The role of octopamine receptor agonists in the synergistic toxicity of certain insect growth regulators (IGRs) in controlling Dengue vector Aedes aegypti (Diptera: Culicidae) mosquito

Accepted Manuscript Title: The role of octopamine receptor agonists in the synergistic toxicity of certain insect growth regulators (IGRs) in controlling Dengue vector Aedes aegypti (Diptera: Culicidae) mosquito Author: Mohamed Ahmed Ibrahim Ahmed Christoph Franz Adam Vogel PII: DOI: Reference:

S0001-706X(15)30162-5 http://dx.doi.org/doi:10.1016/j.actatropica.2015.11.015 ACTROP 3784

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Acta Tropica

Received date: Revised date: Accepted date:

19-8-2015 17-11-2015 27-11-2015

Please cite this article as: Ahmed, Mohamed Ahmed Ibrahim, Vogel, Christoph Franz Adam, The role of octopamine receptor agonists in the synergistic toxicity of certain insect growth regulators (IGRs) in controlling Dengue vector Aedes aegypti (Diptera: Culicidae) mosquito.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2015.11.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The role of octopamine receptor agonists in the synergistic toxicity of certain insect growth regulators (IGRs) in controlling Dengue vector Aedes aegypti (Diptera: Culicidae) mosquito

Mohamed Ahmed Ibrahim Ahmeda,b and Christoph Franz Adam Vogelb,c,*

a

b

Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt

Center for Health and the Environment,

c

Department of Environmental Toxicology, University of

California, One Shields Avenue, Davis, CA 95616, USA

*Corresponding author. Address: Center for Health and the Environment, Environmental Toxicology Department, University of California Davis, Davis, CA 95616, USA Phone no.: +1-530-752-7775 Fax no.: +1-530-752-5300 E-mail address: [email protected]

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Graphical abstract

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Highlights  Pyriproxyfen was the most potent IGR insecticide among the tested insecticides based on the IGR assay.  Diafenthiuron was the most potent lethal insecticide among the Tested IGR according to the lethal toxicity assay.  Amitraz is a promising tool in increasing the potency of the selected insecticides controlling Aedes aegypti mosquito.

Abstract

The synergistic action of octopamine receptor agonists (OR agonists) on many insecticide classes (e.g., organophosphorus, pyrethroids, and neonicotinoids) on Aedes aegypti L. has been reported recently. An investigation of OR agonist’s effect on insect growth regulators (IGRs) was undertaken to provide a better understanding of the mechanism of action. Based on the IGR bioassay, pyriproxyfen was the most potent IGR insecticide tested (EC50= 0.0019 ng/ml). However, the lethal toxicity results indicate that diafenthiuron was the most potent insecticide (LC50=56 ng/cm2) on Aedes aegypti adults after 24 h of exposure. The same trend was true after 48 and 72 h of exposure. Further, the synergistic effects of OR agonists plus amitraz (AMZ) or chlordimeform (CDM) was significant on adults. Among the tested synergists, AMZ increased the potency of the selected IGRs on adults the greatest. As results, OR agonists were largely synergistic with the selected IGRs. OR agonists enhanced the lethal toxicity of IGRs, which is a valuable new tool in the field of Aedes aegypti control. However, further field experiments need to be done to understand the unique potential role of OR agonists and their synergistic action on IGRs.

Keywords: Aedes aegypti, Octopamine receptor agonists, Mosquito control, IGRs, Insecticide resistance, Synergistic ratio

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1- Introduction

Aedes aegypti L., a mosquito vector that transmits Dengue disease, is globally distributed in tropical and subtropical areas, which have increased since 1970 (Chandrashekhar et al., 2015; Govindarajan and Rajeswary, 2015; Poole-Smith et al., 2015). Insecticides play a central role in managing Aedes aegypti (Ahmed and Matsumura, 2012; Bara et al., 2014; Bissinger et al., 2014; Ahmed and Vogel, 2015), such as field controlling of larvae and adults, indoor controlling of adults through direct spraying, residual applications, and use of insecticide coated mosquito bed-nets. An unfortunate result of the extensive reliance on insecticides for mosquito control is the eventual rise in broad-spectrum insecticide resistance (Maestre-Serrano et al, 2014; Pocquet et al., 2014; Saavedra-Rodriguez et al., 2015). Thus, it is very important to employ contrastive methods to avoid or at least reduce the development of insecticide resistance. Insect growth regulators (IGRs) provide this option. IGRs inhibit chitin synthesis during insect development. Various features of IGRs make them attractive as alternatives to broad-spectrum of insecticides, especially conventional insecticides: IGRs are more selective, less harmful to the environment, and more compatible with pest management systems that include biological controls, such as natural predators. Furthermore, intelligent use of IGRs should reduce the likelihood of resistance developing (Brabant and Dobson, 2013; Suman et al., 2013; Thavara et al., 2013; Seccacini et al., 2014).

