Larva Immersion and Square Dip method
Lambda-cyhalothrin
H. armigera larva mortality ranged from 3.3 to 100 percent when exposed to different concentrations (2, 1, 0.5, 0.25, 0.12, 0.0625 and 0.03125μl/ml) of lambda-cyhalothrin (Table 1). Larval mortality of 100, 100, 100 and 90% mortality was obtained at field rate (5.0 x 10-4g. a.i/ml) of the insecticide for Goffa-Sawla, Upper-Awash, Werer, and Gewane population, respectively. Four times lower dose from the recommended rate (1.25 x 10-4g. a.i/ml) resulted in 100% mortality on Gofa-Sawla population, which was higher than field rate dose-mortality (90%) of Gewane and two times lower dose rate mortality percent (96%) Upper-Awash and (86.7%) of Werer Population (Table 1). In squared dip method the field rate lambda-cyhalothrin (5.0 x 10-4g. a.i/ml) resulted in 100, 100, 96.7 and 93.3% mortality on Goffa-Sawla, Upper-Awash, Werer and Gewane populations, respectively (Table 1). The four-times lower dose (1.25 x 10-4 g. a.i/ml) caused 100% mortality while the eight-times lower dose (2.5 x 10-5a.i/ml) 93.3%) mortality on the Goffa-Sawla population while only 83.3% mortality was recorded for Werer to two times-lower doses (2.5 x 10-4g. a.i/ml) and 90% mortality of the Upper-Awash population to field rate dose of Gewane population (Table 1). Both bioassay method showed the Gewane population was less susceptible to lambda-cyhalothrin in larva immersion method and larva susceptibility were in the decline sequence of Goffa-Sawla >Upper-Awash >Werer > Gewane population.
The different concentrations of lambda-cyhalothrin 5% EC resulted in variable levels of, mortality when tested against H. armigera larvae which originated from different locations. (Table 2). In the larva immersion method the Goffa-sawla population had a comparatively low value of LC50 (0.074μl/ml) and LC90 (0.260 μl/ml) with a steep log dose probit slope of the mortality regression line of 2.36. Whereas, high LC50 (0.498μl/ml) and LC90 (2.870 μl/ml) values were obtained for the Gewane population with slope values of 1.69 (Table 2). In the squared dip method P-values in the goodness-of-fit table of Werer (0.9967), Upper-Awash (0.9985), Goffa-Sawla (0.9134) and Gewane (0.9389) for the Pearson chi-square indicate an adequate fit for the model with a normal distribution (Table 2). The Goffa-sawla population had a steep log10 dose probit slope of the line 2.52. Whereas, Werer (1.84), Upper-Awash (1.80), and Gewane (1.78) slope values. Both bioassay method indicates the Goffa Sawla population is much more sensitive to lambda-cyhalothrin compared to other population. Goffa-Sawla population was significantly different (P<0.05) from Werer, Upper-Awash, and Gewane populations without any overlap of 95% CL (Table2).
This study revealed that variation in the level of susceptibility to lambda-cyhalothrin exists in H armigera collected from different locations. Both bioassay methods showed that the tested population had a low level of resistance to lambda-cyhalothrin. The Gewane population has a higher resistance ratio compared to other tested populations. The LC50 of Gewane population in larva immersion and square dip technique recorded 0.498 and 0.447 values, respectively. The RF for the respective larval immersion and square dip study showed 6.73 and 7.45 times more resistance of the Gewane H. armigera population compared to the susceptible Goffa-Sawla population. Both bioassays showed the presence of a low level of resistance to lambda-cyhalothrin in tested locations. Several studies have indicated the development of resistance in H armigera for pyrethroids. A low level of resistance to lambda-cyhalothrin was reported by Karaagac et al. (2013) from Turkey and Avilla and González-Zamora (2010) in Spain. Other studies reported moderate to high-level resistance (Hussain et al. (2014) and high-level resistance (Duraimurugan & Regupathy, 2005) of H. armigera to pyrethroids. This finding contrast with Geremew et al. (2004) who found in larva immersion and squared dip methods.
