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Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae)
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
Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae) Mohamed Ahmed Ibrahim Ahmed a,b, Christoph F.A. Vogel b,c,*, Fumio Matsumura b,c a
Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt Center for Health and the Environment, University of California Davis, CA 95616, USA c Department of Environmental Toxicology, University of California Davis, CA 95616, USA b
A R T I C L E
I N F O
Article history: Received 24 October 2014 Accepted 12 January 2015 Available online Keywords: Aedes aegypti qRT-PCR Formamidines Trehalose Octopamine receptor
A B S T R A C T
We recently reported that formamidine pesticides such as amitraz and chlordimeform effectively synergize toxic actions of certain pyrethroid and neonicotinoid insecticides in some insect species on the 4th instar larvae of Aedes aegypti. Here we studied the biochemical basis of the synergistic actions of the formamidines in amplifying the toxicity of neonicotinoids and pyrethroids such as dinotefuran and thiamethoxam, as well as deltamethrin-fenvalerate type of pyrethroids. We tested the hypothesis that their synergistic actions are mediated by the octopamine receptor, and that the major consequence of octopamine receptor activation is induction of trehalase to increase glucose levels in the hemolymph. The results show that formamidines cause a significant up-regulation of the octopamine receptor and trehalase mRNA expressions. Furthermore, formamidines significantly elevate levels of free glucose when co-treated with dinotefuran, deltamethrin and fenvalerate, but not with permethrin or fenitrothion, which showed no synergistic toxic effects with formamidines. These results support the conclusion that the main mode of synergism is based on the ability to activate the octopamine receptor, which is particularly effective with insecticides causing hyperexcitation-induced glucose release and consequently leading to quick energy exhaustion. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Formamidines belong to a unique group of pesticides that have multiple effects on biological activities of arthropod species [1]. As for their mode of actions, formamidines are known to interact with several types of aminergic receptors. Among those, the agonistic effects of formamidines on the octopamine receptor are now considered to be the most important feature of their insecticidal actions
Abbreviations: AMZ, amitraz; CDM, chlordimeform; DCDM, desmethylchlordimeform; OR, Octopamine Receptor; cAMP, Cyclic Adenosine Monophosphate; DEET, N, N-diethyl-3-methylbenzamide; qRT-PCR, Quantitative Reverse-Transcriptase Polymerase Chain Reaction; HK, hexokinase; G-6-PDH, glucose6-phosphate dehydrogenase; UV–VIS Spectrophotometer, Ultraviolet–visible Spectrophotometer; DNF, dinotefuran; Delta, deltamethrin; Perm, permethrin; Imida, imidacloprid; thiam, thiamethoxam; Fenit, fenitrothion; IRR, Institutional Review Board. * Corresponding author. Department of Environmental Toxicology and Center for Health and the Environment, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA. Fax: +1 530 752 5300. E-mail address: [email protected] (C.F.A. Vogel).
[2]. In agreement with their agonistic action of the aminergic receptors, the formamidines affect monoamine-mediated production of cyclic adenosine monophosphate (cAMP) and induce behavioral changes that are similar to adrenergic responses in treated insects such as the loss of appetite and hyperexcitation [3]. For instance, chlordimeform (CDM) causes a decrease in feeding of several species of lepidopterous and hemipterous insects, which suggests that the resulting mortality observed may be the consequence of starvation rather than a direct toxic action of the insecticide. Beeman and Matsumura [4] previously reported that CDM causes intense anorexia in the American cockroach at doses as low as 1–5 μg/ insect. In agreement with the above conclusion, Lund et al. [5] found that decreasing plant consumption by tobacco hornworm larvae is afforded by a nonlethal mechanism that was to be the result of motor stimulation through its actions on central non-cholinergic systems. In contrast, Beeman and Matsumura [4] found that the phenomenon of anorexia in the American cockroach is accompanied by an increase in hemolymph glucose levels, but not trehalose levels. Indeed in that study it was shown that the combined glucose and trehalose level remained relatively constant. An additional study showed that trehalase activity in the thoracic muscles has
http://dx.doi.org/10.1016/j.pestbp.2015.01.008 0048-3575/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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2
increased as a result of in vivo administration of CDM [6]. This was confirmed by in vitro incubation of isolated thoracic muscles with desmethylchlordimeform (DCDM). Furthermore, the same phenomenon could be reproduced by administering exogenous octopamine in vitro and in vivo. These results provide ample evidence that one of the primary actions of this group of formamidines is to functionally activate the octopamine receptor (OR) in muscles and other non-neural tissues such as fat bodies. Despite those extensive studies, there is no consensus emerging regarding the possible biochemical causes. For instance, there are some suggestion that increasing the rate of uptake of insecticides has been proposed to be a mechanism of CDM synergism on pyrethroids, but the same effect was not produced by amitraz (AMZ) and its metabolite in the same study [7,8]. Furthermore, no report on the action mechanisms of formamidine insecticides on mosquito insects has been found so far, although there have been a number of publications on the existence of the OR in dipterous insects, including in Drosophila melanogaster. In the current study, we have investigated the molecular and biochemical mechanisms of action of AMZ, along with CDM as a positive control, by testing our hypothesis that the molecular basis of synergistic action of AMZ is its agonistic action on the OR in mosquitoes, which results in the up-regulation of the enzymatic activity of trehalase in larvae of Aedes aegypti. 2. Materials and methods 2.1. Ae. aegypti The ROCK (insecticide-susceptible) strain of Ae. aegypti was obtained from Dr. Thomas W. Scott, the University of California Davis and was used for all experiments. The basic rearing regimen was according to the description by Paul et al. [9] with some modifications. 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 was once held under vacuum for 45 min. The vacuum was then released, and 50 mg of larval diet (dog food powder) was added to the water. The hatched larvae were held overnight in the same flask, and then 200 larvae were transferred to each 600 ml liter 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 that emerged were reared in an environmental chamber with a temperature ranging from 22 to 30 °C and 80% RH. The photoperiod, adopted throughout this study, was 14:10 (L:D) h. All adults were held in a 60 by 60 by 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 days. The UCDavis 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. D. melanogaster D. melanogaster (wild type strain Canton S) were raised on standard medium at 25 °C at a density of 20 larvae/7 ml of medium, and adults were synchronized at eclosion. 2.3. Chemicals Octopamine hydrochloride (95%), chlordimeform (99.5%), amitraz (99.4%), fenitrothion (98.6%), permethrin (47.6% cis- 50.4% trans-),
