Abstract: Six strains of German cockroach, Blattella germanica, collected from three major cities in Indonesia which are Jakarta, Bandung and Surabaya were bio-assayed with topical application to monitor insecticide resistance to insecticides propoxur, permethrin and fipronil. In general, compared with VCRU susceptible strain as the standard, all cockroach strains were resistant to all insecticides tested, with Resistance Ratios ranging from 2.11-1013.17 fold. Majority of strains exhibited very high resistance (RR50>50) to permethrin; this study also revealed the existence of extremely high resistance to permethrin (RR50 of 1013.17 fold) in HHB-JKT strain. Resistance levels to both propoxur and fipronil is vary (RR50 ranging from 2.11-44.72 fold). But, in general, the field strains are still susceptible to fipronil. This study is the second report of German cockroach resistance to insecticides in Indonesia which suggest the importance of continued monitoring of insecticides susceptibility for controlling the German cockroach, B. germanica. In addition, the findings can be used in formulating potential strategies for cockroach resistance management.
INTRODUCTION
The German cockroach, Blattella germanica (Dictyoptera: Blattellidae) is unquestionably the most significant urban insect pest. It is known to be the vector of many pathogens that may cause a threat to human health, trigger asthma, vertigo and nausea to humans. Many professional Pest Control Operators (PCOs) as well as home owners have made efforts to control this pest mostly by insecticides. Unfortunately, the intensive and continuous use of insecticides to control this pest has caused the development of resistance of B. germanica to many insecticides (Ahmad et al., 2009; Nasirian et al., 2011). Furthermore, it is believed the German cockroach is able to develop resistance to many types of insecticides, owing to its survival ability, shorter life cycle and ability to digest chemical substances (Buczkowski and Schal, 2001). Resistance in the German cockroach to all the major groups of insecticides viz., organochlorines, organophosphates, carbamates and synthetics pyrethroids is quite common and has been reported in many countries (Hemingway et al., 1993; Jensen, 1993; Lee et al., 1996; Valles and Yu, 1996; Kristensen et al., 2005; Nasirian, 2010). As for Indonesia, the resistance of B. germanica to insecticide is poorly documented and the only available information was published by Ahmad et al. (2009), they reported 95 fold resistance of German cockroach collected from Bandung strain to permethrin and suggested that the detoxifying enzyme Mixed Function Oxidases (MFO) played an important role in the development of resistance.
In Indonesia, synthetic insecticide, particularly pyrethroids and to the some extent of organophosphates and carbamates is commonly used by PCOs to control German cockroach. However, according to recent reports from some PCOs, notably from Jakarta, Bandung and Surabaya, as well as complaints from customers, intensive insecticide treatments did not reduce the population of cockroach into the expected level. This information leads to the possibility that cockroaches in some parts of Indonesia may have developed resistance to insecticides.
Therefore, knowing that the information on the resistance of B. germanica to insecticides from Indonesia is still limited; this report describes the present resistance status of B. germanica collected from Jakarta, Bandung and Surabaya to carbamate, pyrethroids and phenylpyrazole.
MATERIAL AND METHODS
Laboratory experiment
Cockroaches: There were seven strains of B.
germanica used in this experiment (Table 1). The VCRU
(Vector Control Research Unit) strain is a standard susceptible strain, originally
obtained from the Vector Control Research Unit School of Biological Sciences
Universiti Sains Malaysia. The field strains were collected from Jakarta, Bandung
and Surabaya.
All cockroaches were reared without selection pressure in the Laboratory of Entomology, School of Life Sciences and Technology, Bandung Institute of Technology, Indonesia. Rearing and experimental conditions were; temperature of 22-28°C, relative humidity of 52-95% and photoperiod of 12:12 and was provided with water and mixture of fresh minced potatoes, oat meal and bread ad libitum. Rearing methods by Noland et al. (1949) and modification by Ahmad and Suliyat (2011) were applied. For experimental purposes, only male cockroaches were used.
