Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years

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1 Journal of Integrative Agriculture 2012, 11(2): February 2012 RESEARCH ARTICLE Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years Svetlana Kontsedalov 1, Fauzi Abu-Moch 1, Galina Lebedev 1, Henryk Czosnek 2, A Rami Horowitz 3 and Murad Ghanim 1 1 Department of Entomology, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel 2 Institute of Plant Sciences, Faculty of Agriculture, Rehovot 76100, Israel 3 Department of Entomology, Agricultural Research Organization, Gilat Research Center, M.P. Negev 85280, Israel Abstract The sweetpotato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is an extremely polyphagous insect pest that causes significant crop losses in Israel and worldwide. B. tabaci is a species complex of which the B and Q biotypes are the most widespread and damaging worldwide. The change in biotype composition and resistance to insecticide in Israel was monitored during the years to identify patterns in population dynamics that can be correlated with resistance outbreaks. The results show that B biotype populations dominate crops grown in open fields, while Q biotype populations gradually dominate crops grown in protected conditions such as greenhouses and nethouses, where resistance outbreaks usually develop after several insecticide applications. While in previous years, Q biotype populations were widely detected in many regions in Israel, significant domination of the B biotype across populations collected was observed during the year 2010, indicating the instability of the B. tabaci population from one year to another. Reasons for the changing dynamics and the shift in the relative abundance of B. tabaci biotype, and their resistance status, are discussed. Key words: Bemisia tabaci, biotype, insecticide, monitoring, resistance INTRODUCTION The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is classified as one of the top 100 world invasive species ( search.asp?st=100ss). This cosmopolitan insect pest gained its importance as one of the most destructive agricultural pests worldwide owing to its ability to feed on hundreds of plant species, many of which are important agricultural crops (Dinsdale et al. 2010). Detection of B. tabaci developmental stages in agricultural products during international trade is very difficult due to the very small size of the insect, thus increasing its invasiveness. Indeed, B. tabaci is reported from all continents except countries with very cold climates where temperatures are cold enough all year around to prevent one or several generations from being established (Dinsdale et al. 2010). B. tabaci is a phloem-feeder and inflicts damage in many crops due to direct feeding, honeydew secretion and vectoring of plant viruses (Byrne and Bellows 1991; Bedford et al. 1994; Perring 2001; Brown and Czosnek 2002; Horowitz et al. 2004, 2005; Inbar and Gerling 2008). This insect is made up of a complex of biotypes, which vary greatly in their biology, mainly host range, fecundity, and insecticide resistance, and in their ability to transmit plant viruses and induce plant disorders. Received 1 March, 2011 Accepted 29 June, 2011 Correspondence Murad Ghanim, Tel: , Fax: , ghanim@agri.gov.il

2 Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years Members in this species complex, which include more than 10 different biotypes, and differ in their biology, are well defined by microsatellite PCR-based DNA markers (Boykin et al. 2007). Significant differences between genetic groups, which were mainly observed based on mitochondrial DNA molecular markers as well as crossing experiments between some of them, suggest that B. tabaci is a cryptic species complex composed of at least 24 distinct species (De Barro et al. 2011). Based on this new classification, the B biotype is termed Middle East-Asia Minor 1 and the Q biotype is termed Mediterranean (Dinsdale et al. 2010; De Barro et al. 2011). This new terminology, however, is not yet widely accepted, and throughout this manuscript we will use the term biotype for consistency with previous literature. The B and Q biotypes of B. tabaci are the most predominant and damaging biotypes worldwide. The B biotype is defined by high fitness parameters such as high fecundity and fertility (De Barro et al. 2006), while the Q biotype is associated with outbreaks of severe insecticide resistance and have developed resistance to all major insecticide groups (Horowitz et al. 2004, 2005, 2007). Besides these biological differences, the two biotypes do not mate and gene flow between them is unlikely (Perring and Symmes 2006; Elbaz et al. 2010; Sun et al. 2011). Both biotypes can be sympatric on many host plants and were reported to be efficient transmitters of begomoviruses (Sanchez-Campos et al. 1999; Gottlieb et al. 2010). In this manuscript, we summarize biotype and resistance