Insecticide Resistance Studies on Blattella germanica (Dictyoptera: Blattellidæ) from Cuba CRISTINA DIAZ PANTOJA,a,b MIRTHA G. PEREZ,c ERIC CALVO,b MARIA M. RODRIGUEZ,b AND JUAN A. BISSETb bLaboratory
of Toxicology and Genetic, Institute of Tropical Medicine “Pedro Kourí”, Autopista Novia del Mediodía Km 6, A.P 601, Marianao 13, Havana City, Cuba
cProvincial
Unity of Surveillance and Antivectorial Control, Santiago de Cuba
ABSTRACT: We studied the levels of resistance to seven insecticides: malathion, chlorpyrifos, pirimiphos-methyl, propoxur, cypermethrin, deltamethrin, and lambda-cyhalothrin in nine strains of Blattella germanica (Linnaeus, 1717) collected from sites in Santiago de Cuba and Havana City. The strains from Santiago de Cuba, generally had high levels of resistance to malathion, cypermethrin, deltamethrin, and lambda-cyhalothrin, but only low levels of resistance to pirimiphos-methyl, chlorpyrifos, and propoxur. In the strains from Havana City we found a moderate resistance to the organophosphate insecticides, resistance to the pyrethroids (except for three strains that showed susceptibility to lambda-cyhalothrin), and susceptibility to the carbamate insecticide (propoxur).
INTRODUCTION Control of Blattella germanica has been very difficult to achieve. In order to keep it under control, different organoclorine and organophosphate insecticides have been used, as well as carbamate and pyrethroid compounds. Yet, the species has developed resistance to these compounds. At present, resistance has become a substantial problem that contributes to failure of control operations in many countries.1 In Cuba, very little is known about insecticide resistance in pests. In a study carried out with strains of Blattella germanica gathered from two municipalities of Havana City, organophosphates insecticides, carbamates, and pyrethroids were tried. This study revealed a marked resistance to malathion, deltamethrin, and cypermethrin.2 There are many studies on insecticide resistance in different geographic places in the world, but not in Cuba. Yet, despite the fact that a great number of insecticides are used to control Blattella germanica in Cuba, deeper studies on this topic have not been made. Thus, the authors aim to establish the levels of resistance and/or susceptibility to insecticides applied in field-collected strains of German cockroach taken from Havana City and Santiago de Cuba—the largest cities in Cuba. aTelecommunication.
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MATERIALS AND METHODS To develop this research, a total of nine Blattella germanica strains were gathered from the fields of two cities, Santiago de Cuba and Havana City. Details on the origin of each are presented in TABLE 1. The collections were made by trapping according to the regulation stated by vector control in Cuba. They were taken in glass flasks that contained an attractant, a fine coat of vaseline in the upper internal edge, and were externally covered with dark paper. The samples were collected weekly. The collected roaches were sent to the Tropical Medicine Institute “Pedro Kourí”. All cockroach colonies were reared in an insectarium, maintained in glass containers at 27 ± 2°C and a relative humidity of 77 ± 3%. They were bred with rodent feed and water ad libitum. To determine the levels of resistance, seven insecticides were used: malathion (95% [AI]; American Cynamid), chlorpyrifos (94% [AI]; Down Chemical), pirimiphos-methyl (90% [AI]; ZENECA International), propoxur (99% [AI]; Bayer), cypermethrin and lambdacyhalothrin (70% and 98.7% [AI]; Zeneca Public Health), and deltamethrin (98.3% [AI]; Hoescht). The resistance and/or susceptibility bioassays were measured by topical application to the abdomen of adult males taking into account the modifications stated by Scott.3 All tests showed at least three doses that caused between 2 and 98% mortality, three replications of each test were performed, 150 insects each were tested in all cases. Mortality was registered 24 h after insecticide application. Data were analyzed by a probit-log computer program.4 Resistance ratios (RR) were used to express the level of resistance, the values of LC50 were compared with those from a susceptible reference strain.
TABLE 1. Origin of Blattella germanica used for this study Straina
Year Collected
Collection Site
Plaza
1995
apartment
Habana Vieja
1995
restaurant
Marianao
1995
kitchen
Miramar
1996
street
Playa
1996
house trailer
Centro Habana
1996
bar
District Abel Santamaría
1997
camping area
Bayamo
1997
apartment
District 26 de Julio
1997
bar
a In
the municipality of the same name.
