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Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito
ARTICLE IN PRESS Pesticide Biochemistry and Physiology ■■ (2015) ■■–■■
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Pesticide Biochemistry and Physiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p e s t
Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito Mohamed Ahmed Ibrahim Ahmed a,b, Christoph Franz Adam Vogel b,c,* a
Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt Center for Health and the Environment, University of California Davis, Davis, CA 95616, USA c Environmental Toxicology Department, University of California Davis, Davis, CA 95616, USA b
A R T I C L E
I N F O
Article history: Received 9 October 2014 Accepted 19 January 2015 Available online Keywords: Aedes aegypti Octopamine receptor agonists Neonicotinoids IGRs Insecticide resistance
A B S T R A C T
Studying insecticide resistance in mosquitoes has attracted the attention of many scientists to elucidate the pathways of resistance development and to design novel strategies in order to prevent or minimize the spread and evolution of resistance. Here, we tested the synergistic action of piperonyl butoxide (PBO) and two octopamine receptor (OR) agonists, amitraz (AMZ) and chlordimeform (CDM) on selected novel insecticides to increase their lethal action on the fourth instar larvae of Aedes aegypti L. However, chlorfenapyr was the most toxic insecticide (LC50 = 193, 102, and 48 ng/ml, after 24, 48, and 72 h exposure, respectively) tested. Further, PBO synergized all insecticides and the most toxic combinatorial insecticide was nitenpyram even after 48 and 72 h exposure. In addition, OR agonists significantly synergized most of the selected insecticides especially after 48 and 72 h exposure. The results imply that the synergistic effects of amitraz are a promising approach in increasing the potency of certain insecticides in controlling the dengue vector Ae. aegypti mosquito. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The current insecticide-based strategy for vector control of Aedes aegypti L. relies heavily on the use of pyrethroids on bed nets, indoor spraying and larvicidal applications [1–3]. Such a heavy reliance on only one type of insecticide is likely to lead to future problems of resistance development that would create intractable predicaments in the control efforts of Ae. aegypti [4]. Knowing the history of many insecticide resistance problems, most researchers in this field agree that such a scenario is not farfetched in this case. However, it is also necessary to point out that insecticidal chemicals are the best proven approach among other methods of pest control that works well in controlling the dengue vector mosquitoes as shown by the earlier success of DDT [5]. Importantly, there is no question about the great need to continue searching for novel approaches
Abbreviations: PBO, piperonyl butoxide;; AMZ, amitraz; CDM, chlordimeform; IPM, integrated pest management; OR, octopamine receptor; cAMP, cyclic adenosine monophosphate; IGRs, insect growth regulators; IRR, Institutional Review Board; SR, synergistic ratio; LC50, lethal concentration, 50%; 95% CL, 95% confidence limit. * Corresponding author. Center for Health and the Environment, Environmental Toxicology Department, University of California Davis, Davis, CA 95616, USA. Fax: +1 530 752 5300. E-mail address: [email protected] (C.F.A. Vogel).
of mosquito control, such as development of vaccines, biological control measures, and introduction of molecular biological means of mosquito control [6]. Chemical insecticides are developed by pesticide manufacturing companies through mostly empirical screening and biological testing efforts. In more recent years, however, most pesticide companies rely on the known mode of action of each chemical class of insecticides, such as chemicals targeting the sodium channel, the nicotinic acetylcholine receptor, cholinesterase etc., because such knowledge-based approaches increase the chance of discovering the ‘blockbuster’ pesticide more quickly [7]. The major classes of pesticides used today can be distinguished by the biochemical target through which each group of pesticides attacks. Accumulation of knowledge in this regard has helped tremendously in selecting flexible approaches to control pests, such as rotating insecticides with a totally different mode of action to ameliorate the damages resulting from very stubborn cases of recalcitrant resistant pests in the past. Thus, a useful approach to preclude rapid development of resistance has been found to be avoiding excessive, uniform, and region-wide uses of high dose application of the main type of insecticide throughout all stages of the mosquito life cycle [8]. Unfortunately, the heavy reliance on pyrethroids in controlling vector mosquitoes as larvicides and adulticides is now reaching the stage where resistance problems are expected to grow [9–11]. The most effected type of strategy is an integrated pest management (IPM)
http://dx.doi.org/10.1016/j.pestbp.2015.01.014 0048-3575/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
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program in order to mitigate or reduce the chance of rapid development of resistance. In this regard, many different types of efforts have been made to minimize the use of the main insecticide of the control program including limiting the percentage used in terms of the spray areas, using mixtures of insecticides with different modes of action, using the right insecticide formulation and targeting only the critical time or area of vulnerability [12]. There are a number of examples of successful applications of mixtures of pesticides in resistance management [8,13]. At least there is one demonstration of the effectiveness of combinatorial effects (permethrin plus imidacloprid) reducing the number of mosquito bites when topically applied to dogs [14]. The effectiveness of formamidine, type octopamine receptor (OR) agonist, in synergizing the insecticidal actions of pyrethroids on houseflies (dipterous insects) was first reported by Liu and Plapp [15] with the most effective synergistic effect using amitraz. In view of the significant synergism between octopamine and cypermethrin, the molecular base of this synergism is likely activation of the OR signaling. The subject of the mechanism of action of this class of OR agonists, particularly that of formamidines, has been extensively reviewed [16–18]. Briefly in insects octopamine acts like epinephrine, and OR in this regard serves as the receptor mediating the sympathetic route, equivalent of the mammalian autonomic controlling system. As in the case of epinephrine, one of the major consequences of its signaling in insects is excitation coupled with loss of appetite. While there are qualitative differences in the actions between octopamine and epinephrine, the above two basic actions of OR signaling serves as two of the main modes of their pesticidal actions. While studies on the action of octopamine are rare in mosquitoes, in the brain of adult Culex pipiens mosquitoes, octopamine causes activation of cyclic adenosine monophosphate (cAMP), indicating this basic mechanism does indeed operate in this species. Among dipterous insects, Drosophila is the best studied species in which octopamine has been shown to induce cAMP. In some insects elevation of cAMP by octopamine directly causes the loss of appetite [19]. Neonicotinoids are a relatively new group of powerful insecticides that clearly shows a wider spectrum of insect controlling property especially mosquito control [20–22]. The molecular bases of their diverse action spectra are gradually becoming elucidated [23,24]. Their basic mode of action is to attack the nicotinic acetylcholine receptor (nAChR), which is totally different from the action sites of any of the major insecticides frequently used for the control of mosquitoes in the past. While the original types of neonicotinoids were relatively polar, suited mostly on sucking type insect pests, the spectra of effectiveness of neonicotinoids have been steadily expanding including chewing type of insect pests because of their increased hydrophobic properties [25]. Recently, new promising tools have been used in mosquito control. For instance, chlorfenapyr is a protoxin requiring activation by cytochrome P450, mediates its toxic effects by disrupting the production of ATP resulting in cellular death and ultimately mosquito mortality [26]. Another example are insect growth regulators (IGRs) which inhibit chitin synthesis during the development of mosquitoes [27,28]. The advantage of the IGRs is causing less detrimental effects to beneficial insects. For instance they do not affect an insect’s nervous system and are less toxic to beneficial insects, more compatible with pest management systems that use biological control components, and are less likely to become resistant to IGRs [29–32]. Herein, we studied the synergistic action of OR agonists, AMZ and CDM, plus PBO on the insecticidal activity of selected novel insecticides against fourth instar larvae of Ae. aegypti. However, the potential of this study is to shed light on new promising tools to control the dengue vector Ae. aegypti mosquitoes.