monitoring results among B and Q biotype populations during the years , and provide a comprehensive survey for the dynamics of both biotypes in Israel. We show that the dynamics of the two biotypes and their resistance status can drastically change within and between seasons, depending on several factors discussed in the manuscript. RESULTS AND DISCUSSION Dynamics of B and Q biotype populations under protected conditions The two experiments conducted in southern and northern Israel were conducted as described in the Materials and Methods section. The results for the first experiment in the north showed that with the start of the experiment, B. tabaci populations outside the nethouses reached very high levels. The populations trapped outside the nethouses mostly contained B biotype, but sometimes Q biotype was also detected. Inside the nethouses, however, the B biotype was detected one month after the start of the experiment, then both biotypes were detected on August 5 (Fig. 1-A). The sampling done on August 9 detected only Q biotype, and all samplings performed until the end of the experiment detected only Q biotype individuals in all treatments including the control. These results suggest that the chemical treatments were effective in controlling B biotype but were ineffective in controlling the Q biotype and selected for this biotype inside the nethouses. The populations in all treatments, including those that were chemically treated, reached very high levels until the end of the experiment, and showed that once resistant populations are stable, they cannot be treated with chemical insecticides; thus the experiment was terminated. The results in the second experiment conducted in Kikar Sdom, southern Israel showed that one month after the start of the experiment, B and Q biotype B. tabaci individuals were detected in all treatments (Fig. 1-B, June 11), however subsequent collections and biotype tests showed that only Q biotype individuals were detected in all treatments (Fig. 1-B, June 18, June 24, July 3 and July 6), while the B biotype was not detected in experimental tunnels, but was detected in melons and watermelons that surround the experiment. The results in this experiment were somewhat similar to results obtained in the experiment conducted in the Beit Shean Valley (Experiment 1), where dominance of the Q biotype on basil grown in protected conditions, was observed, especially in the responsive treatment in which insecticide applications were given in response to B. tabaci population detection and rise. The dominance of the Q biotype was even observed in the control treatments in both experiments, suggesting that the environmental conditions developed inside the nethouses were in favor of the Q biotype. The main condition that can select for the Q biotype during the summer season is high temperatures as was previously shown (Mahadav et al. 2009). Other factors such as preference to the crop tested (basil) might also affect the results, however, other crops must be compared in simi-

3 314 Svetlana Kontsedalov et al. lar conditions to verify this assumption. In the Beit Shean Valley in the north and Kikar Sdom in the south, temperatures close to 40 C or above it are common during the summer, and thus the Q biotype dominates many of the crops grown during this season in protected conditions. Fig. 1 Development of B. tabaci populations in two different experiments in northern (A) and southern (B) Israel during Average of three traps per treatment was calculated. See text for more details. Biotype composition and resistance status during During late 2008 and 2009, 23 populations were sampled from northern and southern Israel (Table 1). The biotype composition in each population was tested using PCR on 20 individuals. The majority of the populations were collected mainly from fresh herbs, tomatoes, peppers, and other crops grown under greenhouses and nethouses except population 9 from watermelons grown in open field (Table 1). Eight of the 23 collected populations were mixed populations that contained B and Q biotypes (Fig. 2-A). The Q biotype was much more prevalent than the B biotype in the majority of the tested populations. In total, 74% of the tested individuals were from the Q biotype and 26% from the B biotype (Fig. 2-B). Fourteen of the 23 populations contained 100% Q biotype individuals, while only one population contained 100% B biotype individuals. The prevalence of the Q biotype in these collections was somewhat expected and can mainly be attributed to the ability of this biotype to develop resistance to insecticides (Horowitz et al. 2005). Under these conditions, B biotype populations are not able to survive repeated exposure to insecticides, and within several generations, the Q biotype dominates the populations in the greenhouse, and resistance outbreaks and failures to control B. tabaci populations are foreseen. Since minimal immigration of