity, and diel difference disappeared by chlorpyrifos and permethrin treatment in the or strain. This abrupt decrease in the long-range activity in scotophase was also in accordance with that shown in the case of local activity in the or strain (Fig. 3a-d). In the KNIH strain, diel difference appeared differently depending upon the insecticides. Diel difference appeared to persist in the long-range activity in the chlorpyrifos treatment, as shown in the control. In the permethrin treatment, however, the diel difference was generally less obvious (Fig. 4e).
Discussion Response patterns in the long-range and local activities after the insecticide treatment showed some consistencies and differences depending upon the insecticides and insect strains. The over-all decrease in visiting frequency (Figs. 1, 2) suggested that the insecticides had some long-term and systematic effects on reducing small-scale movements around microhabitats. The long-range acti-
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vity, however, tended to be increased in the chlorpyrifos treatment (Figs. 3e, 4e). This clearly demonstrated that the toxic chemicals might differently control the locomotive mechanism of small- and large-scale movements of the German cockroach. Overall, the treatment effect of insecticides clearly appeared in the response of the activity in this study. When ANOVA was conducted on the largescale movement and the local activity at harborage in the different light phases by considering the strain and the insecticide as the two-way treatment factors (Table. 1), the response in the two activities were significantly different in the treatment of insecticides. In the strains, however, the effect was not clearly shown. This trend of the insecticide impacts generally appeared similar in the two photoand scoto-phases, both for the large-scale movement and local activity at harborage. In the largescale movement, however, there was cross effect between the strain and insecticide treatment in scotophase while there was no interacting effect in the local activity at harborage at this light phase. Besides the clear tendency of higher local activity at harborage than at other microhabitats, as well as the general decrease in this local activity after the treatment of insecticides, the detailed responses in the local activity at different microhabitat were diverse, depending upon the experimental conditions such as strains, chemicals and light phases as mentioned before. Visiting frequencies at the microhabitats other than harborage were usually very low and consequently these contributed to lowering the signal to noise ratio, making it difficult to find coherence in the data. Due to this problem, it was not easy to statistically segregate, or to find some regularity in the overall impact of insecticides on the local and large-scale activity in interrelationship of the different microhabitats in this study. Further study, including a modified setting of the experimental condition, the increase in the observation number and physiological investigation, may be needed to verify in detail the systematic effect of insecticides on the activity of the German cockroach in semi -natural conditions. The notable characteristic, clearly and consistently observed in the local activity in observation cages, however, was the highest visiting frequency at harborage among the microhabitats. This trend
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Table 1. Analysis of variance on strains (STRAIN) and insecticide treatments (TREAT) in visiting frequencies at harborage and large-scale activities when male adults of the two German cockroach strains were treated with chlorpyrifos and permethrin insecticides in sublethal doses DF
Mean Square
666.359 8.583 640.679 75.223 75.223 741.581 5,919.307 6,660.889
3 1 2 2 2 5 116 121
222.120 8.583 320.339 37.611 37.611 148.316 51.029 55.049
4.353 .168 6.278 .737 .737 2.907
.006 .682 .003 .481 .481 .016
Scotophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
4,452.023 209.191 4,028.879 267.906 267.906 4,719.928 10,522.550 15,242.479
3 1 2 2 2 5 116 121
1,484.008 209.191 2,014.440 133.953 133.953 943.986 90.712 125.971
16.360 2.306 22.207 1.477 1.477 10.406
.000 .132 .000 .233 .233 .000
Large-scale Activity Photophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
20.125 4.009 14.935 4.930 4.930 25.055 139.470 164.526
3 1 2 2 2 5 110 115
6.708 4.009 7.468 2.465 2.465 5.011 1.268 1.431
5.291 3.162 5.890 1.944 1.944 3.952
.002 .078 .004 .148 .148 .002
Scotophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
19.191 3.303 13.712 9.299 9.299 28.490 95.166 123.656
3 1 2 2 2 5 110 115
6.397 3.303 6.856 4.650 4.650 5.698 .865 1.075
7.394 3.818 7.924 5.374 5.374 6.586
.000 .053 .001 .006 .006 .000
Visiting Frequency at Harborage Photophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
