Resistance to carbamate insecticides in Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan

Crop Protection 20 (2001) 427}432

Resistance to carbamate insecticides in Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan Mushtaq Ahmad *, M. Iqbal Arif
, Zahoor Ahmad
Agricultural Adaptive Research Farm, Vehari, Pakistan
Central Cotton Research Institute, Multan, Pakistan Received 17 April 2000; accepted 22 November 2000

Abstract The status of resistance to four carbamates viz. carbaryl, alanycarb, methomyl and thiodicarb was monitored in Pakistani populations of Helicoverpa armigera (HuK bner) during 1994 to 1999 using an IRAC leaf-dip method. A general trend was that resistance to carbaryl, alanycarb and methomyl was high in 1994 and 1995, low in 1996 and 1997, and moderate to high in 1998 and 1999. Because of limited use of carbamates in Pakistan, this trend is not understood. It may be the consequence of cross-resistance from mechanisms developed for other insecticide groups. However, a broad cross-resistance was evident among carbaryl, alanycarb and methomyl. Surprisingly, a very low level of resistance to thiodicarb was found throughout this six-year study. No correlation of resistance factors existed between thiodicarb and carbaryl or methomyl. This lack of cross-resistance between thiodicarb and other carbamates in H. armigera is very encouraging for devising an insecticide resistance management strategy against this pest.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Helicoverpa armigera; Resistance; Carbamate insecticides; Pakistan

1. Introduction

2. Materials and methods

The American bollworm, Helicoverpa armigera (HuK bner) (Lepidoptera: Noctuidae), is now the most important pest of cotton in Pakistan. Recent outbreaks have occurred in Pakistan in 1997 and 1998. Biologically, it is one of the most successful pests due to its high fecundity, great migration potential, wide host range, and diapausing behaviour to overcome unfavourable environmental conditions. It is considered a &di$cult to control' pest because of its ability to develop resistance against almost all the conventional insecticide chemistries applied for its control. In Pakistan, H. armigera resistance to endosulfan, pyrethroids and organophosphates has already been reported (Ahmad et al., 1995, 1997, 1998a, b, 1999). This paper documents carbamate resistance in "eld populations of H. armigera in Pakistan from 1994 to 1999.

2.1. Insects Fifth or sixth instar larvae of H. armigera were collected from various locations in Pakistan (Fig. 1). Each collection of about 400 larvae was made by walking through a 5-acre block of a particular host crop in a zigzag manner to randomize collections. The larvae were fed in the laboratory on a semi-synthetic diet (modi"ed from Shorey and Hale, 1965), consisting of chickpea #our (300 g), ascorbic acid (4.7 g), methyl-4-hydroxybenzoate (3 g), sorbic acid (1.5 g), streptomycin (1.5 g), corn oil (12 ml), yeast (48 g), agar (17 g) and distilled water (1300 ml) with a vitamin mixture. Adults were fed on a sucrose solution with the addition of vitamins and methyl-4-hydroxybenzoate. 2.2. Insecticides

* Corresponding author. Present address: Central Cotton Research Institute, P.O. Box 572, Multan, Pakistan.

The following commercial formulations of di!erent carbamates were used for bioassays: carbaryl (Sevin, 850 g/kg SP (soluble powder); Rhone-Poulenc, Lyon,

0261-2194/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 0 ) 0 0 1 6 8 - X

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M. Ahmad et al. / Crop Protection 20 (2001) 427}432

Fig. 1. Sampling sites where Helicoverpa armigera larvae were collected (refer to Table 1 for key to sites) in Pakistan.

France), alanycarb (Onic 300 g/l EC (emulsi"able concentrate); Otsuka, Osaka, Japan), methomyl (Lannate 400 g/kg SP; DuPont, Wilmington, DE, USA), and thiodicarb (Larvin 800 g/kg DF (dry #owable); RhonePoulenc). 2.3. Bioassays Newly moulted second instar larvae from F laborat ory cultures were exposed to di!erent insecticides using the leaf-dip technique recommended by the Insecticide Resistance Action Committee (IRAC) (Anonymous, 1990). Serial dilutions as ppm of the active ingredient of the test compounds were prepared using distilled water. Five-centimeter cotton leaf discs were cut and dipped into the test solutions for 10 s with gentle agitation, then allowed to dry on paper towel on both sides. Five larvae were released on to each leaf disc placed in a 5-cmdiameter petri dish with adaxial side up. Eight replicates of "ve larvae were used for each concentration and 6}11 serial concentrations were used for each test insecticide. The same number of leaf discs per treatment was dipped into distilled water as an untreated check. Moistened "lter papers were placed beneath leaf discs to avoid desiccation of leaves in petri dishes. After releasing the larvae, test containers were covered with a piece of black cloth to minimize cannibalism. Before and after the treatment, larvae were maintained at a constant temperature of 25$23C with a photo-period of 14 h.