individuals from outside into the protected greenhouse occurs, the majority of the collected populations shown in Table 1 and Fig. 2 contained 100% Q biotype individuals. Another factor that contributes to the dominance of the Q biotype in protected crops is the ability of this biotype to tolerate high temperatures (Mahadav et al. 2009). As explained above, high temperatures that reach over 40 C inside the greenhouses contribute to the dominance of Q biotype populations, while the B biotype shows lower tolerance and tend to decline under extreme temperatures. The dominance of the B biotype in some populations that were collected from greenhouses and nethouses and were under chemical regime (populations 1 and 21) suggests that although this biotype is known as more susceptible to insecticides, it still has the ability to develop resistance to some insecticides (Horowitz et al. 2005; Crowder et al. 2009). However, this depends on the insecticides applied, the genetic background and the history of exposure to insecticides of the tested B biotype population. The ability of the Q biotype to rapidly develop resistance within few generations seems to be a major factor that enables rapid prevalence over the B biotype under protected conditions (Horowitz et al. 2005). Some of the collected populations during 2009 (Tables 1 and 2) were subjected to resistance test to three insecticides that belong to the neonicotinoid group: thiamethoxam, imidacloprid and acetamiprid. This group of insecticides is extensively used for B. tabaci management in Israel, and previous works have reported the presence of resistant B. tabaci populations to some neonicotinoids (Horowitz et al. 2004, 2005). Information about the tested populations is given in Table 1 and resistance tests results are shown in Table 2. The tested populations exhibited the highest resistance levels to

4 Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years Table 1 B. tabaci collections made during for assessing B. tabaci biotype dynamics and resistance status through several experiments Strain Location Crop Control Collection date North 1 1) Melea Sage Chemical Parazon Sage Chemical ) Magen Shaul Mint Chemical ) Mehula Sage Chemical Mehula Tomato Organic ) Nir David Taragon Chemical ) Revaya Ment Chemical Havat Eden Basil Chemical Rehov Basil Chemical South 8 1) Ein Tamar Pepper Organic Ein Tamar Watermelon Chemical Ein Tamar Pepper Chemical Ein Tamar Kastor bean Organic Yair sation Basil Organic Ein Tamar Pepper Chemical Ein Tamar Basil Chemical ) Ein Tamar Basil Organic ) Yair Station Basil Organic ) Ein Tamar Basil Chemical ) Ein Tamar Basil Organic Hazeva Basil Organic Ein Tamar Basil chemicals Ein Tamar Basil chemicals ) Populations tested for resistance, see Table 2. thiamethoxam, then to imidacloprid and the lowest levels to acetamiprid (Table 2). The resistance levels for each tested insecticide largely varied between the tested populations, suggesting heterogeneity of the populations, and that B. tabaci populations are not uniform across Israel. For example, 11, 50 and 21 fold differences were observed in LC 50 values between the most resistant and most susceptible populations to thiamethoxam, imidacloprid and acetamiprid respectively. This heterogeneity may be attributed to heterogeneity in the exposure to neonicotinoids across Israel in different farming systems and different management programs, or introduction of new populations into the country from neighboring countries, or from international trade. Some of the populations that exhibited very high levels of resistance were collected from organic farms (for example populations 8, 15, 16, and 18), suggesting immigration and establishment of resistant populations into these farms during the season. B. tabaci biotypes dynamics in field and protected crops during 2010 During 2010 we conducted a more comprehensive survey of B. tabaci populations for assessing the relative abundance of both B and Q biotypes in protected crops (greenhouses and nethouses) and also in open fields. Altogether, we collected 44 populations, starting from February 2010 and ending during October 2010, and included major regions in Israel with extensive agriculture such as the Jordan Valley (east), Beit Shean Valley (north), Ayallon Valley (center), Negev and the Arava (south) (Table 3). Thirty-two populations were collected in open fields and 12 populations from protected crops in greenhouses and nethouses (Table 3). The majority of the farms from which we collected the tested populations used chemical insecticides for managing B. tabaci populations. Twenty individuals from each population were tested using PCR on individual whiteflies. Strikingly, the results obtained showed that populations collected in open field contained 98.5% B Fig. 2 B. tabaci populations collected during late 2008 and 2009 and their biotype composition (A), and percentage of B and Q biotypes in all populations (B).