Formamidine insecticides are related to a unique group of insecticides that have numerous mode of actions on insect pest species. Their effect on monoamine-mediated production of cyclic adenosine monophosphate (cAMP) induces adverse behavioral changes in treated insects (Harrison et al., 1973; Hollingworth, 1976; Beeman and Matsumura, 1978; Evans and Gee, 1980; Nathanson and Hunnicutt, 1981). Formamidines are known to interact with several types of receptors by binding to octopamine receptors (OA receptors) and acting as octopaminergic agonists. This has attracted the attention of many scientists to elucidate the pathways of formamidines’ insecticidal actions (Hollingworth and Murdock, 1980; Evans and Maqueira, 2005; Balfanz et al., 2014; Wu et al., 2014). 4   

OA receptor agonists, amitraz (AMZ) and chlordimeform (CDM), have been extensively studied as targets of synergism of many insecticides. However, increasing the rate of uptake of these insecticides has been proposed to be a mechanism of OA receptor agonists that synergistic pyrethroid, organophosphate, and neonicotinoid insecticides. However, they remain a promising target for novel insect pest control agents (Casida and Durkin, 2013; Ahmed et al., 2015).

In this study, we assessed the toxicity of five IGR insecticides to Aedes aegypti fourth instars and the acute toxicity to Aedes aegypti adults. In addition, we investigated the role of OA receptor agonists, AMZ and CDM, on the activity of selected IGRs against Aedes aegypti adults. 2- Material and Methods 2.1 Mosquitoes

The field strain (Fresno, CA) of Aedes aegypti was obtained from the laboratory of Dr. Thomas W. Scott, University of California, Davis, and was used for all experiments. The rearing regime is described in detail by Ahmed and Vogel (2015). Because the UC Davis Institutional Review Board (IRB) ruled that this study did not meet the requirements for human subject research; therefore, IRB approval was not required.

2.2 Chemicals

Chlordimeform (99.8%), amitraz (96.8%), pyriproxyfen (99%), diafenthiuron (99.9%), lufenuron (99.7%), diflubenzuron (98.1%), and novaluron (99.6%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

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2.3 IGR Bioassays

IGR bioassays were conducted with 20 fourth instars larvae placed in 140-ml glass cups containing 99 ml of distilled water and 1 ml of insecticide (pyriproxyfen, diafenthiuron, lufenuron, diflubenzuron, and novaluron) in acetone solution and only 1 ml of acetone for controls. The cups were covered with double layered white mesh cheesecloth to contain emerging adults form escaping. Emergence was determined after 10 d of insecticide exposure because controls had completed emergence at this time. The synergistic effects of CDM and AMZ were not included in the IGR assays because they prevent adult emergence at the concentrations used.

2.4 Adult Bioassays

Adult bioassays were conducted in glass jars (600 ml) with internal surface area of 65 cm2, which was evenly coated with 1 ml of insecticide solution in acetone solution. For controls, the internal surface of the jars was evenly coated with only 1 ml of acetone. Acetone was allowed to evaporate for 30 min, and then 20 adults (5-7 days post-emergence) were placed inside each jar. The jar opening was covered with double layered white mesh cheesecloth. Adults were considered dead if they were ataxic. Mortality was determined after 24, 48, and 72 h of the exposure.