Insecticides: The insecticides used in this study were technical grade i.e., propoxur 92.25%, permethrin 92.00% and fipronil 86.9% [Active Ingredients (AI)]. Both propoxur and permethrin were provided by PT. Inti Everspring, Indonesia, whilst fipronil was provided by PT. Bayer Indonesia. Prior to the tests, these insecticides were diluted with analytical grade acetone.
Insecticide bioassays: Topical tests to detect and quantify the resistance were carried out. The topical tests in which male adults B. germanica were treated with insecticide concentrations followed the methods as described by Lee et al. (1996). One microliter insecticide solution was applied topically on the first abdominal sternite of each carbon dioxide-anesthetized B. germanica using a microsyringe (Valles and Yu, 1996).
| Table 1: | Source of cockroaches, B. germanica |
After treatment, cockroaches were kept in a 1 L volume of plastic jar, fed on slices of fresh peeled potatoes and provided with water. Mortality was observed at 72 h after treatment.
Resistance ratio: The Resistance Ratio (RR) was obtained by comparing the Lethal Dose (LD)50 and of each field strain to the LD50 of susceptible strain (Scott et al., 1990; Lee et al., 1996).
Statistical analysis: The data were analyzed by probit analysis using POLO-PC software program (Leora Software, 2004) to determine the LD50 and LD90.
RESULT AND DISCUSSION
Our findings provide evidence that in general, B. germanica strains collected from Jakarta, Bandung and Surabaya have developed resistance to the three major groups of insecticides as compared to a susceptible laboratory strain (VCRU) (Table 2-4).
| Table 2: | Toxicity of propoxur applied topically to adult cockroaches |
| aStrain name/each collection, RR: Resistance ratio = LD50 resistant strain/LD50 susceptible strain | |
| Table 3: | Toxicity of permethrin applied topically to adult cockroaches |
| aStrain name/each collection, RR: Resistance ratio = LD50 resistant strain/LD50 susceptible strain | |
| Table 4: | Toxicity of fipronil applied topically to adult cockroaches |
| aStrain name/each collection, RR: Resistance ratio = LD50 resistant strain/LD50 susceptible strain | |
Using the classification resistance ratio developed by Lee and Lee (2004) we found that the resistance levels of the field collected strains were low to high (2.13-16.88 fold) for the propoxur, low to very high (2.83-1013.17 fold) for the permethrin and low to high for the fipronil (2.11-44.872 fold). Knowing the practice of regular spraying with permethrin and propoxur to control B. germanica were commonly carried out by the PCOs in Indonesia, this finding is not really surprising, except for the extremely high resistance to the permethrin, i.e., 1013.17 fold. However, the occurrence of fipronil resistance in two of the six strains was really surprising, because the use of fipronil to control cockroach had not been widely used in Indonesia and only available in bait formulations (Ahmad and Suliyat, 2011).
Resistance levels to propoxur: Three out of six strains had resistance ratios >10 (high resistance) to propoxur, i.e., HHB-JKT (16.88 fold) followed by SBY-SBY (12.25 fold) and KRSA-BDG (12.00 fold). The remaining strains (KRSB-BDG, GFB-JKT and GFA-JKT), had low resistance levels, ranging from 2.13-4.00 fold (Table 2). The slope in the field strain varied from 0.09±0.18-2.70±0.56, only the GFB-JKT strain had similar slope with the VCRU, the laboratory susceptible strain. This finding suggests that the majority of the field strains had more heterozygous individuals for resistance. Interestingly, the levels of cockroach resistance to propoxur reported in these experiments were generally lower than those reported from other countries. The highest resistance of B. germanica to propoxur found in this experiment is lower compared to the ones reported in Malaysia, England and Taiwan; 91.6 fold (Lee et al., 1996), 46 fold (Hemingway et al., 1993) and 62.50 fold (Pai et al., 2005), respectively. However, it is still higher compared to the ones reported in United States and Singapore; 16.6 fold (Scharf et al., 1996; Valles and Yu, 1996) and 21.5 fold (Chai and Lee, 2010), respectively. This finding explains the situation in Indonesia; due to concern of propoxur toxicity to humans, propoxur has not been commonly used by the PCOs in Indonesia to control the cockroach. Although interestingly, a few household aerosols, registered to control mosquito, cockroach and house fly contain a combination of propoxur (carbamates) and transfluthrin (pyrethroids) as the active ingredients. In contrast, in peninsular Malaysia resistance to propoxur was prevalent due to the use of propoxur as the primary insecticide to control German cockroach (Chai and Lee, 2010).