5 316 Svetlana Kontsedalov et al. Table 2 Log-dose probit mortality results for B. tabaci populations collected during and tested with imidacloprid, thiamethoxam and acetamiprid Strain ID 1) Number of Slope±SEM LC 50 (CL) whiteflies tested (mg a.i. L -1 ) RR 50 Thamithoxam Ssc ± ( ) ±0.84 NA NA ± ( ) ± ( ) ± ( ) ± ( ) ± ( ) ±0.22 ~ NA NA > ±0.75 ~ > ±1.10 ~7 000 >1 000 Imidacloprid Ssc ± (2-4) ± ( ) ± (85-178) ± (75-302) ± (86-326) ± (3-7) ± (8-25) ± (20-48) ± (55-128) ± (27-102) ± ( ) 83 Acetamiprid Ssc ± ( ) ±0.14 NA NA ± (18-38) ± (5-10) ± (22-43) ± (24-77) ± (15-45) ± (74-532) ± (53-104) ± (57-85) ± (58-91) 37 1) Ssc, strain susceptible to all insecticides. Numbers 1-18, field collected strains tested for resistance (see Table 1). CL, confidence limits; RR, resistance ratio. biotype individuals and 1.5% Q biotype (Fig. 3-A and C), while populations collected from protected crops contained 83% Q biotype individuals and 17% B individuals (Fig. 3-A and B). This clear difference in the distribution of both B and Q biotype between protected and open field crops supports our previous results from , and can be explained by the ability of the Q biotype to survive chemical control regimes inside greenhouses and nethouses, while the B biotype is easily controlled by chemical insecticides under these conditions. The dominance of the B biotype in open fields however, can be explained by the higher fitness of the B biotype over the Q. Although chemical control is also used in open fields, the high fitness rates and the difficulty in covering plants with airplane chemical sprays in open fields, significantly contribute to the domination of B biotype populations in open fields. In conclusion, our recent surveys clearly demonstrate that the Q biotype of B. tabaci is able to dominate crops grown in protected conditions such as greenhouses when chemical control methods are the main management strategy. Both B and Q biotypes invade greenhouses, however, the B biotype rapidly declines owing to its sensitivity to new insecticide groups such as Insect Growth Regulators and neonicotinoids. This biotype is also susceptible to some environmental conditions such as high temperatures, thus within few chemical management rounds and generations, a failure to control these populations is observed. B biotype populations dominate crops grown in open field conditions, and their success to dominate crops over the Q biotype is attributed to the high fitness rate that define the B biotype, and the inefficiency in applying chemical insecticides in open fields. CONCLUSION In this manuscript, the dynamics of the B and Q biotypes was studied in several parts of Israel. The results clearly demonstrate that the B biotype dominates in open fields, while the Q biotype dominates in crops grown under protected conditions including greenhouses and nethouses, and when these crops are chemically controlled, field failures are likely to occur. It is therefore essential to develop Integrated Resistance Management (IRM) programs that take into account this unique ability of the Q biotype to develop resistance to all major insecticide groups. These programs should be based on continuous resistance monitoring surveys, better technologies in insecticide applications and alternations between insecticides with different mode of actions. MATERIALS AND METHODS Whitefly collections Populations of the sweetpotato whitefly B. tabaci were collected during the years across Israel (Tables 1 and 3). To evaluate the resistance status of B. tabaci population and the distribution of both the B and Q biotypes across Israel, we collected a total of 76 populations during the years and tested their biotype com-

6 Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years Table 3 B. tabaci collections made during 2010 for assessing B. tabaci biotype dynamics and resistance status through several experiments Strain ID Location Crop Control Open field/protected Collection date North 4 Eden station Basil Chemical Protected Moledet Watermelon Chemical Open Merhavia Warermelon Chemical Open Kfar Hahoresh Tomato Chemical Open Megido Sunflower Chemical Open Kfar Hahoresh Watermelon Chemical Open Kfar Hahoresh Tomato Chemical Open Kfar Hahoresh Watermelon Chemical Open Kfar Hahoresh Sunflower Chemical Open Kfar Hahoresh Tomato Chemical Open Tel-Yosef Cotton Chemical Open Alonim Cotton Chemical Open Kfar Hahoresh Cotton Chemical Open Kfar Hahoresh Cotton Chemical Open Hahotrim Cotton Chemical Open Rehov Mint Chemical Protected Eden station Basil Chemical Protected Hahotrim Cotton Chemical Open Kfar Masarik Cotton Chemical Open South 3 Ein Tamar Basil Chemical Protected