was observed in both strains, whether the insecticides had been treated or not. This demonstrated that there was a kind of differentiation in resting behavior at different microhabitats in the German cockroach. Although the test insect took a rest at harbo-
F
Significance of F
Sum of Squares
Source of Variation
rage, it had still a tendency to move frequently in and out, while it did not move around much at the other microhabitats where it had a specific object such as drinking or feeding. The tendency of high local activity at harborage after the treatment of
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insecticides may be for its own safety, but the exact mechanism is presently not known. Local activity was also relatively high at the place of neighbors, and this was understandable when considered that the communication with other individuals would probably accompany frequent small-scale movement of the test insect. In the or strain the local activity at W was higher than at F (Fig. 1). In the KNIH strain, however, this was of a similar level at these places. In the or strain the local activity at W was decreased to the similar level of F after the treatment of insecticides. Presently it is not known why these strains showed different activity behaviors at feeding and drinking places, and the activity after the insecticide treatment in the or strain. A further research may be needed in relating to physiological understanding on the reactive mechanism to stimuli. In the or strain, the local activity tended to show diel difference; higher during the scotophase at all microhabitats in the control (Fig. 3). This local activity during the scotophase, however, was greatly decreased after the treatment of insecticides at all microhabitats, both by chlorpyrifos and permethrin. During the photophase, the activ were usually not much decreased. This suggested that the insecticide treatment might strongly affect the mechanism to control circadian rhythm in the or strain. This tendency of affecting diel difference was also observed in the KNIH strain, selectively on permethrin (Fig. 4). In the control the KNIH strain showed diel difference at the microhabitats of W, F and O. This diel difference disappeared after the treatment of permethrin, with a major effect on the activity in scotophase, as shown in the or strain (Fig. 3). In the chlorpyrifos treatment, however, diel difference persisted. This indicated that a complex mechanism might be involved to express diurnal rhythm of the local activity, and the treatment of insecticides may differentially intervene the activation of rhythmic movement of the German cockroach. In the KNIH strain, the diel difference in visiting frequency was not observed exclusively in harborage, H, although it was observed at all other microhabitats (Fig. 4). The reason why this diel difference did not appear in H in the KNIH strain is presently
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not known. Further experiments may be needed to verify this. In this study, we did not actually measure the action of the test insect in specific microhabitats, for example the amount of feeding at F. In the present situation it was difficult to set all the sensors for detecting actual behavior for feeding, drinking, resting, and communicating with neighbor individuals to carry out the continuous observation. A further study may be needed to represent this actual behavior with the improvement of measurement methods. Since cockroach has certain aggregating behavior in some degree in natural conditions, they have been usually observed in groups (e.g., Denzer et al., 1987, 1988a, 1988b). In this study, for the purpose of tracing each individual behavior, however, single individual was observed, and other individuals were provided as neighbors to alleviate the problem caused by single rearing. It appeared that, in terms of responses to the insecticide treatment, the observation results showed consistencies in some degree in local and large-scale activities, as discussed above. However, there was a high degree of variation in individual data in the course of long observation, and dual daily-peaks in activity usually observed in cockroaches were not clearly detected. Also the variability was high in the observed values with larger coefficient of variation, usually larger than 1, it was statistically difficult to differentiate the differences in some cases. In the future, the development of observation techniques may be needed to increase the credibility of observed data, along with the individual tracing in group rearing and other verification tests in physiology.
Acknowledgements - This research was supported by Korea Science and Engineering Foundation, Project No. 941-0600-039-2. We thank the Lucky Central Institute and Hannong Company for providing test insecticides, permethrin and chlorpyrifos, respectively.