0.26
> 25 > 25 > 25 —
46.59 (23.7–58.7) 103.2 (47.1–129.) > 10 > 10 > 10 8.4
Playa
Centro Habana
District Abel Santamaría
Bayamo
District 26 de Julio
Susceptible values.
0.27 (0.19–0.37)
12.3
3.29 (2.05–4.95)
Miramar
a LC , expressed as µg per cockroach. 50 b RR, resistance ratios are based on LC 50
5.5
0.39
0.44
0.98 (0.21)
1.24 (0.54)
1.65 (0.35)
1.59 (0.35)
0.42 (0.29–0.58)
0.34 (0.25–0.45)
1.5 (0.9–2.1)
3.06 (1.7–4.82)
0.29 (0.17–0.44)
0.14 (0.09–0.2)
0.29 (0.19–0.41)
3.77 (2.37–5.87)
1.4 (0.34)
Marianao
0.19
0.16 (0.1–0.23)
LC50a (95% CI)
1.62 (0.66–2.62)
1.5 (0.3)
Slope (± SE)
Habana Vieja
0.17
RRb
1.5 (0.55–2.5)
LC50a (95% CI)
Malathion
Plaza
Strain
—
1.03
1.62
1.34
5.76
11.8
1.1
0.5
1.1
0.6
RRb
Chlorpyrifos
Insecticide
2.41 (0.4)
2.18 (0.41)
3.06 (0.5)
2.8 (0.52)
3.14 (0.7)
1.62 (0.35)
2.4 (0.44)
2.05 (0.37)
2.27 (0.41)
Slope (± SE)
0.05
1.12 (0.86–1.53)
1.24 (0.96–1.7)
0.7 (0.54–0.9)
0.66 (0.39–0.91)
0.17 (0.09–0.25)
0.74 (0.55–0.98)
0.87 (0.55–1.25)
0.6 (0.36–0.83)
0.2 (0.12–0.28)
LC50a (95% CI)
—
22.4
24.8
14.1
13.2
3.4
14.8
17.4
12
4
RRb
Pirimiphos-methyl
TABLE 2. Toxicity of three organophosphate insecticides to nine strains of German cockroach by topical application
2.79 (0.51)
2.94 (0.51)
3.6 (0.98)
1.69 (0.35)
0.87 (0.2)
2.93 (0.7)
1.86 (0.4)
2.29 (0.57)
1.98 (0.37)
Slope (± SE)
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RESULTS AND DISCUSSION The toxicities for the nine field strains of the German cockroach measured by topical application are reported in tabular form as the resistance ratios for each of the seven insecticides (malathion, chlorpyrifos, pirimiphos-methyl, propoxur, cypermethrin, deltamethrin, and lambda-cyhalothrin). Data for origin, year collected, and collection site are presented in T ABLE 1. Six strains from various places of the capital city and three from Santiago de Cuba (the second capital city) were used for this study. The values of medium lethal concentration for three organophosphate insecticides, as well as the resistance ratios (RR) for each insecticide for the various strains are shown in TABLE 2. Two of the three organophosphate insecticides studied (malathion and pirimiphos-methyl) were used to control insects. Malathion resistance ratios (RR) obtained in the strains from Bayamo, Abel Santamaría and 26th of July Districts exceeded 25 ×. Nevertheless, it was not possible to estimate the exact toxicity for malathion to be able to produce 50% mortality at the maximum concentration tested (10 µg per cockroach) in the strain from Santiago de Cuba. Concerning Havana, most strains had resistance ratios less than 1.0, indicating susceptibility to this organophosphate insecticide. In the capital strains the highest RR was 12.3, in the strain collected from Centro Habana. The Playa strain showed intermediate resistance (RR = 5.5). Although both strains presented moderated levels of resistance to malathion, the slope was low, suggesting high heterogeneity in these field-collected strains. In fact, although malathion was the insecticide mostly used (between 1981 and 1986) during the extermination campaign of the Aedes aegypti mosquito in the country, it has not
TABLE 3. Toxicity of one carbamate insecticides to nine strains of German cockroach by topical application Strain
Insecticide Propoxur LC50a
Plaza
(95% CI)
0.08 (0.007–0.16)
RRb
Slope (± SE)
0.3
1.09 (0.35)
Habana Vieja
1.14 (0.7–1.81)
5.4
1.52 (0.35)
Marianao
0.46 (0.28–0.79)
2.1
1.44 (0.35)
Miramar
0.7 (0.52–0.91)
3.3
3.18 (0.83)
Playa
0.38 (0.2–0.57)
1.8
1.9 (0.31)
Centro Habana
0.72 (0.42–1.1)
3.4
4.98 (0.9)
District Abel Santamaría
0.39 (0.22–0.6)
1.84
1.3 (0.23)
Bayamo
0.65 (0.43–0.96)
3.1
1.6 (0.26)
District 26 de Julio
0.84 (0.59–1.2)
3.98
1.86 (0.29)
0.21
—
Susceptible a LC , expressed as µg per cockroach. 50 b RR, resistance ratios are based on LC
50
values.