Ein Tamar Basil Chemical Protected Ein Tamar Melon Chemical Open Ein Tamar Basil Chemical Protected Ein Tamar Basil Chemical Protected Gilat Cotton Chemical Open Sde-Timan Cotton Chemical Open Netiv Haasara Basil Chemical Protected Ein Tamar Melon Chemical Open Ein Tamar Pepper Chemical Protected Ein Tamar Pepper Organic Protected Ein Tamar Melon Organic Open Grupit Pumpkin Chemical Open Grupit Melon Chemical Open Newe Grupit Sunflower Chemical Open Center 36 Revadim Cotton Chemical Open Shaalabim Cotton Chemical Open Hulata Cotton Chemical Open Revadim Cotton Chemical Open Ein Shemer Cotton Chemical Open Shaalabim Cotton Chemical Open Bet Dagan Eucalyptus Chemical Protected Jordan Valley 42 Naama Mint Chemical Protected Gilgal Oregano Chemical Protected Gilgal Sage Organic Protected position and resistance status. We included populations from all parts of the country and from different crops. The whiteflies were collected from plants into glass Pasteur pipettes attached to a hand-held aspirator. Each collected population in each location was collected from different leaves on different plants. Some of the populations were collected in greenhouses, nethouses and from open fields. For testing biotype composition, collected whiteflies were immediately transferred to absolute ethanol for preservation, and were kept at room temperature until tested by PCR as described below. Twenty individuals were tested from each population. For testing the resistance status of each collected population, whiteflies were reared for one or two generations and were then subjected to resistance tests as described below. Dynamics of B and Q biotype populations under protected conditions The plant chosen to conduct these experiments was basil (Ocimum basilicum, family: Lamiaceae; variety: Pery), a favored host plant to B. tabaci. Two experiments were conducted to test the dynamics of both the B and Q bio-

7 318 Svetlana Kontsedalov et al. Fig. 3 B. tabaci populations collected during 2010 and their biotype composition in open field crops (A) and protected crops (B). The percentage of B and Q biotypes in open field or protected crops is given in C. types populations under chemical management program and protected conditions. The first experiment was conducted during summer in the Beit Shaan Valley, northern Israel, and started on June 24, 2010 and terminated on November 1, Four treatments were compared inside nethouses 2 m wide 4 m long 2 m high, with double entrance made from the 50-mesh insect-proof net. The four treatments were the following: i) responsive treatment in which chemical control was first given in response to B. tabaci appearance, using insecticides that can be used 1 wk prior to harvesting (responsive 1 wk), ii) responsive treatment, in which chemical control was first given in response to B. tabaci appearance, using insecticides that can be used 2 wk prior to harvesting (responsive 2 wk), iii) protective treatment, in which chemical control was applied right after planting and continued with a control program as shown in Table 4, and iv) control nethouses in which no sprays against B. tabaci were given. For comparison, the levels of B. tabaci populations were also estimated outside the nethouses. Inside and outside each nethouse, three yellow sticky traps were placed and the number of whiteflies trapped was counted several times along the experiment by replacing the traps, as indicated in Fig. 1-A. The yellow traps were made of Petri dishes 90 mm wide, spread with sticky glue and placed horizontally on a yellow background and supported on a metal stand at the level of the plants. The second experiment was conducted in Kikar Sdom, southern Israel. This experiment was started on May 5, 2010 and terminated on July 7, Four treatments were compared inside tunnels covered with plastic, and were 5 m wide 40 m long 2 m high, with double entrance to each tunnel made from 50-mesh insect-proof nets: i) responsive treatment, in which chemical control was first given in response to B. tabaci appearance (responsive), ii) the same as in the (i) treatment except that plastic with the ability to absorb UV light was used (responsive UV), iii) protective treatment, in which chemical control was applied right after planting and continued with a control program as shown in Table 4, and iv) control treatment, in which no sprays were given against B. tabaci. In this experiment, yellow sticky traps were used to estimate B. tabaci populations as described above. Whitefly population rearing After collection from the field, whitefly adult populations were directly transferred to insect-proof cages containing