2. Materials and methods 2.1. Mosquitoes The FIELD strain (Fresno, CA) of Ae. aegypti was obtained from the laboratory of Dr. Thomas W. Scott, University of California Davis, and was used for all experiments. The rearing regime has been described earlier [8]. Briefly, the eggs were hatched by placing a piece of dried paper towel loaded with previously deposited eggs in a flask filled with 750 ml of distilled water that been held under vacuum for 45 min. The vacuum was released and 50 mg of larval diet (dog food powder) added to the water. The hatched larvae were held overnight in the same flask, and then 200 larvae were transferred to each 600-ml beaker containing 400 ml of distilled water. Larval diet was added to each beaker according to the following regime: day 1, 75 mg; day 3, 38 mg; day 4, 75 mg; day 5, 113 mg; and day 6, 150 mg. Adult mosquitoes were reared in an environmental chamber with a temperature ranging from 22 to 30 °C, 80% relative humidity (RH), and a photoperiod of 14:10 (L:D) h. Adults were held in a 60 × 60 × 60-cm screened cage and provided 10% sucrose ad libitum. Human blood was provided to adults twice a week. Eggs were collected on paper towels lining the rim of water containers. The papers with eggs were air dried at 27 °C and 80% humidity for 24 h and stored in containers with 100% humidity for 3–30 d. The UC Davis Institutional Review Board (IRR) determined that feeding laboratory-reared mosquitoes on people in this experiment did not meet the requirements for human subject research, and thus, did not require IRR approval. 2.2. Chemicals Piperonyl Butoxide (99%), chlordimeform (99.8%), amitraz (96.8%), nitenpyram (99.9%), chlorfenapyr (99.6%), lufenuron (99.7%), diafenthiuron (99.9%), diflubenzuron (98.1%), and novaluron (99.6%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.3. Larval bioassays Twenty of fourth instar larvae were placed in 140-ml glass cups containing 99 ml of distilled water, 1 ml of each insecticide (chlorfenapyr, nitenpyram, diafenthiuron, diflubenzuron, novaluron, and lufenuron) in acetone solution, and 1 ml of acetone as vehicle for controls. Larvae were considered dead if they were unresponsive to touching with a probe or if they could not reach the surface of the water. Because of the slow-acting nature of some of these insecticides, mortality was determined after 24, 48, and 72 h of exposure. 2.4. Synergistic action bioassay The synergistic action bioassay was conducted as described above. Controls received acetone only and were run concurrently with each series of tests. Each series of synergistic action tests was carried out by testing the lethal action of varying concentrations of a test insecticide alone or the test insecticide co-administered with 10 μg/ ml of PBO, AMZ, or CDM. After the addition of the insecticides, the test solution was stirred briefly to ensure uniform mixture. Preliminary tests showed that PBO at 10 μg/ml was the maximum sublethal concentration where no mortality was observed of fourth instar larvae. Previous study on AMZ and CDM, which was published by our lab, demonstrated that concentration of 10 μg/ml did not cause mortality during the 72-h test period on fourth instar larvae of Ae. aegypti [8]. At least five concentrations were used for each bioassay. Every bioassay was held at 25 °C. Percentage mortality was recorded after 24, 48, and 72 h.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
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1.7 4.8* 1.8 0.93 1.1 0.91 4.1 (0.8) 5.0 (0.2) 3.9 (0.7) 4.8 (0.7) 4.1 (0.1) 4.6 (0.9) 114 (42–307 59 (15–108) 236 (62–889) 2603 (983–4751) 4660 (2452–7925) 7131 (4328–9734) 1.9 8.6* 3.2* 2.6* 2.1* 1.8 5.4 (0.4) 4.8 (0.3) 5.3 (0.2) 4.2 (0.3) 3.9 (0.7) 4.4 (0.3) 97 (18–267) 33 (19–93) 130 (44–329) 921 (673–2875) 2440 (1731–5816) 3616 (1972–6180) a
b
n, no. of larvae tested including control. Concentrations are expressed in ng/ml and the response determined after 24 h. c Concentration of synergists was 10 μg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergists. e Larvae exposed to insecticides and synergists simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05).
0.91 4.6* 3.7* 3.4* 2.9* 2.3* 5.1 (0.1) 5.4 (0.3) 4.8 (0.1) 3.9 (0.6) 4.1 (0.2) 4.9 (0.5) 213 (73–559) 62 (19–142) 114 (32–311) 713 (361–1045) 1767 (1242–3595) 2821 (1430–5082) 360 360 360 360 360 360 3.1 (0.2) 3.6 (0.2) 3.2 (0.2) 3.3 (0.1) 3.8 (0.1) 4.0 (0.1)
Slope (SE) LC50 (95% Slope (SE) LC50 (95%
193(62–1461) 284 (105–1490) 421 (136–597) 2411 (542–4753) 5137 (1315–7971) 6491 (1734–7419) Chlorfenapyr Nitenpyram Diafenthiuron Diflubenzuron Novaluron Lufenuron
Slope (SE) LC50 (95%
CL)c
Insecticides + AMZe (ng/ml)
SRd CL)c
Insecticides + PBOe (ng/ml)
na CL)b
Insecticides (ng/ml)
Insecticide resistance of mosquito populations is increasing at a dramatic rate, threatening the efficacy of control programs throughout insecticide-treated areas. Importantly, the combinations of insecticides with different modes of action could make a potential contribution in mosquito control especially where mosquitoes already show high levels of resistance to conventional insecticides [34,35]. The effect of PBO on the toxicity of the selected insecticides was different. However, PBO showed a significantly different degree of synergism with all selected insecticides after 24, 48, and 72 h exposure. For instance, chlorfenapyr showed only moderate synergism
Insecticides
4. Discussion
Table 1 Toxicity of selected insecticides and synergizing action of PBO, AMZ, and CDM on each insecticide on fourth instar larvae of Ae. aegypti after 24-h exposure.