2.5 Synergistic Action bioassay

The synergistic action bioassay was conducted as described above for adult bioassays. Controls received only acetone and were run concurrently with each series of tests. Synergistic action was studied by testing the lethal actions of varying concentrations of tested insecticide alone or with the co-administered with 10 6   

µg/ml of AMZ and CDM, all dissolved in 1 ml of acetone. After the addition of the insecticides, the test solution was allowed to evaporate for 30 min for the adult bioassays. Previous research on AMZ and CDM, published by our lab, demonstrated that a concentration of 10 µg/ml did not cause mortality during the 72-h test period on fourth instar larvae of Aedes aegypti (Ahmed and Matsumura, 2012; Ahmed and Vogel, 2015) Therefore, at least five insecticide concentrations were used for all bioassays, and every bioassay was held at 25oC. Percentage mortality was recorded after 24, 48, and 72 h of exposure.

2.6 Statistical Analysis

The corrected mortality was calculated according to Abbott's formula (Abbott, 1925). Bioassay data (LC50 and 95% CL values) were analyzed by using IBM SPSS Statistics 22.0 software (SPSS Inc., Chicago, IL). Synergistic action was determined to be significant (P ≤ 0.05) when the 95% CIs for the LC50 values for adults exposed to insecticide alone did not overlap with those for larvae exposed to insecticide + synergist mixture. Synergistic ratio (SR) was calculated by dividing the LC50 value of the test insecticide by that of the LC50 obtained for the combined treatment (insecticide + synergist). Plus, toxicity index calculated as [(LC50 of the most toxic tested IGR insecticide/LC50 of the tested IGR insecticide) ×100].

Results

The toxicity of diafenthiuron, novaluron, lufenuron, and diflubenzuron relative to pyriproxyfen is shown in Table 1. Pyriproxyfen was found to be the most potent insecticide (EC50 = 0.0019 ng/ml) followed by diafenthiuron (EC50 = 0.19 ng/ml). Novaluron and lufenuron had moderate toxicity (EC50 = 0.63 and 2.85 ng/ml, respectively). Least toxic was diflubenzuron (EC50 = 5.30 ng/ml). Pyriproxyfen was more toxic than diafenthiuron, novaluron, lufenuron, and diflubenzuron by 100, 333, 1429, and 2500 fold, respectively (Fig. 1). Diafenthiuron was more toxic than novaluron, lufenuron, and diflubenzuron by 3, 14, and 25 fold, respectively. 7   

The lethal toxicity and the synergism of IGRs alone and in combination with AMZ and CDM on adult Aedes aegypti mosquitoes after 24, 48, and 72 h of exposure is shown in Table 2-4. Diafenthiuron was the most potent insecticide (LC50 = 56 ng/cm2) after 24 h of exposure followed by diflubenzuron and novaluron (LC50 = 401 and 832 ng/ cm2, respectively) (Table 2). Lufenuron had the lowest toxicity (LC50 = 1087 ng/cm2) among the IGRs tested with 24 h of exposure. This order of insecticide toxicity held after 48 and 72 h of exposure (Tables 3 and 4, respectively).

In the presence of the OR agonists, most insecticide LC50 values decreased compared to when an insecticide was used alone. Adding AMZ or CDM to the IGRs caused significant synergism, especially after 48 and 72 h exposure (Tables 3 and 4, respectively). Figure 2 presents time-dependent changes in the synergistic ratio (SR) of LC50 as affected by AMZ (Fig. 2A) and CDM (Fig. 2B) on adult mosquitos. The most prominent trend was that synergism was greater when IGRs were co-treated with AMZ compared to CDM. Diafenthiuron plus AMZ increased in toxicity with time: 4.3 fold after 24 h, 6.4-fold after 48 h, and 8.9-fold after 72 h of exposure. This trend was repeated with the other IGRs when combined with AMZ. 3- Discussion OA receptor agonists, AMZ and CDM, are unique synergists with specificity against Aedes aegypti. OA receptor agonists also exhibit a wide variety of possible mode of actions, including inducing behavioral changes, blocking neuromuscular transmission, and inhibiting monoamine oxidase activity (Aziz and Knowles, 1973; Hollingworth, 1976; Beeman and Matsumura, 1978).