Resistance levels to permethrin: The LD50 and RR50 values for permethrin tested against all strains are shown in Table 3. All B. germanica field strain were resistant to permethrin and most of them were considered highly resistance with RR50>50. The HHB-JKT strain exhibited the highest resistance to permethrin (1013.17 fold) followed by SBY-SBY strain (405.50 fold) and GFA-JKT (315.67 fold). Based on the previous report by Ahmad et al. (2009), a cockroach strain from Bandung had a RR50 of 95 for permethrin. It was really surprising to have found such a high resistance to permethrin (1013.17 fold) from HHB-JKT strain. This value of resistance, to the best of our knowledge, is apparently the highest in the world. Report of cockroaches resistance with RR50>50 fold to permethrin is very rare. Some cases of cockroaches resistances to permethrin with RR50>50 fold are Malaysian strain (280 fold) (Lee and Lee, 2004) and Korean strain 109.8 fold (Chang et al., 2010). In Iran, for example, despite continued use of permethrin to control German cockroach for almost 30 years, the resistance ratios of seven field collected strains were relatively low, ranged from 5.26-23.7 fold (Limoee et al., 2006). The finding of RR50>1000 fold in this study is exceptional; hence, it is necessary to make new criteria in classifying resistance level, i.e., extremely high resistance for RR>1000. However, interestingly, the exception was found on GFB-JKT and KRSB-BDG strains which had RR50<10, i.e., 2.83 and 6.83 fold, respectively. The slope in the field strain varied from 1.21±0.29 to 2.67±0.25 which is lower than the VCRU strain (4.92±0.81). This finding indicates that field strains had more heterozygous individuals for resistance, whilst the VCRU strain had more homozygous individuals for susceptible.
The fact that majority of Indonesian strains had very high resistance to permethrin could be explained by the following reasons; first, because permethrin has been used in Indonesia to control various public health pests, including cockroach for more than 25 years. Second, because most of the field technicians of the PCO’s did not have good understanding about insect resistance phenomena to insecticide and how to manage the resistance insects. This condition is supported by the recent survey carried out by Ahmad and Suliyat (2011) in 18 kitchen restaurants around Bandung. They reported that despite every other week of cypermethrin spray by PCO over a period of two years, the survey found that all 18 kitchens had moderate to heavy infestations with the number of cockroaches ranging from 41-243. Even though control failures was rather obvious and resistance seemed to be the problem; for reason that pyrethroids have low toxicity to humans as compare with other insecticides, PCO kept doing the same thing except changing one brand of pyrethroids insecticides with another pyrethroids.
Resistance levels to fipronil: The LD50 and RR50 values for fipronil tested against all strains are shown in Table 4. Based on the LD50 values among fipronil, propoxur (Table 2) and permethrin (Table 3), it is shown that fipronil was the most toxic insecticide with an LD50 of 0.76 ng insect-1 on VCRU strain. This LD50 value shows that fipronil is 79.1 fold and 105.5 fold more toxic than propoxur and permethrin, respectively. The findings of the present study is in agreement with those reported by Scott and Wen (1997) who found that fipronil was 100 fold more toxic to the susceptible cockroach as compared to bendiocarb (carbamate) and chlorpyrifos (organophosphate). Furthermore Nasirian et al. (2006) reported that fipronil was 2233 fold more toxic than permethrin if used topically to the susceptible cockroach. Interestingly, pattern of the slope which measures the heterogeneity of the test population in fipronil is different than those in propoxur and permethrin. In this experiment we find that the slope in the field strain varied from 1.10±0.40 to 4.93±0.89, whereas, the slope for the VCRU was 2.36±0.62. Based on the slopes, the finding suggests that the majority of the field strains, i.e., GFB-JKT, KRSA-BDG and KRSB-BDG had more susceptible homozygous individuals for fipronil.