2.4. Data analysis Larval mortalities were assessed after 48 h. Larvae were considered dead if they failed to make a coordinated movement. Results were expressed as percentage mortalities, corrected for untreated (check) mortalities using Abbott's formula. Data were analyzed on computer using probit analysis (Finney, 1971). Resistance factors (RFs) were determined at LC s and LC s by dividing the   lethal concentration (LC) values of each insecticide by the corresponding LC values for the Bhakkar strain, which showed the lowest LC values. The Bhakkar strain was collected from chickpea in a rain-fed area where pesticide usage is still very low. To interpret resistance patterns among the carbamates tested, pairwise correlations of log LC values of all populations for each insecticide were calculated.

3. Results and discussion 3.1. Baselines Since the Bhakkar population showed the lowest LC values (Table 1), it was used as a reference population. The LC of thiodicarb for the Bhakkar population  (9.5 ppm) was the same as that of the Reading strain (9.8 ppm), which is an established susceptible laboratory strain of H. armigera (Ahmad et al., 1995). The baseline

M. Ahmad et al. / Crop Protection 20 (2001) 427}432

429

Table 1 Resistance of "eld populations of Helicoverpa armigera to carbamate insecticides Insecticide Location

Map reference

Crop

Date collected

No. Slope$SLC (mg l\),  tested E (95% "ducial limits)

RF at LC 

LC (mg l\),  (95% "ducial limits)

RF at LC 

Carbaryl

1 2 3 4 5 7 11 12 13 14 15 1 2 4 5 7 12 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Chickpea Potato Okra Cotton Squash Chickpea Cotton Cotton Squash Cotton Cotton Chickpea Potato Cotton Squash Chickpea Cotton Cotton Cotton Chickpea Potato Okra Cotton Squash Cotton Chickpea Cotton Cotton Okra Cotton Cotton Squash Cotton Cotton Chickpea Potato Okra Cotton Squash Cotton Chickpea Cotton Cotton Okra Cotton Cotton Squash Cotton Cotton

Mar. 94 Dec. 94 June 95 Sep. 95 Apr. 96 Mar. 97 Aug. 98 Oct. 98 Apr. 99 Sep. 99 Oct. 99 Mar. 94 Dec. 94 Sep. 95 Apr. 96 Mar. 97 Oct. 98 Sep. 99 Oct. 99 Mar. 94 Dec. 94 June 95 Sep. 95 Apr. 96 Oct. 96 Mar. 97 Sep. 97 Oct. 97 June 98 Aug. 98 Oct. 98 Apr. 99 Sep. 99 Oct. 99 Mar. 94 Dec. 94 June 95 Sep. 95 Apr. 96 Oct. 96 Mar. 97 Sep. 97 Oct. 97 June 98 Aug. 98 Oct. 98 Apr. 99 Sep. 99 Oct. 99

280 440 400 320 320 280 280 240 280 320 280 320 320 320 360 280 320 280 360 320 400 400 320 320 320 280 320 320 320 280 280 320 320 320 320 360 360 280 280 320 280 360 360 280 280 280 360 320 280

1.0 19 149 343 7.1 2.3 3.7 43 57 103 34 1.0 68 68 5.2 2.6 231 200 69 1.0 57 84 75 9.2 67 4.0 7.8 13 15 15 9.5 49 33 16 1.0 3.4 0.8 4.3 1.8 2.8 3.7 2.4 3.3 7.7 7.6 2.9 8.1 5.8 7.3

110 4500 56 000 51 000 1200 290 790 6100 8000 17000 5000 63 3600 4500 380 110 12 000 9500 5700 26 2400 4500 2000 270 2000 110 250 460 370 420 200 1700 960 470 47 200 59 150 69 140 130 150 170 280 440 98 430 260 300

1.0 41 509 464 11 2.6 7.2 55 73 155 45 1.0 57 71 6.0 1.7 190 151 90 1.0 92 173 77 10 77 4.2 9.6 18 14 16 7.7 65 37 18 1.0 4.3 1.3 3.2 1.5 3.0 2.8 3.2 3.6 6.0 9.4 2.1 9.1 5.5 6.4