The results of 24, 48, and 72 h mortality tests on selected insecticides with or without PBO, AMZ, or CDM and their levels of synergism are presented in Tables 1–3. Chlorfenapyr was the most toxic insecticide (LC50 = 193, 102, and 48 ng/ml, after 24, 48, and 72 h exposure, respectively), followed by nitenpyram (LC50 = 284, 205, and 131 ng/ml, after 24, 48, and 72 h exposure, respectively). However, the IGR insecticides (diafenthiuron, diflubenzuron, novaluron, and lufenuron) were the least toxic insecticides. PBO significantly synergized all insecticides except chlorfenapyr and the most toxic insecticide was nitenpyram even after 48 and 72 h exposure (LC50 values, 62, 28, and 16 ng/ml, respectively). AMZ significantly synergized most of selected insecticides except chlorfenapyr and lufenuron after 24 h exposure, meanwhile, after 48 and 72 h exposure, AMZ synergized significantly all insecticides and the most toxic insecticides after 48 h were nitenpyram (LC50 = 32 ng/ml) and after 72 h diafenthiuron (LC50 = 23 ng/ml). CDM was only synergized by nitenpyram after 24 h exposure whereas after 48 h exposure, 4 insecticides were synergized, chlorfenapyr, nitenpyram, diafenthiuron, and novaluron; however, after 72 h exposure, all insecticides were synergized. Fig. 1 shows the time-dependent changes in the SR of LC50 as affected by PBO (Fig. 1a), AMZ (Fig. 1b), and CDM (Fig. 1c). The most noticeable tendency was the time-dependent rises in SR on all selected insecticides (Fig. 1a). However, nitenpyram showed the most clear-cut time-dependent decrease in SR with both AMZ and CDM (Fig. 1b and c). In general, synergism was more pronounced when insecticides were co-treated with AMZ rather than with PBO or CDM. The toxicity of all insecticides on fourth instar larvae was increased after 48 and 72 h (Fig. 2a). For example, novaluron increased the toxicity from 2.51-fold after 48 h to 8.50-fold after 72 h compared to the toxicity after 24 h, however, in the combination with PBO (Fig. 2b), novaluron increased toxicity from 2.86-fold after 48 h to 12.44-fold after 72 h. This trend continued in fourth instar larvae which increased in toxicity after 48 and 72 h (Fig. 2c and d) after treatment with the selected insecticides in combination with AMZ and CDM. Broadly, a stronger increase in toxicity was found with IGR insecticides in combination with AMZ and CDM.
SRd
3. Results
LC50 (95% CL)c
Insecticides + CDMe (ng/ml)
Data from all tests were corrected for control mortality with Abbott’s formula [33]. Bioassay data were pooled and analyzed (LC50 and 95% CL values) by using IBM SPSS statistics 22 program (SPSS Inc., Chicago, IL). A synergistic action was determined to be significant (P ≤ 0.05) when the 95% CIs for the LC50 values for larvae exposed to insecticide alone did not overlap with those for larvae exposed to insecticide + octopamine receptor agonist mixtures. Synergistic ratio (SR) was calculated by dividing the LC50 value of the test insecticide by that of the LC50 obtained by the combined treatment with insecticide + octopamine receptor agonists.
Slope (SE)
SRd
2.5. Statistical analysis
3
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
4
Insecticides
LC50 (95% Chlorfenapyr Nitenpyram Diafenthiuron Diflubenzuron Novaluron Lufenuron
Insecticides + PBOe (ng/ml)
Insecticides (ng/ml) CL)b
102 (36–291) 205 (79–611) 253 (95–681) 962 (174–2560) 2043 (569–3138) 4105 (1060–7204)
Slope (SE)
na
CL)c
LC50 (95%
3.8 (0.2) 3.6 (0.2) 3.1 (0.2) 3.0 (0.1) 3.9 (0.1) 3.8 (0.1)
360 360 360 360 360 360
88 (26–282) 28 (5–76) 58 (26–185) 246 (92–723) 618 (253–751) 1577 (863–4602)
Insecticides + AMZe (ng/ml)
Slope (SE)
SRd
CL)c
LC50 (95%
3.6 (0.7) 4.8 (0.3) 5.5 (0.8) 4.9 (0.2) 3.8 (0.1) 4.2 (0.4)
1.2 7.3* 4.4* 3.9* 3.3* 2.6*
28 (7–68) 32 (7–82) 56 (14–147) 282 (103–441) 703 (259–2064) 1519 (692–3068)
Insecticides + CDMe (ng/ml)
Slope (SE)
SRd
LC50 (95% CL)c
Slope (SE)
SRd
5.1 (0.8) 4.2 (0.2) 4.9 (0.6) 3.4 (0.7) 4.1 (0.5) 3.8 (0.3)
3.6* 6.4* 4.5* 3.4* 2.9* 2.7*
43 (25–186) 53 (11–130) 121 (37–339) 505 (214–967) 887 (142–2115) 2927 (1089–6485)
4.1 (0.2) 4.8 (0.6) 4.9 (0.2) 4.3 (0.3) 3.6 (0.7) 4.7 (0.1)
2.4 3.9* 2.1* 1.9 2.3* 1.4
a
Table 3 Toxicity of selected insecticides and synergizing action of PBO, AMZ, and CDM on each insecticide on fourth instar larvae of A. aegypti after 72-h exposure. Insecticides
LC50 (95% Chlorfenapyr Nitenpyram Diafenthiuron Diflubenzuron Novaluron Lufenuron a
Insecticides + PBOe (ng/ml)
Insecticides (ng/ml) CL)b
48 (15–96) 131 (47–460) 170 (51–314) 419 (89–740) 604 (161–833) 1847 (462–2729)
Slope (SE)
na
c
LC50 (95% CL)
4.1 (0.2) 3.6 (0.2) 3.1 (0.2) 3.3 (0.1) 3.8 (0.1) 3.6 (0.1)
360 360 360 360 360 360
36 (19–84) 16 (7–41) 33 (13–114) 86 (42–273 142 (94–358) 497 (216–901)
Slope (SE)
LC50 (95%
5.4 (0.8) 4.7 (0.1) 5.2 (0.3) 4.3 (0.6) 3.9 (0.8) 4.2 (0.4)
1.3 8.2* 5.2* 4.9* 4.3* 3.7*
7 (3–20) 27 (32–614) 23 (11–63) 93 (52–321) 194 (74–409) 634 (392–1023)