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been used during the last ten years. Cochran5 similarly reported that the resistance to malathion in Blattella germanica prevails, despite its very limited use at present. In relation to chlorpyrifos, the resistance factor in seven out of the nine strains in the study indicated susceptibility (TABLE 2), only two strains from Havana City presented intermediate levels of resistance (Playa and Centro Habana), but the slope of both were the highest among all strains to chlorpyrifos. These strains were also resistant to chlorpyrifos and malathion. The strains resistant to chlorpyrifos were also resistant to malathion. On the other hand, the strains resistant to malathion were not always resistant to chlorpyrifos. In Cuba chlorpyrifos has not been used to control cockroachs. Therefore, we suggest that all of them have at least one similar resistance mechanism. Pirimiphos-methyl was used during an anti-Aedes aegypti campaign, but not against cockroaches. Results from tests with pirimiphos-methyl showed the highest levels for organophosphate insecticides resistance, RR > 12 (TABLE 2). Plaza and Playa strains exhibited 4 and 3.4 resistance ratios, respectively, indicating increasing levels of resistance. There are a few reports concerning the resistance to this insecticide in this species. The studies known in Cuba on this topic are those made by Díaz et al.,2 which found similar results; the strains studied showed RR > 3. The only carbamate insecticide, propoxur, was used recently in Cuba to control cockroachs. With this insecticide (see TABLE 3) most strains were susceptible or showed low levels of resistance. Only one strain (Habana Vieja) presented an intermediate level of resistance RR = 5.4. Both propoxur and chlorpyrifos insecticides showed lower resistance than others; these materials are seldom used for cockroach control, but at present we have started to apply propoxur in places where resistance problems with other classes of insecticides had appeared. Several of the pyrethroids that were released recently have also been studied. Evidence of resistance to cypermethrin in all the strains was determined (see T ABLE 4). The strains from the oriental region of the country exhibited very high levels (exceeding 306) of resistance to this insecticide. The capital strains tested had RR > 3. The other strains had resistance ratios greater than 10. This insecticide was widely used in the control of (mainly) insects during the control of Cuban Blattella germanica. Cypermethrin insecticide was the first pyrethroid compound used for cockroach control for more than 10 years and it was considered to be generator of resistance to other pyrethroid insecticides.6,7 Results from this study indicated an extremely high resistance to deltamethrin although this compound has not been used to control cockroaches in Cuba. The resistance ratios were between 12 and 250 (T ABLE 4). Cypermethrin and deltamethrin insecticides probably have similar resistance mechanism, because we only used cypermethrin, and the field-strain collected also exhibited resistance to cypermethrin and deltamethrin. In order to control the German cockroach, other insecticides have replaced cypermethrin. A test with lambda-cyhalothrin indicated that nearly half of the strains showed RR > 30 (TABLE 4). Of the remaining strains, all except two are susceptible. The pattern of resistance for this compound was different from that for cypermethrin and deltamethrin insecticides, suggesting that Cuban Blattella germanica develop a different resistance mechanism to lambda-cyhalothrin.
0.04
> 306 > 306 > 306 —
0.38 (0.23–0.43) 0.79 (0.5–0.94) > 15 > 15 > 15 0.04
Playa
Centro Habana
District Abel Santamaría
Bayamo
District 26 de Julio
Susceptible values.