Table 1 Nucleotide mRNA primer sequences used in qRT-PCR approaches for studying the possible target genes of formamidine pesticides on Ae. aegypti larvae. Gene
Sequences
α-ACTIN
FP: 5′-CAACCATGTACCCAGGAATC-3′ RP: 5′-CACCGATCCAGACGGAGTAT-3′ FP: 5′-CAGTGTATAGCGCACGAGGA-3′ RP: 5′-AACACACAATATCCGCACGA-3′ FP: 5′-AAATTTCCTGGCGTACAACG-3′ RP: 5′-ACCAGTGCACCTCCGTTATC-3′
OCTOPAMINE RECEPTOR TREHALASE
deltamethrin (99%), dinotefuran (99.5%), imidacloprid (99.5%), thiamethoxam (99.5%), and DEET (N, N-diethyl-3-methylbenzamide) (98%) were obtained from Chem. Service (West Chester, PA). Hexokinase and glucose-6-phosphate dehydrogenase from baker’s yeast (Saccharomyces cerevisiae) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.4. Drosophila feeding test The Drosophila feeding experiment consisted of 3 newly emerged females and 3 males placed into a vial in which the medium that contained 1 μg/ml of CDM was already set to occupy the lower half of each vial prior to the addition of Drosophila adults. Altogether there were 5 vials for Canton S strain which served as replicates in addition to one control vial containing the Drosophila nutritional media without CDM. 2.5. Long-term Ae. aegypti larval developing test Fourth instar larvae of Ae. aegypti in water were treated with different concentrations (6.25, 12.5, 25, 50 μg/ml) of CDM and the appearance of pupae as well as emergence of adults were monitored for 7 days. Number of mosquitoes at each stage of development was recorded on each day at the same time (7:00 PM) of the day. 2.6. Quantitative real-time reverse transcriptase PCR (qRT-PCR) The oligonucleotide sequences of primers used for detecting all of the marker mRNA expressions are shown in Table 1. Total RNA was isolated from twenty 4th instar larvae treated in vivo by each pesticide (the concentrations that have been used in this study were based on the LC50 values after 48 h exposure from a previous study which was published by our lab). In the same previous study, it was demonstrated that AMZ and CDM at a concentration of 10 μg/ml did not cause mortality during the 72-h test period on fourth instar larvae of Ae. aegypti [10] using a high pure RNA isolation kit (Qiagen), and cDNA synthesis was carried out as described by Vogel et al. [11] with some modifications to fit the mosquito model. Quantitative detection of target gene mRNAs was performed with a LightCycler Instrument (Roche Diagnostics, Mannheim, Germany) using the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and as described by Sciullo et al. [12]. Briefly, DNA-free total RNA (1.0 μg) was reverse-transcribed using 4 U Omniscript reverse transcriptase (Qiagen) and 1 μg oligo (dT)15 in a final volume of 40 μl. The primers for each gene were designed on the basis of the respective cDNA or mRNA sequences using OLIGO primer analysis software, provided by Steve Rozen and Whitehead Institute/MIT Center for Genome Research [13]. PCR amplification was carried out in a total volume of 20 μl, containing 2 μl of cDNA, 10 μl of 2 X QuantiTect SYBR Green PCR Master Mix, and 0.2 M of each primer. The PCR cycling conditions were 95 °C for 15 min followed by 30–40 cycles of 94 °C for 15 s, 60 °C for 20 s, and 72 °C for 10 s. Detection of the fluorescent product was performed at the end of the 72 °C extension period. Negative controls
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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(A)
% of larvae
were run concomitantly to confirm that the samples were not crosscontaminated. A sample with DNase- and RNase-free water instead of RNA was concomitantly examined for each of the reaction units described earlier. To confirm the amplification specificity, the PCR products were subjected to melting curve analysis. All PCR assays were performed in duplicate or triplicate. The intra-assay variability was <7%. For quantification, data were analyzed with the LightCycler analysis software according to the manufacturer’s in structions. The variables were examined by student t-test.
control Larvae 6.25 ppm Larvae 12.5 ppm Larvae 25 ppm Larvae 50 ppm Larvae 1
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2.8. Statistical analysis All experiments were repeated a minimum of three times, and the results were expressed as mean ± standard deviation.
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Fig. 2. Long-term effects of chlordimeform (CDM) treatments (given to water in which larvae were reared) on development of Ae. aegypti (A) larvae into (B) pupa and (C) adults as expressed in percentages of Ae. aegypti at each day of development.
Statistical differences between controls (treated with vehicle only) and pesticides treated samples were determined by student t-test for the analysis of the significance difference between each pair of tests. The statistical significance of the synergistic effect of formamidines against the test insecticide (e.g. deltamethrin) was assessed by using the ANOVA analysis, each case comparing the difference between the effect of the test insecticide alone and that of the insecticide + AMZ (or CDM). 3. Results
0
250 200
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Twenty 4th-instar larvae of Ae. aegypti were placed in glass cups with three replicates tested per concentration (n = 60). Each cup contained 100 ml of water and varying concentrations of each insecticide in acetone. Controls containing only acetone were run concurrently with each test. Fifteen larvae were homogenized in 500 μl saline solution, and then centrifuged in 15,000 g for 5 min. An aliquot of 60 μl of 1 N HCL was added to the 60 μl sample from the supernatant from the above centrifugal preparation, which was treated immediately with 60 μl of 1 N NaOH to neutralize the effect of HC1 (totally 180 μl). To the above preparation, an aliquot of 25 μl of the test reagent mixture [ containing 1M Tris–HCl (pH 7.8), 100 mM Mg acetate, 100 mM NADP, 100 mM ATP, and per test, 2 U each of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH)] was added. The final volume of the reaction was made to 205 μl. The reaction was allowed to proceed at room temperature in square glass cuvette having a 1-cm light path. The total conversion of NADP to NADPH and the change in absorption at A340 were measured 6 min after starting the reaction using UV–VIS Spectrophotometer.