Bhakkar Sahiwal Khanewal Dadu Pakpattan Layyah Kabirwala Mailsi Multan Alipur Faisalabad Alanycarb Bhakkar Sahiwal Dadu Pakpattan Layyah Mailsi Alipur Faisalabad Methomyl Bhakkar Sahiwal Khanewal Dadu Pakpattan Vehari Layyah Lar Khokhran Muzafargarh Kabirwala Mailsi Multan Alipur Faisalabad Thiodicarb Bhakkar Sahiwal Khanewal Dadu Pakpattan Vehari Layyah Lar Khokhran Muzafargarh Kabirwala Mailsi Multan Alipur Faisalabad

2.6$0.2 35 (29}42) 1.5$0.1 660 (520}840) 1.2$0.1 5200 (4000}6900) 2.1$0.2 12 000 (10 000}15 000) 1.9$0.2 250 (200}310) 2.3$0.2 82 (67}99) 1.7$0.2 130 (110}170) 2.1$0.2 1500 (1200}1800) 2.1$0.2 2000 (1600}2500) 1.9$0.2 3600 (2900}4400) 2.0$0.2 1200 (930}1400) 1.9$0.2 13 (11}16) 2.0$0.2 880 (680}1000) 1.8$0.2 880 (710}1100) 1.7$0.1 67 (54}85) 2.4$0.2 34 (28}41) 2.1$0.2 3000 (2500}3700) 2.3$0.2 2600 (2100}3100) 1.6$0.1 900 (710}1100) 2.0$0.2 6.3 (5.1}7.7) 1.6$0.1 360 (280}460) 1.4$0.1 530 (410}690) 2.0$0.2 470 (380}570) 1.9$0.2 58 (46}72) 1.9$0.2 420 (340}520) 2.0$0.2 25 (20}31) 1.8$0.2 49 (40}62) 1.7$0.2 80 (63}100) 2.1$0.2 92 (75}110) 2.0$0.2 94 (76}120) 2.4$0.2 60 (50}73) 1.8$0.2 310 (250}390) 1.9$0.2 210 (170}260) 1.9$0.2 100 (83}130) 1.8$0.2 9.5 (7.6}12) 1.6$0.1 32 (25}40) 1.5$0.1 7.9 (6.1}10) 2.2$0.2 41 (34}50) 2.1$0.2 17 (13}20) 1.8$0.2 27 (22}34) 2.3$0.2 35 (29}43) 1.6$0.1 23 (18}29) 1.8$0.1 31 (25}39) 2.2$0.2 73 (60}89) 1.6$0.2 72 (57}92) 2.4$0.2 28 (23}34) 1.7$0.1 77 (61}97) 1.9$0.2 55 (44}68) 2.0$0.2 69 (56}85)

LC values of carbaryl for the Bhakkar population were the highest followed by alanycarb, thiodicarb and methomyl. The LC values of methomyl, thiodicarb and alanycarb for the Bhakkar population seem reasonably low; whereas LC values of carbaryl for this population were quite high, indicating low intrinsic e$cacy of carbaryl against H. armigera.

(84}150) (3000}6700) (35 000}90 000) (37 000}70 000) (830}1700) (210}400) (510}1200) (4200}8800) (5800}11 000) (12 000}24 000) (3500}7200) (44}91) (2600}5000) (3100}6500) (260}550) (84}150) (8900}17 000) (6900}13 000) (3900}8400) (19}37) (1600}3500) (2900}7000) (1400}2800) (190}390) (1400}2800) (76}150) (170}370) (310}670) (270}510) (290}620) (150}270) (1100}2400) (680}1400) (330}660) (33}68) (140}300) (38}89) (110}210) (49}98) (99}210) (93}170) (100}220) (120}240) (200}380) (280}680) (72}130) (300}630) (180}370) (210}420)

3.2. Carbaryl Carbaryl resistance in 1994 in the Sahiwal population of H. armigera was moderate (19-fold at LC and 40 fold at LC ). The two populations tested in 1995  showed a very high resistance. Subsequently, carbaryl resistance was very low in the Pakpattan, Layyah and

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M. Ahmad et al. / Crop Protection 20 (2001) 427}432