Literature Cited Bang, J.R., H.R. Lee and LW. Kim. 1993. Studies on the
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insecticide resistance of the German cockroach (Blattella germanica L.). II. Resistance development and cross resistance. Korean J. Appl. Entomol. 32: 129-133. Bret, B.L. and M.H. Ross. 1986. Behavioral responses of the German cockroach, Blatella germanica (L.) (Orthoptera: Blattellidae), to a propoxur formulation. J. Econ. Entomol. 79: 426-430. Chon, T.-S., Y.S. Park and M.H. Ross. 1997. Temporal pattern of within-harborage time and visiting frequency in two strains of the German cockroach, Blattella germanica, in semi-natural conditions. Korean J. Appl. Entomol. 36: 299-310. Denzer, D., M.E.A. Fuchs and G. Stein. 1987. Studies on the diurnal rhythm of Blattella germanica L. (Orthopt., Blattellidae). 1. Nymphs and males. J. Appl. Entomol. 104: 495 -503. Denzer, D., M.B.A. Fuchs and G. Stein. 1988a. Studies on the diurnal rhythm of Blattella germanica L. (Orthopt., Blattellidae). 2. Females. J. Appl. Entomol. 105: 174-181. Denzer, D., M.E.A. Fuchs and G. Stein. 1988b. Studies on the diurnal rhythm of Blattella germanica L. (Orthopt., Blattellidae), 3. Mixed population. J. Appl. Entomol. 105:
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262-269. Norusis, M.J. 1986. SPSS/PC+ advanced statistics. SPSS Inc. Chicago. 360p. Ross, M.H. 1993. Comparisons between the response of German cockroach field -collected strains (Dictyoptera: Blattellidae) to vapors and contact with a cyfluthrin formulation. 1. Entomol. Sci. 28: 168-174. Ross, M.H. and D.G. Cochran. 1962. A body color mutation in the German cockroach. Nature 195: 518-519. Scott, J.G., S.B. Ramaswamy, F. Matsumura and K. Tanaka. 1986. Effect of method of application on resistance to pyrethroid insecticides in Blattella germanica (Orthoptera: Blattellidae). J. Econ. Entomol. 79: 571-575. Shim, J.-c. and K.-R. Lee. 1979. Toxicity test of public health insecticides against cockroach (Blattella germanica L.) in Korea. Korean J. Entomol. 9: 23-28. Wooster, M.T. and M.H. Ross. 1989. Sublethal responses of the German cockroach to vapors of commercial insecticide formulations. Entomol. Exp. Appl. 52: 49-56. (ReceivedJanuary 21, 1998;AcceptedMarch 26, 1998)
Activity of the German Cockroach, Blattella germanica (L.) (Orthoptera: Blattellidae), at Different Microhabitats in Semi- natural Conditions when Treated with Sublethal Doses of Chlorpyrifos and Permethrin* Chon, Tae-Soo*, Young Seuk Park and Mary H. Ross' Abstract - Male adults of a mutant, or (orange body), and KNIH (Korea National Institute of Health) strains of the German cockroach, Blattella germanica (L.), were individually treated with chlorpyrifos and permethrin in sublethal doses. Local activity at different microhabitats for harborage, food, water, and other neighbor cockroaches was measured by observing visiting frequency for 4-5 days continuously after the chemical treatment. Also long-range activity was observed by counting the number of crossing imaginary center-lines within the observation cage during the observation period. The local activity was generally decreased after the treatment of chemicals at most microhabitats. Trends of the activity observed in the control also appeared in response to the chemical treatments; local activity was the highest at harborage, and was low at the other microhabitats. The dieI difference observed in the local activity, however, disappeared in the or strain by the treatment of both insecticides. Especially high values in local and long-range activity at scotophase were decreased greatly. In the KNIH strain, the local activity was less affected with chlorpyrifos. Diel difference persisted in the chlorpyrifos treatment, but disappeared in the permethrin treatment. The long-range activity was increased in the chlorpyrifos treatment in the KNIH strain. Key Words - Blattella germanica
(L.), Activity, Semi-natural conditions, Chlorpyrifos, Perme-
thrin
Introduction Although there have been numerous studies on the application of insecticides to the German cockroach, Blattella germanica (L.), few studies have been directly related to the responding behavior after the treatment of chemicals, especially in the long-term observation. Most studies were mainly aimed to determine the dose-mortality relationship, but the long-term behavior after the chemical treatment has been seldom observed, except for some short-term studies on immediate movement (Bret and Ross, 1986), population dispersal (Wooster and