175
a) Larvae
120% 100% 80% 60% 40% 20% 0%
2.7. Determination of free glucose levels
200
3
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3
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5
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9 10 11 12 13 14
Days
Fig. 1. Effect of chlordimeform (CDM) administered with the feeding medium on the development of larvae of wild type D. melanogaster into (A) Pupae and (B) adults. *Statistically different from respective control with P ≤ 0.05.
Since very few studies have been conducted previously on the actions of formamidines on dipterous insects, it was necessary to conduct certain preliminary studies to ascertain that formamidines indeed cause similar, if not identical symptoms, like those observed in other orders of insects. For this purpose, we focused our attention to the developmental changes that are indicative of the effects of formamidines in reducing food intake. For this reason, we chose larval development of Drosophila, since they could be reared on nutritional media similar to that used in assessing the effect of CDM on larvae of Manduca sexta [5,6]. Fig. 1 shows the effects of CDM on D. melanogaster larval development. The speed of development of wild type strain of larvae raised in nutritional media in vials which had been treated with CDM (1 μg/ml) for 17 days was compared to their respective control group, which received only the same volume of the solvent. It is clear that CDM significantly delayed the development of larvae to pupae and adults. The pupal stage started only after day 8 and adults from 13 in CDM-treated group,
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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O.R. mRNA exp., (X control)
(A) 5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00
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(A) O.R. mRNA exp., (x control)
instead of day 5 and day 9, respectively for the control insects. Following these series of tests, there were fewer adults in the treated group. Such an observation suggests that CDM deterred the Drosophila larval development. Our hypothesis on the cause for the developmental delay is the reduction of food consumption by the affected Drosophila larvae due to suppression of their appetite as in the case of CDM treated M. sexta [5]. The effects of CDM, at several concentrations in water, on the larval development of Ae. aegypti were then studied. The results shown in Fig. 2 indicated that CDM caused a delay also in the development of Ae. aegypti, larvae into pupae and then adults, although this trend was not as clear as in the case of Drosophila. Nevertheless the most apparent inhibitory effect of CDM on the development of Ae. aegypti larvae was observed in the timing of pupation ( Fig. 2A). In the control group all larvae completed the process of pupation by day 3, whereas in CDM treated groups, none of them, even those that received the lowest concentration, completed pupation. In the next series of tests, the effects of octopamine agonists on OR mRNA expression, trehalase
O.R. mRNA exp., (X control)
4
12 hrs
24 hrs
Time Fig. 3. Comparative potencies of 3 proposed octopamine receptor agonists: timedependent effect of in vivo treatment of amitraz (AMZ) (10 μg/ml), octopamine (10 μg/ ml), and chlordimeform (CDM) (10 μg/ml) on (A) octopamine receptor mRNA expression, (B) trehalase mRNA expression, and (C) glucose levels in 4th instar of Ae. aegypti. Control containing only acetone. Control value at each time point was set as 1. Control value of glucose concentration is 0.002 μg/μl. *Statistically different from corresponding control with P ≤ 0.05 at each time point.
Fig. 4. Effectiveness of amitraz (AMZ) and chlordimeform (CDM) in synergizing action of (A) deltamethrin and (B) dinotefuran: time-dependent effect of in vivo treatment of deltamethrin (Delta) (0.29 ng/ml) and dinotefuran (DNF) (131 ng/ml) in combination with AMZ (10 μg/ml) and CDM (10 μg/ml) on octopamine receptor (O.R.) mRNA expression level in 4th instar of Ae. aegypti. Control treated with the vehicle acetone only. Control value at each time point was set as 1. *Statistically different from deltamethrin or dinotefuran with P ≤ 0.05 at each time point. #Action of the test insecticide (deltamethrin or dinotefuran in this case) alone statistically different from corresponding control with P ≤ 0.05 at each time point.
mRNA expression, and glucose levels were studied. As shown in Fig. 3A–C, OR expressions increased significantly after treating the larvae (4th instar) with 10 μg/ml of AMZ, octopamine, and CDM, the maximum level of stimulation induced by all 3 compounds being achieved after 30 min (4.40-, 3.91-, and 3.10-fold compared to the control, respectively). When the trehalase mRNA expression was used as the criterion on the action of those 3 OR agonists, the maximum significant level of its up-regulation by AMZ, octopamine, and CDM was not observed at 3 hrs unlike the case of OR mRNA expression; rather they were significantly stimulated at later time points as compared to control assessed at each time point. However, the time course of the changes in free glucose concentration was induced by 3 OR agonists, AMZ, CDM, and octopamine. The results indicate that all 3 OR agonists significantly increased the free glucose levels at 3, 6, 12, and 24 hrs post treatment, when compared to the control. By using the same approach, synergistic actions of formamidines on select list of insecticides were examined one by one. It was found that OR expression was stimulated significantly by deltamethrin alone (Fig. 4A), maximum expression being reached by 30 min. The expression level stimulated by deltamethrin alone at 30 min was 4.24-fold above control, whereas the combination of AMZ and CDM induced OR expression by 6.98- and 5.43-fold, respectively. A similar synergy test was conducted on dinotefuran (Fig. 4B). It was found that in the case of dinotefuran, both AMZ and CDM synergized upregulation of the OR mRNA expression, the highest significant activation being recorded at 30 min and the value for dinotefuran alone was 2.66-fold, and in combination with AMZ and CDM were 3.62- and 3.10-fold, respectively. Statistically significant synergistic effects were observed with dinotefuran alone at 3 hrs as well.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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Trehalase mRNA exp., (X control)
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(Fig. 6E). In contrast to the effects of the neonicotinoids, treatment with fenitrothion alone or in combination with AMZ and CDM did not affect free glucose levels (Fig. 6F). The effect of AMZ (Fig. 7A) at varying concentrations on total glucose level after 12 hrs is shown along with a parallel test with CDM after 6 hrs of treatment (Fig. 7B). The results obtained from Ae. aegypti larvae showed that the maximum level of stimulation by both formamidine was reached at the concentration range between 10 and 50 μg/ml. The maximum level of free glucose achieved by AMZ was higher compared to CDM.