n, no. of larvae tested including control. Concentrations are expressed in ng/ml and the response determined after 72 h. c Concentration of synergists was 10 μg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergists. e Larvae exposed to insecticides and synergists simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). b
Insecticides + AMZe (ng/ml) SRd
CL)c
Insecticides + CDMe (ng/ml)
Slope (SE)
SRd
LC50 (95% CL)c
Slope (SE)
SRd
4.5 (0.6) 4.2 (0.7) 4.9 (0.4) 3.7 (0.2) 4.1 (0.3) 3.6 (0.8)
6.9* 4.9* 7.4* 4.5* 3.1* 2.9*
13 (2–31) 47 (19–132) 49 (23–341) 161 (78–476) 252 (86–427) 880 (361–2135)
4.6 (0.2) 4.7 (0.6) 4.8 (0.2) 4.1 (0.4) 4.9 (0.1) 4.2 (0.3)
3.7* 2.8* 3.5* 2.6* 2.4* 2.1*
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n, no. of larvae tested including control. Concentrations are expressed in ng/ml and the response determined after 48 h. c Concentration of synergists was 10 μg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergists. e Larvae exposed to insecticides and synergists simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). b
M.A.I. Ahmed, C.F.A. Vogel/Pesticide Biochemistry and Physiology ■■ (2015) ■■–■■
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
Table 2 Toxicity of selected insecticides and synergizing action of PBO, AMZ, and CDM on each insecticide on fourth instar larvae of A. aegypti after 48-h exposure.
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Synergistic Ratio (SR 50)
10
a) Insecticides + PBO
9
5
a) Insecticides alone
8 7
Chlorfenapyr
6
Nitenpyram
5
Diafenthiuron
4
Diflubenzuron
3
Novaluron
2
Lufenuron
Times fold increase in toxicity
8 Chlorfenapyr 6
Nitenpyram Diafenthiuron Diflubenzuron
4
Novaluron
1
Lufenuron
2
0 24
48
72
0
Time (h) 10
72
14
9 8
Chlorfenapyr
7
b) Insecticides + PBO
12
Nitenpyram
6 5
Diafenthiuron
4
Diflubenzuron
3
Novaluron
2
Lufenuron
1 0 24
48
10 Nitenpyram Diafenthiuron 6
Diflubenzuron Novaluron
4
Lufenuron 2 0
c) Insecticides + CDM
6
Chlorfenapyr 8
72
Time (h)
24
48
72
Time (h)
5 Chlorfenapyr
4
16
Nitenpyram 3
c) Insecticides + AMZ
14
Diafenthiuron Diflubenzuron
2
Novaluron Lufenuron
1 0 24
48
Times fold increase in toxicity
Synergistic Ratio (SR 50)
48
Time (h)
Times fold increase in toxicity
Synergistic Ratio (SR 50)
24
b) Insecticides + AMZ
72
12 Chlorfenapyr
10
Nitenpyram 8
Diafenthiuron Diflubenzuron
6
Novaluron
4
Lufenuron
Time (h) 2
Fig. 1. Time-dependent changes in the synergistic ratio (SR50) as calculated from LC50 values from Tables 1–3 for combined treatments with (a) PBO, (b) AMZ, and (c) CDM as assessed 24, 48, and 72 h after their initial exposure.
0 24
48
72
Time (h) 20
d) Insecticides + CDM
18 16
Times fold increase in toxicity
compared to the other insecticides due to the role of CYP450s in activation of this insecticide. These results agreed with previous studies on Ae. aegypti [28,36]. The synergistic action contributed by the octopamine receptor agonists showed that AMZ is more effective than CDM and this trend was more conspicuous after 72 h exposure and the time-dependent effect was upturned SR for both, AMZ and CDM, on all selected insecticides except nitenpyram which showed a rapid decline of the SR indicating a similar synergistic mode of action for both OR agonists. In general, these findings support the notion that the basic mechanism of the synergistic effects of OR agonists is based on an increase of the insecticide’s penetration or uptake and/or inactivation of the detoxification enzymes on the selected insecticides [8]. The IGR insecticides tested in this study showed the lowest toxicity on the fourth instar larvae of Ae. aegypt compared to other insecticides despite their lethal action which suggests that these IGRs have another mechanism of action rather than chitin synthesis inhibition regardless of their acute toxicity which is in line with a previous study [28], especially after showing a significant increase in toxicity with PBO and OR agonists (predominately with AMZ, after 48 and 72 h exposure). In summary, we emphasized that PBO and OR agonists significantly synergized the lethal action of certain selected novel insecticides on fourth instar larvae of Ae. aegypti. Furthermore, the
14 Chlorfenapyr
12
Nitenpyram 10
Diafenthiuron
8
Diflubenzuron
6
Novaluron
4
Lufenuron
2 0 24
48
72
Time (h) Fig. 2. Times fold increase in toxicity of selected insecticides (a) alone and with (b) PBO, (c) AMZ, and (d) CDM on 4th larvae of Ae. aegypti after 24, 48, and 72 h exposure.
OR agonists might have a specific mode of action in synergizing certain insecticides and future field experiments must be performed to evaluate their potencies in combination with certain insecticides. Overall, the option of associating insecticides with different modes of action is a promising alternative strategy for resistance management in the future.
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6
Acknowledgments 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 thank Prof. Dr. Thomas W. Scott, Department of Entomology and Nematology, University of California Davis for kindly providing us with Ae. aegypti eggs that were used for this study. This work is dedicated to Prof. Dr. Fumio Matsumura.