In our study, the selected IGRs had significant toxicity on fourth instar larvae, and thus, hold promise in Aedes mosquito control. Pyriproxyfen and diafenthiuron showed the most toxicity against the larvae, and these findings are consistent with previous studies (Dell Chism and Apperson, 2003; Satho et al., 2003; Ritchie et al., 2013; Harburguer et al., 2014). Therefore, despite their slow action compared to common insecticides, IGRs are extraordinary potent insecticides. 8   

Against Aedes aegypti adults, diafenthiuron was the most efficient IGR among the tested insecticides. Thus, the selected IGRs may have another mechanism of action other than the inhibition of chitin synthesis. It has been reported that diafenthiuron is acutely toxic to larvae and adults of Aedes aegypti after 48 h treatment (LC50 = 120 ng/ml and 13 ng/cm2 for larvae and adults, respectively) (Paul et al., 2006). In another study, consistent with our findings, diafenthiuron was the most lethally toxic IGR among the tested IGRs assessed on fourth instar larvae of Aedes aegypti (LC50= 421, 253, and 170 ng/ml after 24,48, and 72 h exposure, respectively) (Ahmed and Vogel, 2015). However, pyriproxyfen did not cause any acute toxicity on the adult Aedes aegypti.

In the presence of OR agonists, IGR toxicity increased dramatically, particularly after 48 and 72 h of exposure. Further, the SR was greater with AMZ than CDM on Aedes aegypti adults. These results correspond well with a previous study that used the same OR agonists as synergists on Aedes aegypti (Ahmed and Matsumura, 2012; Ahmed and Vogel, 2015).

There are numerous physiological explanations for the interaction of insecticides with respective endogenous OR agonists as neurotransmitters, neuromodulators, and neurohormones to modulate multiple physiological and behavioral processes in insect pests (Roeder et al., 2003; Roeder, 2005). The synergistic action may due to elevated blood-sugar levels, which result in strong excitants that cause anorexia. This in turn would lead to accelerated energy exhaustion as shown by initial hyper excitation to the given insecticides (Ismail and Matsumura, 1991; Ismail and Matsumura, 1992; Ahmed et al., 2015) or the continuous burst of mandibular movements resulting in antifeeding in those affected insects (Shimizu and Fukami, 1984).

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4- Conclusion

We conclude that OR agonists significantly synergize the lethal action of select IGRs against Aedes aegypti adults. Further studies to understand the molecular and physiology aspects of OR agonists will help with resistance-management strategies in Aedes aegypti control with respect to the choice of insecticides and pest species. The availability of updated genetic and bioinformatic knowledge will significantly enhance this trend of research.

Acknowledgements

This work was supported and funded by a post-doctoral scholarship for the senior author from the Ministry of Higher Education, the Government of Egypt, grant number, SAB 1918. We are grateful to Prof. Dr. Thomas W. Scott, Department of Entomology and Nematology, University of California, Davis, for kindly providing us with Aedes aegypti eggs that were used for this study.

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Fig. 1. Toxicity index of five IGR insecticides on 4th instar larvae of Aedes aegypti after 10 days of exposure. Toxicity index = [(LC50 of the most toxic tested IGR insecticide/LC50 of the tested IGR insecticide) ×100].

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Synergistic Ratio (SR 50)

A) Insecticides + AMZ

10 9 8 7 6 5 4 3 2 1 0

Diafenthiuron Diflubenzuron Novaluron Lufenuron

24

48

72

Time (h)

B) Insecticides + CDM

Synergistic Ratio (SR 50)

4 3.5 3 2.5

Diafenthiuron

2

Diflubenzuron

1.5

Novaluron

1

Lufenuron

0.5 0 24

48

72

Time (h) Fig. 2. Time-dependent changes in the synergistic ratio (SR50) as calculated from LC50 values from Tables 2-4 for combined treatments with (A) AMZ and (B) CDM on adult Aedes aegypti as assessed 24, 48, and 72 h after their initial exposure.

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Table 1 Toxicity of five IGR insecticides to fourth instars of Aedes aegypti. EC50b a n Compounds Slope (± SE) (95% CL) 0.0019 Pyriproxyfen 3.9 (± 0.28) 360 (0.00044-0.0046) 0.19 Diafenthiuron 4.2 (± 0.53) 360 (0.03-0.57) 0.63 Novaluron 4.7 (± 0.81) 360 (0.03-2.34) 2.85 Lufenuron 4.9 (± 0.34) 360 (0.77-6.68) 5.30 Diflubenzuron 5.1 (± 0.62) 360 (1.39-13.33) a n = number of larvae tested, including control. b Effective concentration (ng/ml) to cause 50% of treated larvae to fail to emerge as adults. The toxicity was evaluated as percentage of adult emergence after 10 d.