Kabirwala populations (2}11-fold) tested in 1996, 1997 and 1998. Then again, the last four populations tested in 1998 and 1999 had moderate to high resistance both at LC s (33}101-fold) and LC s (45}152-fold).   Carbaryl was a commonly used insecticide in Pakistan during 1970s. After the introduction of pyrethroids, its use became negligible during 1980s and 1990s. The reasons for #uctuations of carbaryl resistance during our 6-year study period are not clear. It may just be a chance or it may be a result of cross resistance due to mechanisms selected by other pesticides applied on H. armigera. Carbaryl resistance of H. armigera was earlier reported from Thailand (Ahmad and McCa!ery, 1988; Ahmad, 1994). 3.3. Alanycarb Alanycarb exhibited a high resistance ('50-fold) in the two populations of H. armigera tested in 1994 and 1995. The same populations had moderate to high resistance to carbaryl as well. Pakpattan and Layyah populations tested in 1996 and 1997 showed very low resistance (2}6-fold), also like that of carbaryl. Then, Mailsi, Alipur and Faisalabad populations tested in 1998 and 1999 exhibited very high resistance, which was even higher than that of carbaryl resistance in the same populations. The development of H. armigera resistance to both carbaryl and alanycarb in Pakistan therefore followed the same trend. Alanycarb has never been used in Pakistan, therefore resistance to this insecticide must be a case of cross resistance from other carbamates or pesticides.

carbamates in H. armigera have never been reported before. A maximum of 28-fold resistance of H. armigera was recorded to carbaryl from Thailand (Ahmad and McCa!ery, 1988) and 11-fold resistance to methomyl from Australia (Gunning et al., 1992). A general trend of initial high resistance of H. armigera in 1994 and 1995, a low resistance in 1996, 1997 and in some populations of 1998, and a moderate to high resistance in 1998 and 1999 was the same for carbaryl, alanycarb and methomyl. Alanycarb was never used in Pakistan and the use of carbaryl remained very low during 1980s and 1990s. Even the use of methomyl and thiodicarb is still limited in Pakistan. The application of methomyl and thiodicarb mostly started in 1990s after the development of H. armigera resistance to pyrethroids. The occurrence of a high carbamate resistance in H. armigera, particularly during 1994 and 1995, is very puzzling. This may be due to cross-resistance from other insecticide groups such as organophosphates and pyrethroids. However, cross resistance from pyrethroids seems unlikely, since resistance to pyrethroids in Pakistani populations of H. armigera was quite high during 1996 and 1997 (Ahmad et al., 1998b) vis-a-vis low resistance to carbamates in these years in the present study. Earlier no metabolic cross-resistance was found between pyrethroids and carbaryl in a Thailand strain of H. armigera (Ahmad and McCa!ery, 1991). Whereas H. armigera resistance in Pakistan has been increasing steadily to endosulfan (Ahmad et al., 1998a), pyrethroids (Ahmad et al., 1998b) and organophosphates (Ahmad et al., 1999), it has been erratic to carbamates over the last 5 years. This implies that carbamate resistance may have developed independently of other chemistries.

3.4. Methomyl 3.5. Thiodicarb Except for Pakpattan population, the other four populations of H. armigera tested from 1994 to 1996 showed a high level of resistance to methomyl. The Pakpattan population had a very low resistance to all the carbamates tested herein. Then, methomyl resistance was low in all the six populations tested in 1997 and 1998 and moderate in the three populations tested in 1999. Populations of 1999 likewise exhibited high levels of resistance to carbaryl and alanycarb. The use of methomyl is still low in Pakistan. The existence of a moderate to high level of resistance to methomyl in some Pakistani populations of H. armigera seems to result from cross-resistance by mechanisms produced by other insecticide(s). Methomyl resistance has also been reported in H. armigera from Australia (Gunning et al., 1992) and tobacco budworm, Heliothis virescens (F.) from the United States (Martinez-Carrillo and Reynolds, 1983; Roush and Wolfenbarger, 1985). In our studies, the highest resistance (at LC ) to  carbaryl, alanycarb and methomyl was 345-, 230- and 85-fold, respectively. Such high levels of resistance to