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4. Discussion
Time
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Time Fig. 5. Effectiveness of amitraz (AMZ) and chlordimeform (CDM) in synergizing the action of (A) deltamethrin and (B) dinotefuran: time-dependent effect of in vivo treatment of deltamethrin (0.29 ng/ml) and dinotefuran (131 ng/ml) and in combination with AMZ and CDM on trehalase mRNA expression level in 4th instar of Ae. aegypti. Statistical symbol and other conditions are as shown in Fig. 4.
Interestingly, when the same test was applied with deltamethrin alone on trehalase mRNA expression (Fig. 5A), it was found that this insecticide caused a steady increase in trehalase mRNA expression over the test period. A significant increase of trehalase mRNA by deltamethrin compared to the matched controls was found after 1 hr peaking at 6 hrs after treatment. When AMZ and CDM were co-treated with deltamethrin, it was found that both induced significant synergistic actions at 1 hr and 3 hrs. In the case of dinotefuran tested alone (Fig. 5B), it caused significant stimulation as in the case of deltamethrin. The highest increase of trehalase expression was observed after 6 hrs of treatment. A significant synergistic action of dinotefuran co-treatment with AMZ and CDM on trehalase mRNA expression was found after 1 hr. Treatment with deltamethrin alone caused a significant increase of free glucose levels at 3 hrs compared to the corresponding control (Fig. 6A). Furthermore, the action of deltamethrin on free glucose levels was significantly synergized by formamidines. Particularly noticeable was the synergistic effect of AMZ, which was significant at all points in time tested peaking at 3 hrs (12-fold) and declining to 4-fold after 24 hrs of treatment. In contrast, treatment with permethrin did not lead to significant changes on free glucose levels even in the presence of either formamidine (Fig. 6B). The effects of 3 neonicotinoid insecticides, dinotefuran (Fig. 6C), imidacloprid (Fig. 6D), and thiamethoxam (Fig. 6E) on the expression of free glucose levels were examined next. Dinotefuran, when tested alone, showed modest stimulatory effects on glucose levels at 12 hrs (Fig. 6C); however, co-treatment with AMZ and CDM synergized the action of dinotefuran significantly. Treatment with imidacloprid caused a maximum elevated level of free glucose at 12 hrs (Fig. 6D). The synergistic actions of both formamidines AMZ and CDM were significant after 6 hrs and 12 hrs of treatment. A similar trend was observed after treatment with thiamethoxam in co-treatment with AMZ
Formamidines have attracted the interest of many research groups for their biological activity and pharmacological potential [14]. To date, however, few studies have been conducted in dipterous insects, and, furthermore, practically no data exist on the possible biochemical and molecular mechanism of formamidines on Ae. aegypti. In our study, formamidines showed significant reduction of larval development of Ae. aegypti and Drosophila. In agreement with our observation of Drosophila larvae, Gruntenko et al. [15] showed that an increased level of octopamine was related to a decreased number of vitellogenic (stages 8–10) and mature (stage 14) oocytes in Drosophila virilis as well as a decreased fecundity of D. melanogaster and D. virilis. Our observation that exposure to formamidine suppresses insect feeding is supported by a large body of evidence. For instance, Lund et al. [5] found that CDM causes significant anorexia in M. sexta larvae through its action on the central nervous system, which was supported by the work of Hitoshi and Fukami [16] on other lepidopteran species. In the case of the American cockroach, Periplaneta americana, it is clear that increased hemolymph glucose levels are involved, since the neurotoxic symptoms in this species develop only at CDM doses approximately 1000-fold higher. Ismail and Matsumura [6] observed an increase in the level of both glucose and trehalose in the hemolymph of American cockroaches, following injection of CDM or octopamine itself. The increase in free glucose levels could be explained by the ability of these agents to increase trehalase activity. Ismail and Matsumura [6] also demonstrated that octopamine causes elevation of trehalase in vivo and in vitro in several tissues of the American cockroach including its thoracic muscles. To elucidate the biochemical and molecular mechanism of the action of formamidines on Ae. aegypti larvae, we analyzed the mRNA expression of the OR and trehalase, as well as levels of the free glucose released. Both AMZ and CDM are capable of synergizing the action of a number of pyrethroids and neonicotinoids which is associated with their lethal actions as shown earlier [10]. Interesting to note in this regard is the missing synergistic action of formamidines in co-treatment with permethrin or fenitrothion. Although, in this particular case the conclusion of functional activation of OR is supported by induction of OR expression and formamidineinduced levels of free glucose, in addition to the in vivo observation of the exogenously added octopamine on the mRNA expression of OR and trehalase. The results may provide a molecular and biological mechanism for the correlation between the selective synergistic actions of AMZ and CDM in combination with active pyrethroids and neonicotinoids on Ae. aegypti larvae. Perhaps the most significant finding of the current study is the selective effect to increased glucose levels of deltamethrin-fenvalerate type pyrethroids and dinotefuran-thiamethoxam type neonicotinoids, but not by fenitrothion or permethrin, which was significantly amplified when Ae. aegypti larvae were co-treated with formamidines. Such findings provide a logical explanation for the basic synergistic action mechanism of formamidines to act as OR agonists to cause depletion of trehalose/glucose storage that eventually leads to the exhaustion of energy reserves in the affected insects. Indeed, those
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Fig. 6. Effectiveness of amitraz (AMZ) and chlordimeform (CDM) in synergizing the action of (A) deltamethrin (Delta), (B) permethrin (=Perm), (C) dinotefuran (DNF), (D) imidacloprid (=Imida), (E) thiamethoxam (=thiam), and (F) fenitrothion (=Fenit): time-dependent effects of in vivo treatment of deltamethrin (0.29 ng/ml), permethrin (0.45 ng/ ml), dinotefuran (131 ng/ml), of imidacloprid (360 ng/ml), thiamethoxam (130 ng/ml), and fenitrothion (3.50 ng/ml) and in combination with AMZ and CDM on glucose level in 4th instar larvae. Control value of glucose concentration was 0.002 μg/μl. Statistical symbol and other conditions are as shown in Fig. 4.