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Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
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Pesticide Biochemistry and Physiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p e s t
Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito Mohamed Ahmed Ibrahim Ahmed a,b, Christoph Franz Adam Vogel b,c,* a
Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt Center for Health and the Environment, University of California Davis, Davis, CA 95616, USA c Environmental Toxicology Department, University of California Davis, Davis, CA 95616, USA b
A R T I C L E
I N F O
Article history: Received 9 October 2014 Accepted 19 January 2015 Available online Keywords: Aedes aegypti Octopamine receptor agonists Neonicotinoids IGRs Insecticide resistance
A B S T R A C T
Studying insecticide resistance in mosquitoes has attracted the attention of many scientists to elucidate the pathways of resistance development and to design novel strategies in order to prevent or minimize the spread and evolution of resistance. Here, we tested the synergistic action of piperonyl butoxide (PBO) and two octopamine receptor (OR) agonists, amitraz (AMZ) and chlordimeform (CDM) on selected novel insecticides to increase their lethal action on the fourth instar larvae of Aedes aegypti L. However, chlorfenapyr was the most toxic insecticide (LC50 = 193, 102, and 48 ng/ml, after 24, 48, and 72 h exposure, respectively) tested. Further, PBO synergized all insecticides and the most toxic combinatorial insecticide was nitenpyram even after 48 and 72 h exposure. In addition, OR agonists significantly synergized most of the selected insecticides especially after 48 and 72 h exposure. The results imply that the synergistic effects of amitraz are a promising approach in increasing the potency of certain insecticides in controlling the dengue vector Ae. aegypti mosquito. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The current insecticide-based strategy for vector control of Aedes aegypti L. relies heavily on the use of pyrethroids on bed nets, indoor spraying and larvicidal applications [1–3]. Such a heavy reliance on only one type of insecticide is likely to lead to future problems of resistance development that would create intractable predicaments in the control efforts of Ae. aegypti [4]. Knowing the history of many insecticide resistance problems, most researchers in this field agree that such a scenario is not farfetched in this case. However, it is also necessary to point out that insecticidal chemicals are the best proven approach among other methods of pest control that works well in controlling the dengue vector mosquitoes as shown by the earlier success of DDT [5]. Importantly, there is no question about the great need to continue searching for novel approaches
Abbreviations: PBO, piperonyl butoxide;; AMZ, amitraz; CDM, chlordimeform; IPM, integrated pest management; OR, octopamine receptor; cAMP, cyclic adenosine monophosphate; IGRs, insect growth regulators; IRR, Institutional Review Board; SR, synergistic ratio; LC50, lethal concentration, 50%; 95% CL, 95% confidence limit. * Corresponding author. Center for Health and the Environment, Environmental Toxicology Department, University of California Davis, Davis, CA 95616, USA. Fax: +1 530 752 5300. E-mail address: [email protected] (C.F.A. Vogel).
of mosquito control, such as development of vaccines, biological control measures, and introduction of molecular biological means of mosquito control [6]. Chemical insecticides are developed by pesticide manufacturing companies through mostly empirical screening and biological testing efforts. In more recent years, however, most pesticide companies rely on the known mode of action of each chemical class of insecticides, such as chemicals targeting the sodium channel, the nicotinic acetylcholine receptor, cholinesterase etc., because such knowledge-based approaches increase the chance of discovering the ‘blockbuster’ pesticide more quickly [7]. The major classes of pesticides used today can be distinguished by the biochemical target through which each group of pesticides attacks. Accumulation of knowledge in this regard has helped tremendously in selecting flexible approaches to control pests, such as rotating insecticides with a totally different mode of action to ameliorate the damages resulting from very stubborn cases of recalcitrant resistant pests in the past. Thus, a useful approach to preclude rapid development of resistance has been found to be avoiding excessive, uniform, and region-wide uses of high dose application of the main type of insecticide throughout all stages of the mosquito life cycle [8]. Unfortunately, the heavy reliance on pyrethroids in controlling vector mosquitoes as larvicides and adulticides is now reaching the stage where resistance problems are expected to grow [9–11]. The most effected type of strategy is an integrated pest management (IPM)
http://dx.doi.org/10.1016/j.pestbp.2015.01.014 0048-3575/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
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program in order to mitigate or reduce the chance of rapid development of resistance. In this regard, many different types of efforts have been made to minimize the use of the main insecticide of the control program including limiting the percentage used in terms of the spray areas, using mixtures of insecticides with different modes of action, using the right insecticide formulation and targeting only the critical time or area of vulnerability [12]. There are a number of examples of successful applications of mixtures of pesticides in resistance management [8,13]. At least there is one demonstration of the effectiveness of combinatorial effects (permethrin plus imidacloprid) reducing the number of mosquito bites when topically applied to dogs [14]. The effectiveness of formamidine, type octopamine receptor (OR) agonist, in synergizing the insecticidal actions of pyrethroids on houseflies (dipterous insects) was first reported by Liu and Plapp [15] with the most effective synergistic effect using amitraz. In view of the significant synergism between octopamine and cypermethrin, the molecular base of this synergism is likely activation of the OR signaling. The subject of the mechanism of action of this class of OR agonists, particularly that of formamidines, has been extensively reviewed [16–18]. Briefly in insects octopamine acts like epinephrine, and OR in this regard serves as the receptor mediating the sympathetic route, equivalent of the mammalian autonomic controlling system. As in the case of epinephrine, one of the major consequences of its signaling in insects is excitation coupled with loss of appetite. While there are qualitative differences in the actions between octopamine and epinephrine, the above two basic actions of OR signaling serves as two of the main modes of their pesticidal actions. While studies on the action of octopamine are rare in mosquitoes, in the brain of adult Culex pipiens mosquitoes, octopamine causes activation of cyclic adenosine monophosphate (cAMP), indicating this basic mechanism does indeed operate in this species. Among dipterous insects, Drosophila is the best studied species in which octopamine has been shown to induce cAMP. In some insects elevation of cAMP by octopamine directly causes the loss of appetite [19]. Neonicotinoids are a relatively new group of powerful insecticides that clearly shows a wider spectrum of insect controlling property especially mosquito control [20–22]. The molecular bases of their diverse action spectra are gradually becoming elucidated [23,24]. Their basic mode of action is to attack the nicotinic acetylcholine receptor (nAChR), which is totally different from the action sites of any of the major insecticides frequently used for the control of mosquitoes in the past. While the original types of neonicotinoids were relatively polar, suited mostly on sucking type insect pests, the spectra of effectiveness of neonicotinoids have been steadily expanding including chewing type of insect pests because of their increased hydrophobic properties [25]. Recently, new promising tools have been used in mosquito control. For instance, chlorfenapyr is a protoxin requiring activation by cytochrome P450, mediates its toxic effects by disrupting the production of ATP resulting in cellular death and ultimately mosquito mortality [26]. Another example are insect growth regulators (IGRs) which inhibit chitin synthesis during the development of mosquitoes [27,28]. The advantage of the IGRs is causing less detrimental effects to beneficial insects. For instance they do not affect an insect’s nervous system and are less toxic to beneficial insects, more compatible with pest management systems that use biological control components, and are less likely to become resistant to IGRs [29–32]. Herein, we studied the synergistic action of OR agonists, AMZ and CDM, plus PBO on the insecticidal activity of selected novel insecticides against fourth instar larvae of Ae. aegypti. However, the potential of this study is to shed light on new promising tools to control the dengue vector Ae. aegypti mosquitoes.