8 Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years Table 4 Control programs against B. tabaci populations developed in both experiments conducted in the North and South during the year ) North Jun 24 Jul 8 Jul 10 Jul 11 Jul 17 Aug 23 Aug 29 Aug 31 Sep 6 Sep 12 Sep 16 Sep 20 Sep 22 Sep 28 Oct 5 Protective AB 1) CE AB CD CE CD CD CD CE CD CD CD Responsive 1 wk CD CD CD CD CD CE CE CD CD CD Responsive 2 wk CD CD CD AB CD CD AB CD AB CD South May 5 May 15 May 20 May 25 Jun 1 Jun 10 Jun 18 Jun 21 Jun 24 Jun 25 Jul 1 Protective AE AB CD F CD G CD FG E CD CD Responsive F CD G CD FG E CD CD Responsive UV F CD G CD FG E CD CD 1) The spray dates and treatments are shown. A, Confidor (imidacloprid); B, Evisect (thiocyclam hydrogen oxalate); C, Totach (neem oil+pyrehtrum natural); D, Knimat (potassium salts); E, Zohar LQ 215 (fatty acid potassium salts); F, Calipso (thiacloprid); G, Botanigard (Beauveria bassiana); H, EOS (mineral oil). For each insecticide, the commercial name is given first and the active ingredient in parenthesis. cotton cv. Acala seedlings (obtained from Zeraim Gedera, Israel). These adults were given a week to lay eggs and to establish a colony. The colonies were then maintained in the laboratory under standard conditions ((26±2) C, 60% RH, 14:10 h L:D). Identification of B. tabaci biotypes Biotypes were identified using microsatellite markers based on a standard PCR reaction with the primer pair Bem23 which distinguishes between B and Q biotypes based on the fragment size amplified. The primers are Bem23-F 5 - CGGAGCTTGCGCCTTAGTC-3 and Bem23-R5 -CGGC TTTATCATAGCTCTCGT-3. The product size obtained from B biotype was 200 bp and from the Q biotype was 400 bp. The PCR conditions were recently described (Skaljac et al. 2010). To verify the identity of the B and Q biotypes we amplified and sequenced 850 bp of the mitochondrial (mt) COI gene by PCR with the primers C1-J TTGATTTTTTGGTCATCCAGAAGT-3 and L2-N TCCAATGCACTAATCTGCCATATTA-3. The PCR conditions for amplifying mtcoi and the microsatellite markers were those previously described (Skaljac et al. 2010). Resistance bioassays The susceptibility of some of the collected populations to three insecticides of the neonicotinoid group (thiamethoxam, imidacloprid and acetamiprid) was tested. The effect of the three tested insecticides was scored on adults. Because the insecticides differ in their application, different bioassays were employed. The assay for thiamethoxam and acetamiprid was performed by dipping cotton seedlings (20-25 cm high, with two true leaves) for 20 s in the test compound, and then allowing the plant to air-dry in a fume hood under laboratory conditions for 2 h (Horowitz et al. 2004, 2005; Kontsedalov et al. 2008). Adult whitefly females (15-20 per replicate) were then confined on the treated seedlings using clip-on leaf cages for 48 h, after which their mortality was scored. For imidacloprid, cotton stems with two true leaves were inserted in plastic vials containing the compound in the desired concentration for 24 h, and Petri dishes, 45 mm diameter with cotton leaf discs with the same diameter were prepared as previously described (Kontsedalov et al. 2008). Adult mortality was determined after 48 h of whitefly exposure to the treated leaf discs. Deionized water was used as a control for all experiments. Each bioassay was performed with a minimum of five replicates. Insecticide For the resistance monitoring the following insecticides were used: acetamiprid: commercial name Mospilan 200 g active ingredient kg -1, produced by Nippon Soda Co., Tokyo, Japan; thiamethoxam: commercial name Actara 25WG 250 g active ingredient kg -1 produced by Syngenta AG, Basel Switzerland; imidacloprid: commercial name Confidor 350 g active ingredient kg -1, produced by Bayer Crop Science, Germany. Other insecticides used in the control programs in the north and the south are detailed in Table 4. Data analysis Comparisons of adult mortality were performed using Student s t-test with unequal sample sizes, and percentage data were transformed (angular transformation) before analysis. Probit analyses of the concentration-dependent mortality data were performed using POLO-PC (1987) (LeOra software) after correction with Abbott s formula (Abbott 1925) which takes into account the mortality in the control experiment run in parallel with the treatment. Control experiments were performed independently for each insecticide tested. Failure of 95% LC to overlap at a particular lethal concentration indicated a significant difference. Acknowledgements We wish to thank the chief scientist of the Ministry of

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