Ross, 1989) and avoidance (e.g., Ross, 1993) after the chemical treatment. Recently Chon et al. (1997) continuously observed resting behavior of adult males of the German cockroach in harborage in two strains, or (a mutant having an orange body) and KNIH. In overall terms the within-harborage time, quiescent phase of the resting behavior, showed diel difference in both strains. The active phase of the resting behavior, represented by visiting frequency at harborage, however, responded differently between the two strains; while no clear diurnal difference was observed in the KNIH strain, diurnal difference appeared in the or strain. This suggested that the quie-
*To whom correspondence should be addressed. Department of Biology, Pusan National University, Pusan, 609-735, Korea. E-mail: [email protected] I Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0319, USA
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scent and active phase could separately represent the time pattern of the resting behavior of the German cockroach and this resting behavior could be differentiated in the systematic study of behavioral study, with the possibility of genetic difference. Chon et al. (Unpublished observation) subsequently treated these two strains with chlorpyrifos and permethrin in sublethal doses individually, and investigated how these two phases in resting behavior would be extrapolated under the effect of toxic chemicals in a relatively long period. Although individual variation was high in these observations, the response showed some consistencies and differences. Visiting frequency and within-harborage time were generally decreased in both strains with the chlorpyrifos treatment. With the permethrin treatment, however, the two strains showed different responses. Those were decreased in the KNIH strain, while within-harborage time was not changed in the or strain. Also visiting frequency in scotophase was selectively decreased in a large amount by the chemical treatment. This study indicated that the chemical treatment would impose a wide spectrum of resting behavior, and could reveal the different mechanism of activity in the German cockroach. The systematic measurements in the continuous observation could produce indicators cha-
Adult males about 7 days after emergence were treated with the insecticides and observed individually for 4-5 days in a rectangle-shape cage (200 mm x 200 mm x 7 mm), made of transparent acrylic board (2 mm in thickness) where microhabitats for harborage (H), feeding (F), drinking (W) and other individuals (0), were provided at each comer of the cage (see Chon et al., 1997). An organophosphate, chlorpyrifos, and a pyrethroid, permethrin, were treated to each insect in sublethal doses equivalent to LD lO • Based on Scott et al. (1986), permethrin was applied with 118 ng per male in this test, while chlorpyrifos was treated with 92 ng per male according to Shim and Lee (1979). With this amount of sublethal doses, 4 individuals were killed out of 24 individuals in 48 hours after the chlorpyrifos treatment for both strains, while two and three individuals were dead out of 20 individuals, respectively, for the or and KNIH strain in the permethrin treatment. The applied dose of LD lO based on other sources still showed the similar level of mortality response in this study also. The chemicals, dissolved in acetone (2111), were applied to the ventral part of the insect abdomen for testing. Sixteen to twenty eight individuals were tested for each treatment of the chemicals and for the control. The other detailed method for the rearing and observation could be
racterizing these response behavior.
referred to Chon et al. (1997).
In the present study we tried to investigate further how these response patterns in the local activity at harborage could be extended to other microhabitats in the observation cage such as feeding, drinking and neighbor individuals in the chemical treatment. We also measured the relationship of their local activity with the long-range activity.