Table 1. Percent of mortality of 3rd instar H. armigera larvae in different concentration of lambda-cyhalothrin 5% EC 72 hours after treatment with larva immersion bioassay and 48 hours after treatment in squared dip bioassay (29+ 20C & 48+4% RH) on Gofa Sawla, Upper Awash, Werer and Gewane populations (N=30).
larva immersion
|
squared dip
|
Concentration (μl/ml)
|
Percent mortality
|
Concentration (μl/ml)
|
Percent mortality
|
Gofa Sawla
|
Upper Awash
|
Werer
|
Gewane
|
Gofa Sawla
|
Upper Awash
|
Werer
|
Gewane
|
2
|
100
|
100
|
100
|
90.0
|
2
|
100
|
100
|
96.7
|
93.3
|
1
|
100
|
96.7
|
86.7
|
70.0
|
1
|
100
|
90.0
|
83.3
|
73.3
|
0.5
|
100
|
83.3
|
70.0
|
53.3
|
0.5
|
100
|
76.7
|
63.3
|
53.3
|
0.25
|
83.3
|
63.3
|
46.7
|
26.7
|
0.25
|
93.3
|
56.7
|
46.7
|
33.3
|
0.12
|
66.7
|
40.0
|
23.3
|
13.3
|
0.12
|
73.3
|
36.7
|
23.3
|
16.7
|
0.0625
|
50.0
|
23.3
|
10.0
|
6.7
|
0.0625
|
56.7
|
20.0
|
10.0
|
3.3
|
0.03125
|
16.7
|
10.0
|
10.0
|
3.3
|
0.03125
|
23.3
|
6.7
|
3.3
|
3.3
|
Control
|
6.7
|
0
|
6.7
|
3.3
|
Control
|
3.3
|
3.3
|
6.7
|
6.7
|
Table 2. Comparative toxicity of lambda-cyhalothrin 5% EC to H. armigera populations in larva immersion and squared dip study.
Larva immersion
|
Location
|
N
|
LC50 μl/ml
|
95% CL
(lower-upper)
|
LC90 μl/ml
|
95%CL
(lower-upper)
|
The fit of probit analysis
|
RR
|
Slope + SE
|
χ2 (df)
|
P
|
Gofa-Sawla
|
180
|
0.074
|
(0.057 -0.094)
|
0.260
|
(0.192- 0.415)
|
2.36+ 0.333
|
2.778 (4)
|
0.5957
|
_
|
Upper Awash
|
180
|
0.153
|
(0.118 - 0.199)*
|
0.693
|
(0.476 - 1.226)
|
1.96 + 0.250
|
0.512 (4)
|
0.9723
|
2.07
|
Werer
|
180
|
0.264
|
(0.199 - 0.361)*
|
1.419
|
(0.886 -3.022)
|
1.75+ 0.236
|
2.15 (4)
|
0.7089
|
3.57
|
Gewane
|
180
|
0.498
|
(0.364 - 0.763)
|
2.870
|
(1.578 - 8.204)
|
1.69 + 0.256
|
0.622 (4)
|
0.9606
|
6.73
|
Squared dip
|
Location
|
N
|
LC50 μl/ml
|
95% CL
(lower-upper)
|
LC90 μl/ml
|
95%CL
(lower-upper)
|
The fit of probit analysis
|
|
RR
|
Slope + SE
|
χ2 (df)
|
P
|
|
Gofa-Sawla
|
180
|
0.060
|
(0.044 -0.075)
|
0.193
|
(0.144 - 0.306)
|
2.52+0.384
|
0.976 (4)
|
0.9134
|
_
|
Upper Awash
|
180
|
0.194
|
(0.147 -0.258)*
|
1.007
|
(0.657-1.969)
|
1.80 +0.237
|
0.113 (4)
|
0.9985
|
3.25
|
Werer
|
180
|
0.302
|
(0.230 - 0.41)*
|
1.505
|
(0.949-3.162)
|
1.84 +0.249
|
0.168 (4)
|
0.9967
|
5.03
|
Gewane
|
180
|
0.447
|
(0.334 - 0.651)*
|
2.338
|
(1.364-5.869)
|
1.78 +0.261
|
0.797 (4)
|
0.9389
|
7.45
|
N= total number of larva used for probit analysis, LC50 = median lethal concentration, LC90= the lethal concentration which killed 90% of the test H. armigera population, 95% CL= the lower and the higher confidence limits at which the LC50 and LC90 value can fall at 95% probability, SE= standard Error, χ2 =Chi-square, RR (Resistance Ratio) = LC50 of the field population / LC50 of Goffa-Sawla population, superscript denoted astric*=the collected H. armigera populations were not significantly different (P<0.05) among each other in their susceptibility to lambda-cyhalothrin insecticide.