2. Materials and methods 2.1. Mosquitoes The FIELD strain (Fresno, CA) of Ae. aegypti was obtained from the laboratory of Dr. Thomas W. Scott, University of California Davis, and was used for all experiments. The rearing regime has been described earlier [8]. Briefly, the eggs were hatched by placing a piece of dried paper towel loaded with previously deposited eggs in a flask filled with 750 ml of distilled water that been held under vacuum for 45 min. The vacuum was released and 50 mg of larval diet (dog food powder) added to the water. The hatched larvae were held overnight in the same flask, and then 200 larvae were transferred to each 600-ml beaker containing 400 ml of distilled water. Larval diet was added to each beaker according to the following regime: day 1, 75 mg; day 3, 38 mg; day 4, 75 mg; day 5, 113 mg; and day 6, 150 mg. Adult mosquitoes were reared in an environmental chamber with a temperature ranging from 22 to 30 °C, 80% relative humidity (RH), and a photoperiod of 14:10 (L:D) h. Adults were held in a 60 × 60 × 60-cm screened cage and provided 10% sucrose ad libitum. Human blood was provided to adults twice a week. Eggs were collected on paper towels lining the rim of water containers. The papers with eggs were air dried at 27 °C and 80% humidity for 24 h and stored in containers with 100% humidity for 3–30 d. The UC Davis Institutional Review Board (IRR) determined that feeding laboratory-reared mosquitoes on people in this experiment did not meet the requirements for human subject research, and thus, did not require IRR approval. 2.2. Chemicals Piperonyl Butoxide (99%), chlordimeform (99.8%), amitraz (96.8%), nitenpyram (99.9%), chlorfenapyr (99.6%), lufenuron (99.7%), diafenthiuron (99.9%), diflubenzuron (98.1%), and novaluron (99.6%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.3. Larval bioassays Twenty of fourth instar larvae were placed in 140-ml glass cups containing 99 ml of distilled water, 1 ml of each insecticide (chlorfenapyr, nitenpyram, diafenthiuron, diflubenzuron, novaluron, and lufenuron) in acetone solution, and 1 ml of acetone as vehicle for controls. Larvae were considered dead if they were unresponsive to touching with a probe or if they could not reach the surface of the water. Because of the slow-acting nature of some of these insecticides, mortality was determined after 24, 48, and 72 h of exposure. 2.4. Synergistic action bioassay The synergistic action bioassay was conducted as described above. Controls received acetone only and were run concurrently with each series of tests. Each series of synergistic action tests was carried out by testing the lethal action of varying concentrations of a test insecticide alone or the test insecticide co-administered with 10 μg/ ml of PBO, AMZ, or CDM. After the addition of the insecticides, the test solution was stirred briefly to ensure uniform mixture. Preliminary tests showed that PBO at 10 μg/ml was the maximum sublethal concentration where no mortality was observed of fourth instar larvae. Previous study on AMZ and CDM, which was published by our lab, demonstrated that concentration of 10 μg/ml did not cause mortality during the 72-h test period on fourth instar larvae of Ae. aegypti [8]. At least five concentrations were used for each bioassay. Every bioassay was held at 25 °C. Percentage mortality was recorded after 24, 48, and 72 h.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
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1.7 4.8* 1.8 0.93 1.1 0.91 4.1 (0.8) 5.0 (0.2) 3.9 (0.7) 4.8 (0.7) 4.1 (0.1) 4.6 (0.9) 114 (42–307 59 (15–108) 236 (62–889) 2603 (983–4751) 4660 (2452–7925) 7131 (4328–9734) 1.9 8.6* 3.2* 2.6* 2.1* 1.8 5.4 (0.4) 4.8 (0.3) 5.3 (0.2) 4.2 (0.3) 3.9 (0.7) 4.4 (0.3) 97 (18–267) 33 (19–93) 130 (44–329) 921 (673–2875) 2440 (1731–5816) 3616 (1972–6180) a
b
n, no. of larvae tested including control. Concentrations are expressed in ng/ml and the response determined after 24 h. c Concentration of synergists was 10 μg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergists. e Larvae exposed to insecticides and synergists simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05).
0.91 4.6* 3.7* 3.4* 2.9* 2.3* 5.1 (0.1) 5.4 (0.3) 4.8 (0.1) 3.9 (0.6) 4.1 (0.2) 4.9 (0.5) 213 (73–559) 62 (19–142) 114 (32–311) 713 (361–1045) 1767 (1242–3595) 2821 (1430–5082) 360 360 360 360 360 360 3.1 (0.2) 3.6 (0.2) 3.2 (0.2) 3.3 (0.1) 3.8 (0.1) 4.0 (0.1)
Slope (SE) LC50 (95% Slope (SE) LC50 (95%
193(62–1461) 284 (105–1490) 421 (136–597) 2411 (542–4753) 5137 (1315–7971) 6491 (1734–7419) Chlorfenapyr Nitenpyram Diafenthiuron Diflubenzuron Novaluron Lufenuron
Slope (SE) LC50 (95%
CL)c
Insecticides + AMZe (ng/ml)
SRd CL)c
Insecticides + PBOe (ng/ml)
na CL)b
Insecticides (ng/ml)
Insecticide resistance of mosquito populations is increasing at a dramatic rate, threatening the efficacy of control programs throughout insecticide-treated areas. Importantly, the combinations of insecticides with different modes of action could make a potential contribution in mosquito control especially where mosquitoes already show high levels of resistance to conventional insecticides [34,35]. The effect of PBO on the toxicity of the selected insecticides was different. However, PBO showed a significantly different degree of synergism with all selected insecticides after 24, 48, and 72 h exposure. For instance, chlorfenapyr showed only moderate synergism
Insecticides
4. Discussion
Table 1 Toxicity of selected insecticides and synergizing action of PBO, AMZ, and CDM on each insecticide on fourth instar larvae of Ae. aegypti after 24-h exposure.