Even though the majority of field strains showed low resistance to fipronil (<5), two out of six strains, i.e., GFA-JKT and HHB-JKT exhibited high resistance to fipronil (>10 to = 50). There is a possibility that the high resistance levels found in GFA-JKT and HHB-JKT strains is due to the cross-resistance between similar modes of actions of the previously applied insecticides. This possibility is based on earlier reports which showed that cyclod ienes resistant insects were more tolerant to fipronil (Scott and Wen, 1997; Kristensen et al., 2005). For example, German cockroach strains Zo960302 and Ga021001 from Europe were 1270 and 2030 fold resistant to dieldrin (cyclodienes) and 15 and 14 fold resistant to fipronil. Even though when the experiments took place, fipronil had never been used in Europe to control cockroach and the use of dieldrin (cyclodienes) to control cockroach was stopped 20 years earlier (Kristensen et al., 2005). Furthermore the cross resistance of German cockroach is not only between fipronil and cyclodienes but also with pyrethroids as reported by Wei et al. (2001) that the Apyr-R strain had 480 fold resistance to deltamethrin and two-fold resistance to fipronil.
This finding in the present study is rather surprising and alarming as well. First, it is surprising because fipronil usage as the active ingredient of gel bait being applied to control cockroach in Indonesia is quite recently (about 5 years). In addition to that, due to their expensive price, the use of gel baits to control cockroaches has not been widely used. Second, it is alarming because fipronil is considered as the best insecticide (in bait formulations) available to control the insecticide resistant cockroach. Ahmad and Suliyat (2011), reported that laboratory-made bait containing 0.03% fipronil was very effective in controlling the population of pyrethroids resistant German cockroach found in 18 kitchens, >90% reduction was achieved after 2 weeks of treatment and 100% reduction was achieved after 5 weeks of treatment, with the initial number of cockroach per kitchen ranging from 41-243.
In conclusion, present findings show that all B. germanica strains collected from Jakarta, Bandung and Surabaya were resistant to the three groups of insecticides (multiple resistance to propoxur, permethrin and fipronil) with resistance ratios ranging from 2.11-1013.17 fold. Since majority of B. germanica had developed a very high resistance levels to permethrin and one strain had developed an extremely high resistance level to permethrin (RR>1000) it is imperative to take preventive steps. A permethrin halt for a certain of period should be carried out to avoid further control failure in the field. For the control of B. germanica, propoxur and especially fipronil are suitable replacement for permethrin, except for those strains which had developed very high resistance to the compounds. Our findings also suggest that for informed decisions of which insecticide(s) is to be used, monitoring of insecticide susceptibility needs to be continued with more samples from all over Indonesia. Nonetheless, unless alternative control strategies is available and PCOs have adequate knowledge on how to manage insect resistance, we have reasons to worry that it is only a matter of time before resistance to any new insecticides is developed. Although this is not surprising seen from the ecological and evolutionary perspectives, as insect will develop resistance to any insecticide applied as a means of survival.
ACKNOWLEDGMENTS
We thank PT. Inti Everspring Indonesia and PT. Bayer Indonesia for providing us with the insecticides used in this experiment, Prof. Chow Yang Lee for VCRU strain (susceptible strain) and to Hilda Lionata for English correction. This study was partially funded by DIPA ITB (2008-2009) to Intan Ahmad.