pyrethroids and neonicotinoids, which show significant synergistic actions with formamidines in terms of their lethal actions, are the ones that are regarded as strong neuro-excitants, meaning that they are expected to increase the release of free glucose, which consequently leads to the exhaustion of the insect. An interesting finding is the lack of such an action by permethrin or fenitrothion, despite their known action on the insect nervous system: to cause dysfunction of the sodium channel and to inhibit cholinesterase, respectively. Particularly intriguing in this regard is the case of permethrin, which has been regarded as the prototype of Type II pyrethroids such as cypermethrin. Perhaps, at least in the case of Ae. aegypti larvae, the action mechanism of permethrin might be very different from that of deltamethrin-fenvalerate type pyrethroids. In conclusion, the current study provides evidence that formamidines increase trehalase activity in vivo, which results in elevated levels of free glucose through their primary action of ac-
tivating the OR. Since increases in blood glucose has been suggested to cause anorexia in other animals [6,17,18], it is likely that such an action of formamidines to increase the glucose level serves as one of the major causes of anorexia as well as depletion of energy leading to the eventual exhaustion of the larvae of Ae. aegypti.
Acknowledgments Supported by the Hatch Fund, from the California Agricultural Experiment Station, College of Agricultural and Environmental Sciences, University of California Davis, and by a scholarship support for the senior author from the Oversea Doctoral Scholarship Fund, Ministry of Higher Education, the Government of Egypt grant number GM 670. We thank Dr. Frank Zalom, Department of Entomology and Nematology for reading this manuscript.
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(A) 0.016
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Fig. 7. Concentration-dependent effect of (A) amitraz (AMZ) treatment on the levels of free glucose on 4th instar of Ae. aegypti larvae in vivo as assessed after 12 hours of treatment and (B) the effects of chlordimeform (CDM) after 6 hours of treatment. Note that control value of glucose concentration in this series of tests was higher than that is shown in the previous testes (Fig. 6) because of the use of a higher number of larvae. *Statistically different from respective control with P ≤ 0.05.
References [1] P.D. Evans, J.D. Gee, Action of formamidine pesticides on octopamine receptors, Nature (London) 287 (1980) 60–62. [2] R.M. Hollingworth, L.L. Murdock, Formamidine pesticides: octopamine-like actions in a firefly, Science 208 (1980) 74–76.
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[3] J.A. Nathanson, E.J. Hunnicutt, N-desmethyl-chlordimeform: a potent partial agonist of octopamine-sensitive adenylate cyclase, Mol. Pharmacol. 20 (1981) 68–75. [4] R.W. Beeman, F. Matsumura, Anorectic effect of chlordimeform in the American cockroach, J. Econ. Entomol. 71 (1978) 859–861. [5] A.E. Lund, R.M. Hollingworth, D.L. Shankland, Chlordimeform: plant protection by a sub lethal, noncrystalline action on the central nervous system, Pestic. Biochem. Physiol. 11 (1979) 117–128. [6] S.M.M. Ismail, F. Matsumura, Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the American cockroach P. americana, Pestic. Biochem. Physiol. 39 (1991) 219–231. [7] M.F. Treacy, J.H. Benedict, K.M. Schmidt, R.M. Anderson, T.L. Wagner, Behavior and spatial distribution patterns of tobacco budworm (Lepidoptera: Noctuidae) larvae on chlordimeform treated cotton plants, J. Econ. Entomol. 80 (1987) 1149–1151. [8] T.C. Sparks, B.R. Leonard, J.B. Graves. Formamidine metabolism and pyrethroid interaction in pyrethroid resistant tobacco budworms. 1989 Proceedings of the Beltwide Cotton Production and Research Conferences. National Cotton Council of America, Memphis, Tenn., 1989, pp. 330–333. [9] A. Paul, L.C. Harrington, J.G. Scott, Evaluation of novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae), J. Med. Entomol. 43 (2006) 55–60. [10] M.A.I. Ahmed, F. Matsumura, Synergistic actions of formamidine insecticides on the activity of pyrethroids and neonicotinoids against Aedes aegypti (Diptera: Culicidae), J. Med. Entomol. 49 (2012) 1405–1410. [11] C.F. Vogel, E. Sciullo, F. Matsumura, Activation of inflammatory mediators and potential role of ah-receptor ligands in foam cell formation, Cardiovasc. Toxicol. 4 (2004) 363–373. [12] E.M. Sciullo, C.F. Vogel, D. Wu, A. Murakami, H. Ohigashi, F. Matsumura, Effects of selected food phytochemicals in reducing the toxic actions of TCDD and p,p’-DDT in U937 macrophages, Arch. Toxicol. 84 (2010) 957–966. [13] S. Rozen, H.J. Skaletsky, Primer3 on the www for general users and for biologist programmers, in: S. Krawetz, S. Misener (Eds.), Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, NJ, 2003, pp. 365–386. [14] D.D. Diaz, M.G. Finn, Formamidine urea as tunable electrophoresis, J. Eur. Chem. 10 (2004) 303–309. [15] N.E. Gruntenko, E.K. Karpova, A.A. Alekseev, N.A. Chentsova, E.V. Bogomolova, M. Bownes, et al., Effects of octopamine on reproduction, juvenile hormone metabolism, dopamine, and 20-Hydroxyecdysone contents in Drosophila, Arch. Insect Biochem. 65 (2007) 85–94. [16] W. Hitoshi, J. Fukami, Stimulating action of chlordimeform and desmethylchlordimeform on motor discharges of Armyworm, Leucania separata Walker (Lepidoptera: Noctuidae), J. Pestic. Sci. 2 (1977) 297–302. [17] E.D. Hensley, L.H. Conch, D.J. Tassel, E.S. Horton, R.E. Musty, Behavioral and metabolic effects of sucrose-supplemented feeding in hyperactive rats, Am. J. Physiol. 253 (1987) 434–444. [18] S.M.M. Ismail, F. Matsumura, Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the tobacco hornworm Mandela septa, Insect Biochem. Mol. Biol. 22 (1992) 713–720.