The local and long-range activities were observed through CCTV and analyged with computeraided data analysis system. The long-range activity, representing a large-scale movement of the test insect inside the observation cage, was measured by counting the number of the cockroaches crossing the imaginary center-lines (horizontal and vertical) in the cage every 3 hrs under 12L112D regime. The local activity was represented by a small-scale movement in and out of microhabitats for harborage, feeding, drinking and other neighbor individuals. Visiting frequency was defined as the number of entering the area of the harborage during each 3 hr time block. Each measurement representing activity was first averaged for each individual during the observation period, and subsequently averaged again for all the observed individuals for each 3 hr time block. The detailed measurement process was reported in Chon et al. (1997). For quantitative analysis of the observed data, ANOVA
Materials and Methods A mutant, insecticide susceptible strain, orange body (or) (Ross and Cochran, 1962), and the KNIH strain (Bang et al., 1993), have been reared at temperature of 27 ± 1°C and relative humidity of 60 ± 10 %, under the light regime of 12L/12D (at 1,500 lux of fluorescence in photophase and less than 10 lux of red light at scotophase). The KNIH strain had been reared in the Korea National Institute of Health since 1978 without exposure to insecticides.
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(Norusis, 1986) was conducted. Probability for significance in ANOVA was expressed up to three significant digits after the decimal point in this report (e.g., p= 0.000).
20
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Local Activity In general, local activity was decreased after the treatment of the chemicals. In the or strain nontreated, the highest freguency of visiting was observed at harborage (H), followed by the microhabitats of drinking (W) and then other individuals (0) (Fig. 1). Although visiting frequencies were consistently decreased after the treatment of the insecticides, pattern of the activity observed in the control persisted. Visiting frequency at harborage was the highest among all the microhabitats, as observed in the control. Local activity at the other microhabitats was generally low. Visiting frequencies at drinking place and other individuals were decreased in higher degrees after the treatment of the insecticides. With the treatment of chlorpyrifos, visiting frequencies were generally decreased at all microhabitats (Fig. 1). There was no apparent activity difference between drinking and feeding places after the treatment of the insecticides on the average. The response in activity to the permethrin treatment was
20
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Fig. 2. Local activity at different microhabitats expressed by visiting frequency (number of visits during 3 hrs) when the KNIH strain was treated with chlorpyrifos and permethrin in sublethal doses.
generally similar to that of the treatment of chlorpyrifos with visiting frequency consistently decreased at all microhabitats (Fig. 1). In the KNIH strain the local activity tended to be lower when compared with the or strain in the control, and the general trend of local activity was also similar to that of the or strain (Fig. 2). It was the highest at H, and lower at other microhabitats. At the microhabitat W, however, the visiting frequency was not as high as in the or strain, and the local activity at Wand F were similar in average values in contrast to the or strain. Local activity at 0 appeared generally high, as shown in the case of the or strain. The effect of insecticide treatment to the KNIH strain was in general also similar to that of the or strain; local activity was decreased at most microhabitats with few exceptions. When treated with chlorpyrifos, visiting frequency at H, for example, was 10.0, while it was 17.1 for the control (Fig. 2). At 0, however, the visiting frequency appeared to be slightly increased after the treatment. At the other microhabitats, visiting frequencies were lower. The impact of the treatment of permethrine was similar to that of the chlorpyrifos treatment, being decreased in a larger amount consistently at all microhabitats.Visiting frequency at H was highest, and was low at the other microhabitats.
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Diel Difference The or strain showed diel difference in their local activity; higher during scotophase at all microhabitats (Fig. 3). At H, for example, visiting frequency was higher at scotophase with 22.1, and lower at photophase 16.0 (Fig. 3a). This local activity in scotophase, however, was abruptly decreased after the treatment of chemicals at all microhabitats, both by
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chlorpyrifos and permethrin. In most cases, after the decrease in visiting frequency in scotophase, local activity showed similar levels in the two phases. At W, visiting frequency was decreased in photophase after the treatment of insecticides. The diel difference in visiting frequency was not clear in the KNIH strain in the control at harborage, H (Fig. 4a), while it appeared at all other micro-
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Fig. 3. Daily temporal patterns of local activity (a-d) (number of visits during 3 hrs) at different microhabitats (a; harborage, b; other individuals, c; drinking and d; feeding) and long-range activity (e) when the or strain was treated with ch1orpyrifos and permethrin in sublethal doses. The activity was measured every 3 hrs during the photophase (P) and the scotophase (5).