The results of 24, 48, and 72 h mortality tests on selected insecticides with or without PBO, AMZ, or CDM and their levels of synergism are presented in Tables 1–3. Chlorfenapyr was the most toxic insecticide (LC50 = 193, 102, and 48 ng/ml, after 24, 48, and 72 h exposure, respectively), followed by nitenpyram (LC50 = 284, 205, and 131 ng/ml, after 24, 48, and 72 h exposure, respectively). However, the IGR insecticides (diafenthiuron, diflubenzuron, novaluron, and lufenuron) were the least toxic insecticides. PBO significantly synergized all insecticides except chlorfenapyr and the most toxic insecticide was nitenpyram even after 48 and 72 h exposure (LC50 values, 62, 28, and 16 ng/ml, respectively). AMZ significantly synergized most of selected insecticides except chlorfenapyr and lufenuron after 24 h exposure, meanwhile, after 48 and 72 h exposure, AMZ synergized significantly all insecticides and the most toxic insecticides after 48 h were nitenpyram (LC50 = 32 ng/ml) and after 72 h diafenthiuron (LC50 = 23 ng/ml). CDM was only synergized by nitenpyram after 24 h exposure whereas after 48 h exposure, 4 insecticides were synergized, chlorfenapyr, nitenpyram, diafenthiuron, and novaluron; however, after 72 h exposure, all insecticides were synergized. Fig. 1 shows the time-dependent changes in the SR of LC50 as affected by PBO (Fig. 1a), AMZ (Fig. 1b), and CDM (Fig. 1c). The most noticeable tendency was the time-dependent rises in SR on all selected insecticides (Fig. 1a). However, nitenpyram showed the most clear-cut time-dependent decrease in SR with both AMZ and CDM (Fig. 1b and c). In general, synergism was more pronounced when insecticides were co-treated with AMZ rather than with PBO or CDM. The toxicity of all insecticides on fourth instar larvae was increased after 48 and 72 h (Fig. 2a). For example, novaluron increased the toxicity from 2.51-fold after 48 h to 8.50-fold after 72 h compared to the toxicity after 24 h, however, in the combination with PBO (Fig. 2b), novaluron increased toxicity from 2.86-fold after 48 h to 12.44-fold after 72 h. This trend continued in fourth instar larvae which increased in toxicity after 48 and 72 h (Fig. 2c and d) after treatment with the selected insecticides in combination with AMZ and CDM. Broadly, a stronger increase in toxicity was found with IGR insecticides in combination with AMZ and CDM.
SRd
3. Results
LC50 (95% CL)c
Insecticides + CDMe (ng/ml)
Data from all tests were corrected for control mortality with Abbott’s formula [33]. Bioassay data were pooled and analyzed (LC50 and 95% CL values) by using IBM SPSS statistics 22 program (SPSS Inc., Chicago, IL). A synergistic action was determined to be significant (P ≤ 0.05) when the 95% CIs for the LC50 values for larvae exposed to insecticide alone did not overlap with those for larvae exposed to insecticide + octopamine receptor agonist mixtures. Synergistic ratio (SR) was calculated by dividing the LC50 value of the test insecticide by that of the LC50 obtained by the combined treatment with insecticide + octopamine receptor agonists.
Slope (SE)
SRd
2.5. Statistical analysis
3
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
4
Insecticides
LC50 (95% Chlorfenapyr Nitenpyram Diafenthiuron Diflubenzuron Novaluron Lufenuron
Insecticides + PBOe (ng/ml)
Insecticides (ng/ml) CL)b
102 (36–291) 205 (79–611) 253 (95–681) 962 (174–2560) 2043 (569–3138) 4105 (1060–7204)
Slope (SE)
na
CL)c
LC50 (95%
3.8 (0.2) 3.6 (0.2) 3.1 (0.2) 3.0 (0.1) 3.9 (0.1) 3.8 (0.1)
360 360 360 360 360 360
88 (26–282) 28 (5–76) 58 (26–185) 246 (92–723) 618 (253–751) 1577 (863–4602)
Insecticides + AMZe (ng/ml)
Slope (SE)
SRd
CL)c
LC50 (95%
3.6 (0.7) 4.8 (0.3) 5.5 (0.8) 4.9 (0.2) 3.8 (0.1) 4.2 (0.4)
1.2 7.3* 4.4* 3.9* 3.3* 2.6*
28 (7–68) 32 (7–82) 56 (14–147) 282 (103–441) 703 (259–2064) 1519 (692–3068)
Insecticides + CDMe (ng/ml)
Slope (SE)
SRd
LC50 (95% CL)c
Slope (SE)
SRd
5.1 (0.8) 4.2 (0.2) 4.9 (0.6) 3.4 (0.7) 4.1 (0.5) 3.8 (0.3)
3.6* 6.4* 4.5* 3.4* 2.9* 2.7*
43 (25–186) 53 (11–130) 121 (37–339) 505 (214–967) 887 (142–2115) 2927 (1089–6485)
4.1 (0.2) 4.8 (0.6) 4.9 (0.2) 4.3 (0.3) 3.6 (0.7) 4.7 (0.1)
2.4 3.9* 2.1* 1.9 2.3* 1.4
a
Table 3 Toxicity of selected insecticides and synergizing action of PBO, AMZ, and CDM on each insecticide on fourth instar larvae of A. aegypti after 72-h exposure. Insecticides
LC50 (95% Chlorfenapyr Nitenpyram Diafenthiuron Diflubenzuron Novaluron Lufenuron a
Insecticides + PBOe (ng/ml)
Insecticides (ng/ml) CL)b
48 (15–96) 131 (47–460) 170 (51–314) 419 (89–740) 604 (161–833) 1847 (462–2729)
Slope (SE)
na
c
LC50 (95% CL)
4.1 (0.2) 3.6 (0.2) 3.1 (0.2) 3.3 (0.1) 3.8 (0.1) 3.6 (0.1)
360 360 360 360 360 360
36 (19–84) 16 (7–41) 33 (13–114) 86 (42–273 142 (94–358) 497 (216–901)
Slope (SE)
LC50 (95%
5.4 (0.8) 4.7 (0.1) 5.2 (0.3) 4.3 (0.6) 3.9 (0.8) 4.2 (0.4)
1.3 8.2* 5.2* 4.9* 4.3* 3.7*
7 (3–20) 27 (32–614) 23 (11–63) 93 (52–321) 194 (74–409) 634 (392–1023)
n, no. of larvae tested including control. Concentrations are expressed in ng/ml and the response determined after 72 h. c Concentration of synergists was 10 μg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergists. e Larvae exposed to insecticides and synergists simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). b
Insecticides + AMZe (ng/ml) SRd
CL)c
Insecticides + CDMe (ng/ml)