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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
Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae) Mohamed Ahmed Ibrahim Ahmed a,b, Christoph F.A. Vogel b,c,*, Fumio Matsumura b,c a
Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt Center for Health and the Environment, University of California Davis, CA 95616, USA c Department of Environmental Toxicology, University of California Davis, CA 95616, USA b
A R T I C L E
I N F O
Article history: Received 24 October 2014 Accepted 12 January 2015 Available online Keywords: Aedes aegypti qRT-PCR Formamidines Trehalose Octopamine receptor
A B S T R A C T
We recently reported that formamidine pesticides such as amitraz and chlordimeform effectively synergize toxic actions of certain pyrethroid and neonicotinoid insecticides in some insect species on the 4th instar larvae of Aedes aegypti. Here we studied the biochemical basis of the synergistic actions of the formamidines in amplifying the toxicity of neonicotinoids and pyrethroids such as dinotefuran and thiamethoxam, as well as deltamethrin-fenvalerate type of pyrethroids. We tested the hypothesis that their synergistic actions are mediated by the octopamine receptor, and that the major consequence of octopamine receptor activation is induction of trehalase to increase glucose levels in the hemolymph. The results show that formamidines cause a significant up-regulation of the octopamine receptor and trehalase mRNA expressions. Furthermore, formamidines significantly elevate levels of free glucose when co-treated with dinotefuran, deltamethrin and fenvalerate, but not with permethrin or fenitrothion, which showed no synergistic toxic effects with formamidines. These results support the conclusion that the main mode of synergism is based on the ability to activate the octopamine receptor, which is particularly effective with insecticides causing hyperexcitation-induced glucose release and consequently leading to quick energy exhaustion. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Formamidines belong to a unique group of pesticides that have multiple effects on biological activities of arthropod species [1]. As for their mode of actions, formamidines are known to interact with several types of aminergic receptors. Among those, the agonistic effects of formamidines on the octopamine receptor are now considered to be the most important feature of their insecticidal actions
Abbreviations: AMZ, amitraz; CDM, chlordimeform; DCDM, desmethylchlordimeform; OR, Octopamine Receptor; cAMP, Cyclic Adenosine Monophosphate; DEET, N, N-diethyl-3-methylbenzamide; qRT-PCR, Quantitative Reverse-Transcriptase Polymerase Chain Reaction; HK, hexokinase; G-6-PDH, glucose6-phosphate dehydrogenase; UV–VIS Spectrophotometer, Ultraviolet–visible Spectrophotometer; DNF, dinotefuran; Delta, deltamethrin; Perm, permethrin; Imida, imidacloprid; thiam, thiamethoxam; Fenit, fenitrothion; IRR, Institutional Review Board. * Corresponding author. Department of Environmental Toxicology and Center for Health and the Environment, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA. Fax: +1 530 752 5300. E-mail address: [email protected] (C.F.A. Vogel).
[2]. In agreement with their agonistic action of the aminergic receptors, the formamidines affect monoamine-mediated production of cyclic adenosine monophosphate (cAMP) and induce behavioral changes that are similar to adrenergic responses in treated insects such as the loss of appetite and hyperexcitation [3]. For instance, chlordimeform (CDM) causes a decrease in feeding of several species of lepidopterous and hemipterous insects, which suggests that the resulting mortality observed may be the consequence of starvation rather than a direct toxic action of the insecticide. Beeman and Matsumura [4] previously reported that CDM causes intense anorexia in the American cockroach at doses as low as 1–5 μg/ insect. In agreement with the above conclusion, Lund et al. [5] found that decreasing plant consumption by tobacco hornworm larvae is afforded by a nonlethal mechanism that was to be the result of motor stimulation through its actions on central non-cholinergic systems. In contrast, Beeman and Matsumura [4] found that the phenomenon of anorexia in the American cockroach is accompanied by an increase in hemolymph glucose levels, but not trehalose levels. Indeed in that study it was shown that the combined glucose and trehalose level remained relatively constant. An additional study showed that trehalase activity in the thoracic muscles has
http://dx.doi.org/10.1016/j.pestbp.2015.01.008 0048-3575/© 2015 Elsevier Inc. All rights reserved.