Deltamethrin
Helicoverpa armigera populations of Werer, Upper-Awash, Goffa-Sawla, and Gewane populations exposed to different concentrations of deltamethrin 2.5% EC experienced a varying level of mortality. At field rate (3 x 10-4g. a.i/ml) deltamethrin gave 100, 93.3, 86.7, and 80.0% mortality 72 hours after larvae were immersed for Goffa-Sawla, Upper-Awash, Gewane, and Werer populations, respectively. In squared dip method at field rate (3.0 x 10-4 g. a.i/ml) deltamethrin gave 100, 90, 83.3 and 80.0% larval mortality after 48 hours in square dip method of Goffa-Sawla, Upper-Awash, Gewane and Werer population, respectively (Table 3). The field-collected H. armigera larva from Goffa-Sawla experienced 100% mortality at two times lower dose (1.5 x 10-4 g. a.i/ml) of deltamethrin which was higher than the field rate mortality of Werer, Upper-Awash, and Gewane populations, respectively (Tables 3). Werer population showed the lowest susceptibility to deltamethrin in both bioassay methods.
The P-values in the goodness-of-fit table of Werer, Upper-Awash, Goffa-Sawla, and Gewane for the Pearson chi-square indicates an adequate fit for the model with the normal distribution (Table 4). The LC50 values indicate that Werer, Upper-Awash, and Gewane populations were not significantly different among each other but differ (P<0.05) from the Goffa-Sawla population with no overlapping 95% CL (Table 4). The LC50 of Gewane population both in larva immersion (0.900) and square dip method (1.171) was lower than the Werer population of the respective LC50 values of 1.257 and 1.435 (Table 4). In the larva immersion method, the probit analysis showed that the Werer population is 8.79 times and Gewane populations 6.45 times more resistant to the susceptible Goffa-Sawla population (Table 4). Similarly, the square dip method also showed that the Werer and Gewane populations are 9.25 and 7.55 more resistant to the susceptible Goffa-Sawla population (Table 4). These indicate that there is a high resistance development in H. armigera for deltamethrin at Werer and Gewane. According to the resistance grouping of Torres-Vila et al. (2002a, b) H. armigera in Middle Awash, Ethiopia showed a low level of resistance to deltamethrin. Deltamethrin has been used to control H. armigera and sucking pests in cotton for a long time. Recently, due to lack of the ultra-low volume (ULV) formulation, the emulsify concentrate (EC) formulation of deltamethrin has been applied like ULV by mixing with a small volume of water to save time and labor (Personal communication). Such misuse of an insecticide against H. armigera, may result in the selection of resistant forms of the pest population. Development of low to high-level resistance in different strains of H. armigera for deltamethrin reported by Faheem et al. (2013) and Hussain et al., (2014) in Pakistan
Table 3. Percent of mortality of 3rd instar H. armigera larvae in different concentrations of deltamethrin 2.5% EC 72 hours after treatment with larva immersion bioassay and 48 hours after treatment in squared dip bioassay(29 + 20C & 48 + 4% RH) on Gofa-Sawla, Upper Awash, Werer and Gewane population (N= 30).