Table 2 Lethal toxicity of select IGRs and synergizing action of AMZ and CDM on each insecticide to Aedes aegypti adult after 24-h of exposure. Insecticides (ng/cm2) Insecticides

Diafenthiuron Diflubenzuron Novaluron Lufenuron

Insecticides + AMZ e (ng/cm2)

LC50 (95% CL)b

Slope (±SE)

na

LC50 (95% CL) c

56 (34-92)

3.9 (±0.71)

360

13 (7-36)

4.6 (±0.35)

360

4.1 (±0.62)

360

4.7 (±0.41)

360

401 (289732) 832 (4761109) 1087 (7021475)

108 (72156) 297 (112709) 572 (312963)

Slope (±SE) 4.8 (±0.51) 4.3 (±0.24) 4.9 (±0.39) 5.2 (±0.76)

Insecticides + CDM e (ng/cm2)

SR d

LC50 (95% CL) c

4.3*

27 (13-64)

3.7* 2.8* 1.9

223 (154580) 858 (4741183) 1310 (9752106)

Slope (±SE) 3.8 (±0.27) 4.1 (±0.62) 3.9 (±0.48) 4.4 (±0.21)

SR d

2.1* 1.8 0.97 0.83

n, no. of adults tested including control. Concentrations are expressed in ng/cm2,and the response determined after 24 h. c Concentration of synergist was 10 µg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergist. e Adults exposed to insecticide and synergist simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). a b

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Table 3 Lethal toxicity of select IGRs and synergizing action of AMZ and CDM on each insecticide to Aedes aegypti adult after 48-h of exposure. Insecticides (ng/cm2) Insecticides

Diafenthiuron Diflubenzuron Novaluron Lufenuron

Insecticides + AMZ e (ng/cm2)

LC50 (95% CL)b

Slope (±SE)

na

LC50 (95% CL) c

32 (19-74)

4.1 (±0.18)

360

5 (2-9)

4.8 (±0.52)

360

44 (23-93)

4.7 (±0.83)

360

3.8 (±0.49)

360

206 (186519) 537 (324856) 895 (4011252)

173 (84405) 407 (286811)

Slope (±SE) 5.1 (±0.42) 4.5 (±0.32) 4.7 (±0.63) 4.2 (±0.29)

Insecticides + CDM e (ng/cm2)

SR d

LC50 (95% CL) c

6.4*

11 (4-36)

4.7*

98 (69-132)

3.1* 2.2*

283 (195407) 526 (371864)

Slope (±SE) 4.3 (±0.73) 4.9 (±0.41) 4.2 (±0.64) 3.8 (±0.51)

SR d

2.9* 2.1* 1.9 1.7

n, no. of adults tested including control. Concentrations are expressed in ng/cm2, and the response determined after 48 h. c Concentration of synergist was 10 µg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergist. e Adults exposed to insecticide and synergist simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). a b

Table 4 Lethal toxicity of select IGRs and synergizing action of AMZ and CDM on each insecticide to Aedes aegypti adult after 72-h of exposure. Insecticides (ng/cm2) Insecticides

Diafenthiuron Diflubenzuron Novaluron Lufenuron

Insecticides + AMZ e (ng/cm2)

LC50 (95% CL)b

Slope (±SE)

na

LC50 (95% CL) c

17 (9-32)

4.3 (±0.64)

360

1.9 (0.74-13)

4.7 (±0.42)

360

23 (16-49)

3.8 (±0.71)

360

68 (38-127)

4.1 (±0.83)

360

116 (84-208)

134 (81347) 287 (113490) 439 (318631)

Slope (±SE) 4.0 (±0.37) 3.9 (±0.56) 4.4 (±0.30) 4.9 (±0.78)

Insecticides + CDM e (ng/cm2)

SR d

LC50 (95% CL) c

8.9*

4.7 (1.6-22)

5.8*

46 (18-73)

4.2* 3.8*

120 (79214) 209 (131416)

Slope (±SE) 4.2 (±0.74) 4.8 (±0.19) 4.7 (±0.76) 3.6 (±0.42)

SR d

3.6* 2.9* 2.4* 2.1*

n, no. of adults tested including control. Concentrations are expressed in ng/cm2, and the response determined after 72 h. c Concentration of synergist was 10 µg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergist. e Adults exposed to insecticide and synergist simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). a b

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