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increased as a result of in vivo administration of CDM [6]. This was confirmed by in vitro incubation of isolated thoracic muscles with desmethylchlordimeform (DCDM). Furthermore, the same phenomenon could be reproduced by administering exogenous octopamine in vitro and in vivo. These results provide ample evidence that one of the primary actions of this group of formamidines is to functionally activate the octopamine receptor (OR) in muscles and other non-neural tissues such as fat bodies. Despite those extensive studies, there is no consensus emerging regarding the possible biochemical causes. For instance, there are some suggestion that increasing the rate of uptake of insecticides has been proposed to be a mechanism of CDM synergism on pyrethroids, but the same effect was not produced by amitraz (AMZ) and its metabolite in the same study [7,8]. Furthermore, no report on the action mechanisms of formamidine insecticides on mosquito insects has been found so far, although there have been a number of publications on the existence of the OR in dipterous insects, including in Drosophila melanogaster. In the current study, we have investigated the molecular and biochemical mechanisms of action of AMZ, along with CDM as a positive control, by testing our hypothesis that the molecular basis of synergistic action of AMZ is its agonistic action on the OR in mosquitoes, which results in the up-regulation of the enzymatic activity of trehalase in larvae of Aedes aegypti. 2. Materials and methods 2.1. Ae. aegypti The ROCK (insecticide-susceptible) strain of Ae. aegypti was obtained from Dr. Thomas W. Scott, the University of California Davis and was used for all experiments. The basic rearing regimen was according to the description by Paul et al. [9] with some modifications. 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 was once held under vacuum for 45 min. The vacuum was then released, and 50 mg of larval diet (dog food powder) was added to the water. The hatched larvae were held overnight in the same flask, and then 200 larvae were transferred to each 600 ml liter 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 that emerged were reared in an environmental chamber with a temperature ranging from 22 to 30 °C and 80% RH. The photoperiod, adopted throughout this study, was 14:10 (L:D) h. All adults were held in a 60 by 60 by 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 days. The UCDavis 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. D. melanogaster D. melanogaster (wild type strain Canton S) were raised on standard medium at 25 °C at a density of 20 larvae/7 ml of medium, and adults were synchronized at eclosion. 2.3. Chemicals Octopamine hydrochloride (95%), chlordimeform (99.5%), amitraz (99.4%), fenitrothion (98.6%), permethrin (47.6% cis- 50.4% trans-),
Table 1 Nucleotide mRNA primer sequences used in qRT-PCR approaches for studying the possible target genes of formamidine pesticides on Ae. aegypti larvae. Gene
Sequences
α-ACTIN
FP: 5′-CAACCATGTACCCAGGAATC-3′ RP: 5′-CACCGATCCAGACGGAGTAT-3′ FP: 5′-CAGTGTATAGCGCACGAGGA-3′ RP: 5′-AACACACAATATCCGCACGA-3′ FP: 5′-AAATTTCCTGGCGTACAACG-3′ RP: 5′-ACCAGTGCACCTCCGTTATC-3′
OCTOPAMINE RECEPTOR TREHALASE
deltamethrin (99%), dinotefuran (99.5%), imidacloprid (99.5%), thiamethoxam (99.5%), and DEET (N, N-diethyl-3-methylbenzamide) (98%) were obtained from Chem. Service (West Chester, PA). Hexokinase and glucose-6-phosphate dehydrogenase from baker’s yeast (Saccharomyces cerevisiae) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.4. Drosophila feeding test The Drosophila feeding experiment consisted of 3 newly emerged females and 3 males placed into a vial in which the medium that contained 1 μg/ml of CDM was already set to occupy the lower half of each vial prior to the addition of Drosophila adults. Altogether there were 5 vials for Canton S strain which served as replicates in addition to one control vial containing the Drosophila nutritional media without CDM. 2.5. Long-term Ae. aegypti larval developing test Fourth instar larvae of Ae. aegypti in water were treated with different concentrations (6.25, 12.5, 25, 50 μg/ml) of CDM and the appearance of pupae as well as emergence of adults were monitored for 7 days. Number of mosquitoes at each stage of development was recorded on each day at the same time (7:00 PM) of the day. 2.6. Quantitative real-time reverse transcriptase PCR (qRT-PCR) The oligonucleotide sequences of primers used for detecting all of the marker mRNA expressions are shown in Table 1. Total RNA was isolated from twenty 4th instar larvae treated in vivo by each pesticide (the concentrations that have been used in this study were based on the LC50 values after 48 h exposure from a previous study which was published by our lab). In the same previous study, it was demonstrated that AMZ and CDM at a concentration of 10 μg/ml did not cause mortality during the 72-h test period on fourth instar larvae of Ae. aegypti [10] using a high pure RNA isolation kit (Qiagen), and cDNA synthesis was carried out as described by Vogel et al. [11] with some modifications to fit the mosquito model. Quantitative detection of target gene mRNAs was performed with a LightCycler Instrument (Roche Diagnostics, Mannheim, Germany) using the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and as described by Sciullo et al. [12]. Briefly, DNA-free total RNA (1.0 μg) was reverse-transcribed using 4 U Omniscript reverse transcriptase (Qiagen) and 1 μg oligo (dT)15 in a final volume of 40 μl. The primers for each gene were designed on the basis of the respective cDNA or mRNA sequences using OLIGO primer analysis software, provided by Steve Rozen and Whitehead Institute/MIT Center for Genome Research [13]. PCR amplification was carried out in a total volume of 20 μl, containing 2 μl of cDNA, 10 μl of 2 X QuantiTect SYBR Green PCR Master Mix, and 0.2 M of each primer. The PCR cycling conditions were 95 °C for 15 min followed by 30–40 cycles of 94 °C for 15 s, 60 °C for 20 s, and 72 °C for 10 s. Detection of the fluorescent product was performed at the end of the 72 °C extension period. Negative controls
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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(A)
% of larvae
were run concomitantly to confirm that the samples were not crosscontaminated. A sample with DNase- and RNase-free water instead of RNA was concomitantly examined for each of the reaction units described earlier. To confirm the amplification specificity, the PCR products were subjected to melting curve analysis. All PCR assays were performed in duplicate or triplicate. The intra-assay variability was <7%. For quantification, data were analyzed with the LightCycler analysis software according to the manufacturer’s in structions. The variables were examined by student t-test.
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Fig. 2. Long-term effects of chlordimeform (CDM) treatments (given to water in which larvae were reared) on development of Ae. aegypti (A) larvae into (B) pupa and (C) adults as expressed in percentages of Ae. aegypti at each day of development.