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Time block Fig. 4. Daily temporal pattern of local activity (a-d) (number of visit during 3 hrs) at different microhabitats and longrange activity (e) when the KNIH strain was treated with chlorpyrifos and permethrin in sublethal doses. (a; harborage, b; other individuals, c; drinking and d; feeding).
habitats of W, F and a (Fig. 4b-d), as shown in the or strain (Fig. 3); lower at photophase and higher at scotophase. The KNIH strain responded differently to the treatment of different insecticides. The visiting frequency was decreased consistently in the case of the permethrin treatment, similar to the case
of the or strain. In the chlorpyrifos treatment, however, the impact was relatively low and the diel difference tended to persist, similar to that of the control except H. At H, the visiting frequency was also decreased by chlorpyrifos.
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Long-range Activity The long-range activity appeared to be higher in the or strain than in the KNIH strain in the control (Figs. 3e and 4e). The total number of crossing lines per 3 hrs in the or strain was 37.3 while that of the KNIH strain was 25.4. Diurnal difference appeared clearly in the long range activity in both strains, higher at scotophase and lower at photophase. The insecticide treatment also caused substantial difference in the response of the long-range activity. With the permethrin treatment the activity was decreased in both strains in total; from 37.3 to 17.9 in the or strain (Fig. 3e), and from 25.4 to 16.7 in the KNIH strain (Fig. 4e). With the chloropyrifos treatment, this activity actually appeared to be increased from 25.4 to 45.8 in the KNIH strain. In the or strain, the response was about the same level to the control, both showing 37.2-38.8 in average values. The insecticide treatment also elicited different responses in different light phases. The or strain showed no clear difference in the long-range activity during the photophase, when treated with insecticides. During the scotophase, however, there was a great difference in the long-range activity, and diel difference disappeared by chlorpyrifos and permethrin treatment in the or strain. This abrupt decrease in the long-range activity in scotophase was also in accordance with that shown in the case of local activity in the or strain (Fig. 3a-d). In the KNIH strain, diel difference appeared differently depending upon the insecticides. Diel difference appeared to persist in the long-range activity in the chlorpyrifos treatment, as shown in the control. In the permethrin treatment, however, the diel difference was generally less obvious (Fig. 4e).
Discussion Response patterns in the long-range and local activities after the insecticide treatment showed some consistencies and differences depending upon the insecticides and insect strains. The over-all decrease in visiting frequency (Figs. 1, 2) suggested that the insecticides had some long-term and systematic effects on reducing small-scale movements around microhabitats. The long-range acti-
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vity, however, tended to be increased in the chlorpyrifos treatment (Figs. 3e, 4e). This clearly demonstrated that the toxic chemicals might differently control the locomotive mechanism of small- and large-scale movements of the German cockroach. Overall, the treatment effect of insecticides clearly appeared in the response of the activity in this study. When ANOVA was conducted on the largescale movement and the local activity at harborage in the different light phases by considering the strain and the insecticide as the two-way treatment factors (Table. 1), the response in the two activities were significantly different in the treatment of insecticides. In the strains, however, the effect was not clearly shown. This trend of the insecticide impacts generally appeared similar in the two photoand scoto-phases, both for the large-scale movement and local activity at harborage. In the largescale movement, however, there was cross effect between the strain and insecticide treatment in scotophase while there was no interacting effect in the local activity at harborage at this light phase. Besides the clear tendency of higher local activity at harborage than at other microhabitats, as well as the general decrease in this local activity after the treatment of insecticides, the detailed responses in the local activity at different microhabitat were diverse, depending upon the experimental conditions such as strains, chemicals and light phases as mentioned before. Visiting frequencies at the microhabitats other than harborage were usually very