larva immersion
|
squared dip
|
Concentration (μl/ml)
|
Percent mortality
|
Concentration (μl/ml)
|
Percent mortality
|
Gofa Sawla
|
Upper Awash
|
Werer
|
Gewane
|
Gofa Sawla
|
Upper Awash
|
Werer
|
Gewane
|
3
|
100
|
93.3
|
80.0
|
86.7
|
3
|
100
|
90.0
|
80.0
|
83.3
|
1.5
|
100
|
76.7
|
56.7
|
66.7
|
1.5
|
100
|
76.7
|
50.0
|
60,0
|
0.75
|
93.3
|
50.0
|
30.0
|
43.3
|
0.75
|
96.7
|
56.7
|
23.3
|
33.3
|
0.375
|
76.7
|
26.7
|
13.3
|
20.0
|
0.375
|
76.7
|
40.0
|
3.3
|
6.7
|
0.1875
|
53.3
|
13.3
|
3.3
|
6.7
|
0.1875
|
56.7
|
20.0
|
3.3
|
3.3
|
0.09375
|
30.0
|
3.3
|
0
|
0
|
0.09375
|
26.7
|
6.7
|
0
|
0
|
0.046875
|
13.3
|
0
|
0
|
0
|
0.046875
|
6.7
|
0
|
0
|
0
|
Control
|
3.3
|
6.7
|
6.7
|
6.7
|
Control
|
6.7
|
6.7
|
0
|
6.7
|
Table 4. Comparative toxicity of deltamethrin 2.5% EC to H. armigera populations in larva immersion and squared dip study.
Larva immersion
|
Location
|
N
|
LC50 μl/ml
|
95% CL
(lower-upper)
|
LC90 μl/ml
|
95%CL
(lower-upper)
|
The fit of probit analysis
|
RR
|
Slope + SE
|
χ2 (df)
|
P
|
Gofa-Sawla
|
150
|
0.143
|
(0.104- 0.246)
|
0.572
|
(0.430- 0.966)
|
2.59 + 0.563
|
0.517 (3)
|
0.915
|
_
|
Upper Awash
|
150
|
0.690
|
(0.533 - 0.890)*
|
2.690
|
(1.863 - 4.894)
|
2.17 + 0.313
|
0.433 (3)
|
0.933
|
4.83
|
Werer
|
150
|
1.257
|
(0.980 - 1.690)*
|
4.814
|
(3.146 - 9.990)
|
2.20 + 0.331
|
0.044 (3)
|
0.998
|
8.79
|
Gewane
|
150
|
0.922
|
(0.717 -1.207)*
|
3.633
|
(2.446 - 7.017)
|
2.15 + 0.314
|
0.203 (3)
|
0.977
|
6.45
|
Squared dip
|
Location
|
N
|
LC50 μl/ml
|
95% CL
(lower-upper)
|
LC90 μl/ml
|
95%CL
(lower-upper)
|
The fit of probit analysis
|
|
RR
|
Slope + SE
|
χ2 (df)
|
P
|
Gofa-Sawla
|
150
|
0.155
|
(0.097 - 0.234)
|
0.515
|
(0.391 - 0.870)
|
2.74 + 0.626
|
0.884 (3)
|
0.829
|
_
|
Upper Awash
|
150
|
0.563
|
(0.400 - 0.758)
|
3.111
|
(1.970 - 7.063)
|
1.727 + 0.287
|
0.104 (3)
|
0.9913
|
3.63
|
Werer
|
150
|
1.435
|
(1.137- 1.899)*
|
4.712
|
(3.199- 9.103)
|
2.48 + 0.371
|
1.689 (3)
|
0.639
|
9.25
|
Gewane
|
150
|
1.171
|
(0.935- 1.504)*
|
3.751
|
(2.643- 6.632)
|
2.53 + 0.359
|
0.865 (3)
|
0.834
|
7.55
|
N= total number of larva used for probit analysis, LC50 = median lethal concentration, LC90= the lethal concentration which killed 90% of the test H. armigera population, 95%CL= the lower and the higher confidence limits at which the LC50 and LC90 value can fall at 95% probability, SE= standard Error, χ2 =Chi-square, RR (Resistance Ratio) = LC50 of the field population / LC50 of Goffa-Sawla population, superscript denoted astric*=the collected H. armigera populations were not significantly different (P<0.05) among each other in their susceptibility to deltamethrin insecticide.