> 10
16.1
3.18 (2.22–4.33)
Miramar
a LC , expressed as µg per cockroach. 50 b RR, resistance ratios are based on LC 50
7.7
7.7
24.4
1.26 (0.33)
2.11 (0.36)
2.31 (0.4)
2.69 (0.47)
> 10
> 10
0.4 (0.29–0.61)
0.32 (0.29–0.53)
0.76 (0.003–1.94)
0.84 (0.47–1.28)
2.30 (1.42–3.29)
1.2 (0.91–1.6)
2.09 (0.39)
Marianao
26.3
0.48 (0.34–0.66)
LC50 (95% CI)
1.29 (0.92–1.87)
1.83 (0.36)
Slope (± SE)
a
Habana Vieja
5.5
RRb
Cypermethrin
0.27 (0.17–0.39)
LC50 (95% CI)
a
Plaza
Strain
—
> 250
> 250
> 250
100
80
19
1.32 (0.35)
1.87 (0.25)
0.71 (0.3)
1.57 (0.38)
1.9 (0.36)
> 250 21
2.32 (0.44)
Slope (± SE)
12
RRb
Deltamethrin
Insecticide
0.09
25.1 (10.2–42.8)
8.7 (4.43–31.4)
19.2 (7.5–24.9)
0.17 (0.092–0.3)
0.14 (0.085–0.3)
0.47 (0.25–0.81)
2.85 (1.14–5.32)
0.23 (0.14–0.32)
—
79
97
213
2.8
2.3
5.2
31.6
2.5
4.4
RRb
Lambda-cyhalothrin
0.4 (0.3–0.57)
LC50 (95% CI)
a
TABLE 4. Toxicity of three pyrethroid insecticides to nine strains of German cockroach by topical application
0.96 (0.28)
0.91 (0.21)
0.77 (0.2)
1.14 (0.39)
1.79 (0.36)
1.35 (0.24)
1.07 (0.33)
1.99 (0.37)
2.11 (0.39)
Slope (± SE)
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Although another pattern could exist, data from this study are not sufficient to reveal it. It is necessary examine additional strains and synergism studies, penetration studies, in vivo and in vitro metabolisms, and enzyme assays in order to better understand insecticide resistance in Blattella germanica.
ACKNOWLEDGMENTS We thank Dr. J.G. Scott for his critical review of this manuscript. Thanks are expressed to Rafael Benavides, who provided Santiago de Cuba field collected strains for use in this study. Thanks are also extended to Mario Fresneda and Alain Soca for technical assistance. REFERENCES 1. A TKINSON, T.H., R.H. W ADLERGH, P.G. K OEHLER & R. P ATTERSON. 1991. Pyrethroid resistance and synergism in a field strain of the German cockroach (Dictyoptera: Blattellidae) J. Econ. Entomol. 84(4): 1247–1250. 2. C OCHRAN, D.G. 1989. Monitoring for insecticide resistance in field-collected strains of the German cockroach (Dictyoptera: Blattellidae). J. Econ. Entomol. 82(2): 336– 341. 3. C OCHRAN, D.G. 1991. Extended selections for pyrethroid resistance in German cockroach (Dictyoptera: Blattellidae). J. Econ. Entomol. 84(5): 1412–1416. 4. D ÍAZ, C., J. B ISSET, T. G ONZALEZ & M.M. R ODRIGUEZ. 1994. Resistencia a insecticidas organofosforados, carbamatos y piretriodes en Blattella germanica (Dictyoptera Blattellidae) de 2 municipios de Ciudad de la Habana, Cuba. Rev. Med. Trop. 46(2): 130–132. 5. R AYMOND, M. 1985. Presentation d’un programme d’analyse log-probit pour microordinateur. Cahiers ORSTROM Sér. Ent. Med. Parasitol. 23: 117–121. 6. S COTT, J.G., D.G. C OCHRAN & B.D. S IEGFRIED. 1990. Insecticide toxicity, synergism and resistance in the German cockroach (Dictyoptera: Blattellidae). J. Econ. Entomol. 83: 1693–1703. 7. S IEGFRIED, B.D. & J.G. S COTT. 1992. Insecticide resistance. Mechanisms in the German cockroach, Blattella germanica (L.). Symposium Series No. 505 American Chemical Society. 218–229.