low and consequently these contributed to lowering the signal to noise ratio, making it difficult to find coherence in the data. Due to this problem, it was not easy to statistically segregate, or to find some regularity in the overall impact of insecticides on the local and large-scale activity in interrelationship of the different microhabitats in this study. Further study, including a modified setting of the experimental condition, the increase in the observation number and physiological investigation, may be needed to verify in detail the systematic effect of insecticides on the activity of the German cockroach in semi -natural conditions. The notable characteristic, clearly and consistently observed in the local activity in observation cages, however, was the highest visiting frequency at harborage among the microhabitats. This trend
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Table 1. Analysis of variance on strains (STRAIN) and insecticide treatments (TREAT) in visiting frequencies at harborage and large-scale activities when male adults of the two German cockroach strains were treated with chlorpyrifos and permethrin insecticides in sublethal doses DF
Mean Square
666.359 8.583 640.679 75.223 75.223 741.581 5,919.307 6,660.889
3 1 2 2 2 5 116 121
222.120 8.583 320.339 37.611 37.611 148.316 51.029 55.049
4.353 .168 6.278 .737 .737 2.907
.006 .682 .003 .481 .481 .016
Scotophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
4,452.023 209.191 4,028.879 267.906 267.906 4,719.928 10,522.550 15,242.479
3 1 2 2 2 5 116 121
1,484.008 209.191 2,014.440 133.953 133.953 943.986 90.712 125.971
16.360 2.306 22.207 1.477 1.477 10.406
.000 .132 .000 .233 .233 .000
Large-scale Activity Photophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
20.125 4.009 14.935 4.930 4.930 25.055 139.470 164.526
3 1 2 2 2 5 110 115
6.708 4.009 7.468 2.465 2.465 5.011 1.268 1.431
5.291 3.162 5.890 1.944 1.944 3.952
.002 .078 .004 .148 .148 .002
Scotophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
19.191 3.303 13.712 9.299 9.299 28.490 95.166 123.656
3 1 2 2 2 5 110 115
6.397 3.303 6.856 4.650 4.650 5.698 .865 1.075
7.394 3.818 7.924 5.374 5.374 6.586
.000 .053 .001 .006 .006 .000
Visiting Frequency at Harborage Photophase Main effects STRAIN TREAT 2-way interactions STRAIN TREAT Explained Residual Total
was observed in both strains, whether the insecticides had been treated or not. This demonstrated that there was a kind of differentiation in resting behavior at different microhabitats in the German cockroach. Although the test insect took a rest at harbo-
F
Significance of F
Sum of Squares
Source of Variation
rage, it had still a tendency to move frequently in and out, while it did not move around much at the other microhabitats where it had a specific object such as drinking or feeding. The tendency of high local activity at harborage after the treatment of
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insecticides may be for its own safety, but the exact mechanism is presently not known. Local activity was also relatively high at the place of neighbors, and this was understandable when considered that the communication with other individuals would probably accompany frequent small-scale movement of the test insect. In the or strain the local activity at W was higher than at F (Fig. 1). In the KNIH strain, however, this was of a similar level at these places. In the or strain the local activity at W was decreased to the similar level of F after the treatment of insecticides. Presently it is not known why these strains showed different activity behaviors at feeding and drinking places, and the activity after the insecticide treatment in the or strain. A further research may be needed in relating to physiological understanding on the reactive mechanism to stimuli. In the or strain, the local activity tended to show diel difference; higher during the scotophase at all microhabitats in the control (Fig. 3). This local activity during the scotophase, however, was greatly decreased after the treatment of insecticides at all microhabitats, both by chlorpyrifos and permethrin. During the photophase, the activ were usually not much decreased. This suggested that the insecticide treatment might strongly affect the mechanism to control circadian rhythm in the or strain. This tendency of affecting diel difference was also observed in the KNIH strain, selectively on permethrin (Fig. 4). In the control the KNIH strain showed diel difference at the microhabitats of W, F and O. This diel difference disappeared after the treatment of permethrin, with a major effect on the activity in scotophase, as shown in the or strain (Fig. 3). In the chlorpyrifos treatment, however, diel difference persisted. This indicated that a complex mechanism might be involved to express diurnal rhythm of the local activity, and the treatment of insecticides may differentially intervene the activation of rhythmic movement of the German cockroach. In the KNIH strain, the diel difference in visiting frequency was not observed exclusively in harborage, H, although it was observed at all other microhabitats (Fig. 4). The reason why this diel difference did not appear in H in the KNIH strain is presently