Slope (SE)
SRd
LC50 (95% CL)c
Slope (SE)
SRd
4.5 (0.6) 4.2 (0.7) 4.9 (0.4) 3.7 (0.2) 4.1 (0.3) 3.6 (0.8)
6.9* 4.9* 7.4* 4.5* 3.1* 2.9*
13 (2–31) 47 (19–132) 49 (23–341) 161 (78–476) 252 (86–427) 880 (361–2135)
4.6 (0.2) 4.7 (0.6) 4.8 (0.2) 4.1 (0.4) 4.9 (0.1) 4.2 (0.3)
3.7* 2.8* 3.5* 2.6* 2.4* 2.1*
ARTICLE IN PRESS
n, no. of larvae tested including control. Concentrations are expressed in ng/ml and the response determined after 48 h. c Concentration of synergists was 10 μg/ml. d SR, synergistic ratio. Calculated by dividing the LC for insecticide by the LC of insecticide + synergists. e Larvae exposed to insecticides and synergists simultaneously. * SR significantly different from control without synergist (=1.0) at (P ≤ 0.05). b
M.A.I. Ahmed, C.F.A. Vogel/Pesticide Biochemistry and Physiology ■■ (2015) ■■–■■
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
Table 2 Toxicity of selected insecticides and synergizing action of PBO, AMZ, and CDM on each insecticide on fourth instar larvae of A. aegypti after 48-h exposure.
ARTICLE IN PRESS M.A.I. Ahmed, C.F.A. Vogel/Pesticide Biochemistry and Physiology ■■ (2015) ■■–■■
Synergistic Ratio (SR 50)
10
a) Insecticides + PBO
9
5
a) Insecticides alone
8 7
Chlorfenapyr
6
Nitenpyram
5
Diafenthiuron
4
Diflubenzuron
3
Novaluron
2
Lufenuron
Times fold increase in toxicity
8 Chlorfenapyr 6
Nitenpyram Diafenthiuron Diflubenzuron
4
Novaluron
1
Lufenuron
2
0 24
48
72
0
Time (h) 10
72
14
9 8
Chlorfenapyr
7
b) Insecticides + PBO
12
Nitenpyram
6 5
Diafenthiuron
4
Diflubenzuron
3
Novaluron
2
Lufenuron
1 0 24
48
10 Nitenpyram Diafenthiuron 6
Diflubenzuron Novaluron
4
Lufenuron 2 0
c) Insecticides + CDM
6
Chlorfenapyr 8
72
Time (h)
24
48
72
Time (h)
5 Chlorfenapyr
4
16
Nitenpyram 3
c) Insecticides + AMZ
14
Diafenthiuron Diflubenzuron
2
Novaluron Lufenuron
1 0 24
48
Times fold increase in toxicity
Synergistic Ratio (SR 50)
48
Time (h)
Times fold increase in toxicity
Synergistic Ratio (SR 50)
24
b) Insecticides + AMZ
72
12 Chlorfenapyr
10
Nitenpyram 8
Diafenthiuron Diflubenzuron
6
Novaluron
4
Lufenuron
Time (h) 2
Fig. 1. Time-dependent changes in the synergistic ratio (SR50) as calculated from LC50 values from Tables 1–3 for combined treatments with (a) PBO, (b) AMZ, and (c) CDM as assessed 24, 48, and 72 h after their initial exposure.
0 24
48
72
Time (h) 20
d) Insecticides + CDM
18 16
Times fold increase in toxicity
compared to the other insecticides due to the role of CYP450s in activation of this insecticide. These results agreed with previous studies on Ae. aegypti [28,36]. The synergistic action contributed by the octopamine receptor agonists showed that AMZ is more effective than CDM and this trend was more conspicuous after 72 h exposure and the time-dependent effect was upturned SR for both, AMZ and CDM, on all selected insecticides except nitenpyram which showed a rapid decline of the SR indicating a similar synergistic mode of action for both OR agonists. In general, these findings support the notion that the basic mechanism of the synergistic effects of OR agonists is based on an increase of the insecticide’s penetration or uptake and/or inactivation of the detoxification enzymes on the selected insecticides [8]. The IGR insecticides tested in this study showed the lowest toxicity on the fourth instar larvae of Ae. aegypt compared to other insecticides despite their lethal action which suggests that these IGRs have another mechanism of action rather than chitin synthesis inhibition regardless of their acute toxicity which is in line with a previous study [28], especially after showing a significant increase in toxicity with PBO and OR agonists (predominately with AMZ, after 48 and 72 h exposure). In summary, we emphasized that PBO and OR agonists significantly synergized the lethal action of certain selected novel insecticides on fourth instar larvae of Ae. aegypti. Furthermore, the
14 Chlorfenapyr
12
Nitenpyram 10
Diafenthiuron
8
Diflubenzuron
6
Novaluron
4
Lufenuron
2 0 24
48
72
Time (h) Fig. 2. Times fold increase in toxicity of selected insecticides (a) alone and with (b) PBO, (c) AMZ, and (d) CDM on 4th larvae of Ae. aegypti after 24, 48, and 72 h exposure.
OR agonists might have a specific mode of action in synergizing certain insecticides and future field experiments must be performed to evaluate their potencies in combination with certain insecticides. Overall, the option of associating insecticides with different modes of action is a promising alternative strategy for resistance management in the future.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014
ARTICLE IN PRESS M.A.I. Ahmed, C.F.A. Vogel/Pesticide Biochemistry and Physiology ■■ (2015) ■■–■■
6
Acknowledgments 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 thank Prof. Dr. Thomas W. Scott, Department of Entomology and Nematology, University of California Davis for kindly providing us with Ae. aegypti eggs that were used for this study. This work is dedicated to Prof. Dr. Fumio Matsumura.
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Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph Franz Adam Vogel, Synergistic action of octopamine receptor agonists on the activity of selected novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae) mosquito, Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.014