Alphacypermethrin
Helicoverpa armigera larva from different locations had varied mortality when exposed to different concentrations of alphacypermethrin (Table 5). Alphacypermethrin caused 100% larva mortality at field rate (1.0 x 10-3g. a.i/ml) on Werer, Upper-Awash, and Gewane populations in both bioassay methods (Table 5). Except for Werer the two times-lower concentration alphacypermethrin resulted in 100% mortality. The four-time lower concentration (2.5 x 10-4 a.i/ml) resulted in 100% mortality only for the Goffa-Sawla population (5). Subsequent dilutions of the insecticide resulted in lower percent mortality of larva to alphacypermethrin (Tables 5). Both bioassay methods showed effective control of bollworm larva was achieved by alphacypermethrin compared with other insecticides tested. According to the current study the order of importance of pyrethroids used to combat H. armigera damage on cotton was: alphacypermethrin >lambda-cyhalotrin>deltamethrin.
Based on LC50 values, and the probit analysis Goffa-Sawla population was significantly different (P <0.05) from Werer, Upper-Awash, and Gewane population with non-overlapping 95% CL (Table 6). In this study, the probit analysis indicated showed resistance ratio in the range of 1.86-1.93 in the larval immersion method (Table 6) and 1.76-1.94 in the square dip method (Table 6). As a result, the level of resistance to alphacypermethrin was comparatively lower compared with other compounds of the pyrethroids group (lambda-cyhalothrin and deltamethrin) tested, which indicates that there is no resistance to the insecticide in all populations tested. The toxicity of alphacypermethrin was high compared to lambda-cyhalothrin and deltamethrin.
Alphacypermethrin insecticide is used for control of cotton bollworm in Middle Awash,. Because of its broad spectrum mode of action, typically it is applied one time during peak squaring and flowering period. That could be the reason for a high level of H. armigera mortality compared to other insecticides evaluated in this study. Alphacypermethrin is a newer insecticide in the study areas and has not been widely used compared to the other tested insecticides. Alpha-cypermethrin, a third-generation pyrethroid is now one of the top-selling insecticides globally (BASF Chemical Company, 2014). Therefore, alphacypermethrin could be used for the resistance management program as one of the insecticides in the alternation scheme.
Table 5. Percent of mortality of 3rd instar H. armigera larvae in different concentrations of alphacypermethrin 100G/L 72 hours after treatment with larva immersion bioassay and 48 hours in squared dip bioassay (29 + 20C 48+4% RH) on Gofa Sawla, Upper Awash, Werer and Gewane populations (N=30).
