Susceptibility of African Bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) to selected pyrethroid Insecticides on Cotton

Helicoverpa armigera is a major threat for all cotton production areas in Ethiopia. Pests control with insecticides from a single chemistry group is common practice in most cotton farms, which may help to development of insecticide resistance. The studies aimed to determine the susceptibility of field population of H. armigera to pyrethroid insecticides. The experiment was carried out at Werer Agricultural Research Center under the laboratory condition using larva immersion and square dip methods. The selected insecticides were tested in seven dilutions levels. In each dilution 30 larvae of 3 rd instars, H. armigera were treated in three replications along with pure water. A low level of resistance was detected for all tested locations to alphacypermethrin and a high resistance ratio to lambda-cyhalothrin and deltamethrin for Gewane and Werer populations. Aplhacypermethrin was the most toxic insecticide and its LC 50 was low compared to other tested synthetic pyrethroids. Whereas, deltamethrin was the least toxic insecticide with high LC 50 . The LC 50 value of the Goffa-Sawla population was significantly different among the populations for Werer, Upper-Awash, and Gewane in both bioassay methods . The study concluded that Helicoverpa armigera might have resistant to deltamethrin in Werer and Gewane populations. Further studies on the monitoring of resistance are recommended. 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 LC 50 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.

For decades, cotton farmers have primarily been using chemical pesticides to control pests in Ethiopia. More than six sprays have been done per one cropping season for controlling a different cotton pest; four-round sprays are allotted for control of cotton bollworms and their effect on the environment has not been quantified (EIAR, 2016;Geremew, 2004).
Controlling these pests with the available insecticides has become difficult (Geremew, 2004).
Pests control method with insecticides from a single chemistry group is common practice in most cotton farms. In the past, development of resistance as in the case of lambda-cyhalothrin for H. armigera species at Dubti (Germew, 2004), dimethoate for aphid species at the Middle Awash (IAR, 1990), and carbamate group (carbosulfan, furathiocarb, and pirimicarb) for aphid species resistance to at Arbaminch, Dubti and Werer (Ermias, 2006). Efficacy reduction of endosulfan at Werer area (WARC, 1998) and commercial farms in Ethiopia (Geremew and Surachate, 2005). Currently, the commonly used synthetic pyrethroid insecticides, lambda-cyhalothrine, and deltamethrine have shown efficacy reduction in controlling African bollworm in the Middle Awash area (Personal communication). This might be due to the development of insecticide resistance by African bollworms (H. armigera). Because of these, the present study was undertaken to determine the suscetebility of field population H. armigera to commonly used synthetic pyrethroid insecticides under laboratory conditions.

Material and methods
The experiment was conducted at Werer Agricultural Research Center (WARC), Amibara District, Gebresu zone of Afar National Regional State during the 2017 cropping seasons under laboratory conditions.

Laboratory Experiments
African bollworm (Helicoverpa armigera) larva collection and rearing The larvae of African bollworm were collected from unsprayed cotton farms in Middle  (Geremew and Surachate, 2003). In each adult rearing, cage one plastic cup plugged with cotton wool immersed in the sugar solution was kept for the adults to feed. The adults were allowed to lay eggs on cheese close or a detached cotton branch placed inside the cage.
The eggs hatch after three or four days. The hatched larvae were collected and reared on cotton leaves. Starting the second instar stage, larvae were separated and held singly in Petri dish with cotton leaves. The experiment was conducted on the third instar larvae. Larvae of ABW were collected from chickpea fields of small-scale farms at Gofa-Sawla area (1260m.a.s.l, E 036 0 56 ' & N 06 0 19'), Southern Ethiopia, with no insecticide use history in the last six years were brought to Werer Agricultural Research Center and used for comparison with African bollworm collected from cotton farms which are heavily sprayed for many years.

Laboratory Bioassay Methodology
The bioassay was conducted using the newly molted F 1 generation of 3 rd instar larva by using the square dip and larval immersion bioassay procedure recommended by Geremew et al. (2004). The experiments were laid with a completely randomized design (CRD) with three replications. For each replicate of a serial dilution, ten larvae were used.

Experiment 1. Larval Immersion Method
Thirty larvae were used in each treatment and each treatment was replicated three times. For each treatment, ten 3 rd instar larvae per replication were used. The larvae were dipped into individual dilutions for ten seconds and placed on tissue paper padded trays for absorbing excessive liquid from the body. Larvae were transferred into glass Petri dish with insecticidefree cotton a square. The check treatment was treated with pre-water. The mortality rate was assessed 24 hours after placing the larvae by probing the larvae with a fine camel hairbrush.
If the larvae respond for probing it was considered alive or dead otherwise.

Experiment 2. Square Dip Method
Medium size cotton squares which weigh 700-1000 milligrams were collected from the unsprayed cotton field and dipped into individual dilutions of insecticides for ten seconds and transferred onto a paper padded tray for air-drying. After 60 minutes of drying, single dipped squares were kept in glass Petri dishes and a single 3 rd instar larva was introduced for feeding on the treated squares. The check treatment was treated with pre-water. The mortality rate was assessed 24 hours after placing the larvae by probing the larvae with a fine camel hairbrush. If the larvae respond for probing it was considered alive or dead otherwise.

Data Collected
The dose-mortality larvae were recorded after 24, 48, and 72 hours of treatment for larval immersion bioassay while after 24, 36, and 48 hours of treatment for square dip method bioassay. Larvae were regarded as dead if they are not able to move when probed with a blunt probe or brush. Results were expressed as percentage mortality. The daily minimum and maximum temperature and RH of the laboratory were recorded.

Statistical Analysis
Data from a bioassay were corrected for control mortality using Abbott's formula (Abbott, 1925): The results obtained from the dose-mortality experiments were estimated by probit analysis (Finney, 1971) using the SAS software version 9.0 (SAS Institute, 1999

Results and Discussion
Larva Immersion and Square Dip method
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 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 (

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 -3 g. 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 LC 50 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.

Conclusions
The current study confirmed a reduction in efficacy and the development of a low level of resistance in the H. armigera population to lambda-cyhalothrin at Werer and Gewane tested locations. The efficacy of deltamethrin was moderately reduced and had a higher resistance ratio compared to lambda-cyhalothrin in Werer and Gewane locations. Helicoverpa armigera might have resistant to deltamethrin; thus, there is a need to replace it with new insecticides with a different mode of action.
These insecticides were used for a long time to control cotton bollworm and sucking pest.
Alphacypermethrin insecticide could be used for the resistance management program as one of the insecticides in the alternation scheme. The study included a limited number of insecticides out of the commercially registered cotton H. armigera control in Ethiopia. Future studies are needed to monitor the level of insecticide resistance