Resistance to thiodicarb remained very low in all the 15 populations tested from 1994 through 1999. Earlier, a very low resistance to thiodicarb was also found in Pakistani populations of H. armigera monitored from 1991 to 1993 (Ahmad et al., 1995). It clearly indicates that there may be little cross-resistance between thiodicarb and other carbamates in the Pakistani populations of H. armigera, in spite of the fact that thiodicarb and methomyl are closely related compounds. Similarly, no H. armigera resistance was found to thiodicarb in Australia, and even a methomyl-selected strain with a RF of 23 to methomyl showed no resistance to thiodicarb (Gunning et al., 1992). It seems that the sulphur bond between the two methomyl molecules, which gives rise to thiodicarb, probably makes thiodicarb innocuous to enzymatic attack in H. armigera. Thiodicarb was also not cross-resistant to endosulfan, cypermethrin and organophosphates in Pakistani H. armigera (Ahmad et al., 1995, 1998a). In fact thiodicarb is rare among the conventional chemistries in that it is

M. Ahmad et al. / Crop Protection 20 (2001) 427}432

431

Table 2 Pairwise correlations between LC values for the carbamates tested on each of Helicoverpa armigera populations referred to in Table 1 (superscripts denote signi"cance of the regression)

H. armigera but not in a resistant one from Thailand. Mechanisms responsible for carbamate resistance in Pakistani populations of H. armigera need further study.

Insecticide

Methomyl

3.8. Resistance management When choices for H. armigera control are already limited due to development of resistance, the erosion in e!ectiveness of another important insecticide class, i.e. carbamates, that was previously e!ective against this pest is of serious concern. Concurrently, the existence of a negligible resistance to thiodicarb in Pakistani populations of H. armigera is very encouraging and needs to be preserved. If thiodicarb is used injudiciously, the resistant genes found in H. armigera at low frequencies at present will be selected to high frequencies. The best-bet strategy to conserve susceptibility to thiodicarb is to limit its use to a single application, alone or in mixture, per season when Helicoverpa pressures are high. Thiodicarb is one of a few insecticides that can still provide an e!ective control of H. armigera with a long residual action and thus can be a useful tool to manage resistance. Since there is no evidence of its cross-resistance to pyrethroids or organophosphates (Ahmad et al., 1998a), thiodicarb can be alternated with these two groups to have a good rotation for the control of H. armigera. Thiodicarb may be best suited for application towards the end of season when resistance levels are the highest to other groups of insecticides. Its overuse may be avoided by adopting nonchemical IPM practices, rotating thiodicarb with new chemistries and making its correct applications at early instars.

Alanycarb LC 

Carbaryl 0.87  Methomyl 0.78  Thiodicarb 0.71 

Carbaryl LC 

LC 

LC 

LC 

LC 

0.90  0.73  0.69

0.85  0.19

0.87  0.18

0.24

0.31

still e$cacious in the "eld against H. armigera in Pakistan (Ahmad et al., 1998a). 3.6. Associations between resistance factors of carbamates Paired comparisons of the log LC s and LC s for all   the populations tested showed positive and signi"cant correlations among carbaryl, alanycarb and methomyl, indicating a broad cross-resistance within these carbamates (Table 2). A signi"cant positive correlation existed between thiodicarb and alanycarb at LC but not at  LC . However, no correlation was found between  thiodicarb and carbaryl or methomyl resistance. 3.7. Mechanisms of carbamate resistance A variety of resistance mechanisms have been implicated in carbamate resistance in di!erent insects. Microsomal cytochrome P -dependent monooxy genases were found responsible for carbaryl resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith) (McCord and Yu, 1987). Enhanced hydrolase activity was shown to play a major role in carbaryl resistance in German cockroach, Blatella germanica (L.) (Ku and Bishop, 1967) and peach-potato aphid, Myzus persicae (Sulz.) (Devonshire and Moores, 1982). Modi"ed acetylcholinesterase exhibiting reduced sensitivity to carbamate inhibition was documented as a cause of resistance in green rice leafhopper, Nephotettix cincticeps Uhler (Iwata and Hama, 1972), mosquito, Anopheles albimanus Weid. (Ayad and Georghiou, 1975) and H. virescens (Kanga and Plapp, 1994). Carbamate resistance due to reduced penetration was demonstrated in B. germanica (Ku and Bishop, 1967), Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Hanna and Atallah, 1971) and Colorado potato beetle, Leptinotarsa decemlineata Say (Rose and Brindley, 1985). Synergism studies by PBO (piperonyl butoxide) and DEF (tribufos) inferred that methomyl resistance in Australian H. armigera might be attributable to monooxygenase and esterase detoxi"cation (Gunning et al., 1992). Ahmad and McCa!ery (1991) found that PBO and DEF both synergized carbaryl in a susceptible strain of

Acknowledgements We thank M.R. Attique, Central Cotton Research Institute, Multan, Pakistan for sharing his laboratory facilities.

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