larva immersion
|
squared dip
|
Concentration (μl/ml)
|
Percent mortality
|
Concentration (μl/ml)
|
Percent mortality
|
Gofa Sawla
|
Upper Awash
|
Werer
|
Gewane
|
Gofa Sawla
|
Upper Awash
|
Werer
|
Gewane
|
1.5
|
100
|
100
|
100
|
100
|
1.5
|
100
|
100
|
100
|
100
|
0.75
|
100
|
100
|
96.7
|
100
|
0.75
|
100
|
93.3
|
96.7
|
90.0
|
0.375
|
100
|
90.0
|
83.3
|
83.3
|
0.375
|
100
|
80.0
|
83.3
|
76.7
|
0.1875
|
90.0
|
73.3
|
73.3
|
63.3
|
0.1875
|
86.7
|
76.7
|
66.7
|
56.7
|
0.09375
|
76.7
|
60.0
|
53.3
|
46.7
|
0.09375
|
70.0
|
60,0
|
53.3
|
43.3
|
0.046875
|
56.7
|
43.3
|
40.0
|
30.0
|
0.046875
|
53.3
|
36.7
|
36.7
|
26.7
|
0.0234375
|
26.7
|
16.7
|
16.7
|
10.0
|
0.0234375
|
23.3
|
16.7
|
16.7
|
10,0
|
Control
|
0
|
0
|
6.7
|
6.7
|
Control
|
10.0
|
0
|
3.3
|
6.7
|
Table 6. Comparative toxicity of alphacypermethrin 100G/L to H. armigera populations in larva immersion and squared dip study.
Larva immersion
|
Location
|
N
|
LC50 μl/ml
|
95% CL
(lower-upper)
|
LC90 μl/ml
|
95%CL
(lower-upper)
|
The fit of probit analysis
|
RR
|
Slope + SE
|
χ2 (df)
|
P
|
Gofa-Sawla
|
180
|
0.043
|
(0.031 - 0.055)
|
0.157
|
(0.114- 0.265)
|
2.28 + 0.366
|
0.992 (3)
|
0.803
|
_
|
Upper Awash
|
180
|
0.070
|
(0.051- 0.091)*
|
0.335
|
(0.232 - 0.591)
|
1.88 + 0.261
|
2.039 (4)
|
0.729
|
1.62
|
Werer
|
180
|
0.080
|
(0.057 - 0.107)*
|
0.471
|
(0.310 - 0.922)
|
1.66 + 0.236
|
0.978 (4)
|
0.913
|
1.86
|
Gewane
|
180
|
0.083
|
(0.078 - 0.133)*
|
0.459
|
(0.318 - 0.806)
|
1.97 + 0.256
|
2.62 (4)
|
0.620
|
1.93
|
Squared dip
|
Location
|
N
|
LC50 μl/ml
|
95% CL
(lower-upper)
|
LC90 μl/ml
|
95%CL
(lower-upper)
|
The fit of probit analysis
|
|
RR
|
Slope + SE
|
χ2 (df)
|
P
|
|
Gofa-Sawla
|
180
|
0.049
|
(0.036 - 0.063)
|
0.186
|
(0.134 - 0.320)
|
2.21 + 0.347
|
1.666 (3)
|
0.664
|
_
|
Upper Awash
|
180
|
0.079
|
(0.055 -0.107)*
|
0.528
|
(0.338- 1.096)
|
1.55+ 0.228
|
1.648 (4)
|
0.8001
|
1.62
|
Werer
|
180
|
0.086
|
(0.062 -0.115)*
|
0.516
|
(0.336-1.029)
|
1.65 + 0.234
|
0.977 (4)
|
0.9133
|
1.76
|
Gewane
|
180
|
0.095
|
(0.100- 0.185)*
|
0.852
|
(0.527- 1.871)
|
1.61+ 0.226
|
0.743 (4)
|
0.9459
|
1.94
|
N= total number of larva used for probit analysis, LC50 = median lethal concentration, LC90= the lethal concentration which killed 90% of the test H. armigera population, 95%CL= the lower and the higher confidence limits at which the LC50 and LC90 value can fall at 95% probability, SE= standard Error, χ2 =Chi-square, RR (Resistance Ratio) = LC50 of the field population/LC50 of Goffa-Sawla population, superscript denoted astric*=the collected H. armigera populations were not significantly different (P <0.05) among each other in their susceptibility to alphacypermethrin insecticide