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not known. Further experiments may be needed to verify this. In this study, we did not actually measure the action of the test insect in specific microhabitats, for example the amount of feeding at F. In the present situation it was difficult to set all the sensors for detecting actual behavior for feeding, drinking, resting, and communicating with neighbor individuals to carry out the continuous observation. A further study may be needed to represent this actual behavior with the improvement of measurement methods. Since cockroach has certain aggregating behavior in some degree in natural conditions, they have been usually observed in groups (e.g., Denzer et al., 1987, 1988a, 1988b). In this study, for the purpose of tracing each individual behavior, however, single individual was observed, and other individuals were provided as neighbors to alleviate the problem caused by single rearing. It appeared that, in terms of responses to the insecticide treatment, the observation results showed consistencies in some degree in local and large-scale activities, as discussed above. However, there was a high degree of variation in individual data in the course of long observation, and dual daily-peaks in activity usually observed in cockroaches were not clearly detected. Also the variability was high in the observed values with larger coefficient of variation, usually larger than 1, it was statistically difficult to differentiate the differences in some cases. In the future, the development of observation techniques may be needed to increase the credibility of observed data, along with the individual tracing in group rearing and other verification tests in physiology.
Acknowledgements - This research was supported by Korea Science and Engineering Foundation, Project No. 941-0600-039-2. We thank the Lucky Central Institute and Hannong Company for providing test insecticides, permethrin and chlorpyrifos, respectively.
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insecticide resistance of the German cockroach (Blattella germanica L.). II. Resistance development and cross resistance. Korean J. Appl. Entomol. 32: 129-133. Bret, B.L. and M.H. Ross. 1986. Behavioral responses of the German cockroach, Blatella germanica (L.) (Orthoptera: Blattellidae), to a propoxur formulation. J. Econ. Entomol. 79: 426-430. Chon, T.-S., Y.S. Park and M.H. Ross. 1997. Temporal pattern of within-harborage time and visiting frequency in two strains of the German cockroach, Blattella germanica, in semi-natural conditions. Korean J. Appl. Entomol. 36: 299-310. Denzer, D., M.E.A. Fuchs and G. Stein. 1987. Studies on the diurnal rhythm of Blattella germanica L. (Orthopt., Blattellidae). 1. Nymphs and males. J. Appl. Entomol. 104: 495 -503. Denzer, D., M.B.A. Fuchs and G. Stein. 1988a. Studies on the diurnal rhythm of Blattella germanica L. (Orthopt., Blattellidae). 2. Females. J. Appl. Entomol. 105: 174-181. Denzer, D., M.E.A. Fuchs and G. Stein. 1988b. Studies on the diurnal rhythm of Blattella germanica L. (Orthopt., Blattellidae), 3. Mixed population. J. Appl. Entomol. 105:
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262-269. Norusis, M.J. 1986. SPSS/PC+ advanced statistics. SPSS Inc. Chicago. 360p. Ross, M.H. 1993. Comparisons between the response of German cockroach field -collected strains (Dictyoptera: Blattellidae) to vapors and contact with a cyfluthrin formulation. 1. Entomol. Sci. 28: 168-174. Ross, M.H. and D.G. Cochran. 1962. A body color mutation in the German cockroach. Nature 195: 518-519. Scott, J.G., S.B. Ramaswamy, F. Matsumura and K. Tanaka. 1986. Effect of method of application on resistance to pyrethroid insecticides in Blattella germanica (Orthoptera: Blattellidae). J. Econ. Entomol. 79: 571-575. Shim, J.-c. and K.-R. Lee. 1979. Toxicity test of public health insecticides against cockroach (Blattella germanica L.) in Korea. Korean J. Entomol. 9: 23-28. Wooster, M.T. and M.H. Ross. 1989. Sublethal responses of the German cockroach to vapors of commercial insecticide formulations. Entomol. Exp. Appl. 52: 49-56. (ReceivedJanuary 21, 1998;AcceptedMarch 26, 1998)