Statistical differences between controls (treated with vehicle only) and pesticides treated samples were determined by student t-test for the analysis of the significance difference between each pair of tests. The statistical significance of the synergistic effect of formamidines against the test insecticide (e.g. deltamethrin) was assessed by using the ANOVA analysis, each case comparing the difference between the effect of the test insecticide alone and that of the insecticide + AMZ (or CDM). 3. Results
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Twenty 4th-instar larvae of Ae. aegypti were placed in glass cups with three replicates tested per concentration (n = 60). Each cup contained 100 ml of water and varying concentrations of each insecticide in acetone. Controls containing only acetone were run concurrently with each test. Fifteen larvae were homogenized in 500 μl saline solution, and then centrifuged in 15,000 g for 5 min. An aliquot of 60 μl of 1 N HCL was added to the 60 μl sample from the supernatant from the above centrifugal preparation, which was treated immediately with 60 μl of 1 N NaOH to neutralize the effect of HC1 (totally 180 μl). To the above preparation, an aliquot of 25 μl of the test reagent mixture [ containing 1M Tris–HCl (pH 7.8), 100 mM Mg acetate, 100 mM NADP, 100 mM ATP, and per test, 2 U each of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH)] was added. The final volume of the reaction was made to 205 μl. The reaction was allowed to proceed at room temperature in square glass cuvette having a 1-cm light path. The total conversion of NADP to NADPH and the change in absorption at A340 were measured 6 min after starting the reaction using UV–VIS Spectrophotometer.
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Fig. 1. Effect of chlordimeform (CDM) administered with the feeding medium on the development of larvae of wild type D. melanogaster into (A) Pupae and (B) adults. *Statistically different from respective control with P ≤ 0.05.
Since very few studies have been conducted previously on the actions of formamidines on dipterous insects, it was necessary to conduct certain preliminary studies to ascertain that formamidines indeed cause similar, if not identical symptoms, like those observed in other orders of insects. For this purpose, we focused our attention to the developmental changes that are indicative of the effects of formamidines in reducing food intake. For this reason, we chose larval development of Drosophila, since they could be reared on nutritional media similar to that used in assessing the effect of CDM on larvae of Manduca sexta [5,6]. Fig. 1 shows the effects of CDM on D. melanogaster larval development. The speed of development of wild type strain of larvae raised in nutritional media in vials which had been treated with CDM (1 μg/ml) for 17 days was compared to their respective control group, which received only the same volume of the solvent. It is clear that CDM significantly delayed the development of larvae to pupae and adults. The pupal stage started only after day 8 and adults from 13 in CDM-treated group,
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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O.R. mRNA exp., (X control)
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instead of day 5 and day 9, respectively for the control insects. Following these series of tests, there were fewer adults in the treated group. Such an observation suggests that CDM deterred the Drosophila larval development. Our hypothesis on the cause for the developmental delay is the reduction of food consumption by the affected Drosophila larvae due to suppression of their appetite as in the case of CDM treated M. sexta [5]. The effects of CDM, at several concentrations in water, on the larval development of Ae. aegypti were then studied. The results shown in Fig. 2 indicated that CDM caused a delay also in the development of Ae. aegypti, larvae into pupae and then adults, although this trend was not as clear as in the case of Drosophila. Nevertheless the most apparent inhibitory effect of CDM on the development of Ae. aegypti larvae was observed in the timing of pupation ( Fig. 2A). In the control group all larvae completed the process of pupation by day 3, whereas in CDM treated groups, none of them, even those that received the lowest concentration, completed pupation. In the next series of tests, the effects of octopamine agonists on OR mRNA expression, trehalase
O.R. mRNA exp., (X control)
4
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Time Fig. 3. Comparative potencies of 3 proposed octopamine receptor agonists: timedependent effect of in vivo treatment of amitraz (AMZ) (10 μg/ml), octopamine (10 μg/ ml), and chlordimeform (CDM) (10 μg/ml) on (A) octopamine receptor mRNA expression, (B) trehalase mRNA expression, and (C) glucose levels in 4th instar of Ae. aegypti. Control containing only acetone. Control value at each time point was set as 1. Control value of glucose concentration is 0.002 μg/μl. *Statistically different from corresponding control with P ≤ 0.05 at each time point.
Fig. 4. Effectiveness of amitraz (AMZ) and chlordimeform (CDM) in synergizing action of (A) deltamethrin and (B) dinotefuran: time-dependent effect of in vivo treatment of deltamethrin (Delta) (0.29 ng/ml) and dinotefuran (DNF) (131 ng/ml) in combination with AMZ (10 μg/ml) and CDM (10 μg/ml) on octopamine receptor (O.R.) mRNA expression level in 4th instar of Ae. aegypti. Control treated with the vehicle acetone only. Control value at each time point was set as 1. *Statistically different from deltamethrin or dinotefuran with P ≤ 0.05 at each time point. #Action of the test insecticide (deltamethrin or dinotefuran in this case) alone statistically different from corresponding control with P ≤ 0.05 at each time point.
mRNA expression, and glucose levels were studied. As shown in Fig. 3A–C, OR expressions increased significantly after treating the larvae (4th instar) with 10 μg/ml of AMZ, octopamine, and CDM, the maximum level of stimulation induced by all 3 compounds being achieved after 30 min (4.40-, 3.91-, and 3.10-fold compared to the control, respectively). When the trehalase mRNA expression was used as the criterion on the action of those 3 OR agonists, the maximum significant level of its up-regulation by AMZ, octopamine, and CDM was not observed at 3 hrs unlike the case of OR mRNA expression; rather they were significantly stimulated at later time points as compared to control assessed at each time point. However, the time course of the changes in free glucose concentration was induced by 3 OR agonists, AMZ, CDM, and octopamine. The results indicate that all 3 OR agonists significantly increased the free glucose levels at 3, 6, 12, and 24 hrs post treatment, when compared to the control. By using the same approach, synergistic actions of formamidines on select list of insecticides were examined one by one. It was found that OR expression was stimulated significantly by deltamethrin alone (Fig. 4A), maximum expression being reached by 30 min. The expression level stimulated by deltamethrin alone at 30 min was 4.24-fold above control, whereas the combination of AMZ and CDM induced OR expression by 6.98- and 5.43-fold, respectively. A similar synergy test was conducted on dinotefuran (Fig. 4B). It was found that in the case of dinotefuran, both AMZ and CDM synergized upregulation of the OR mRNA expression, the highest significant activation being recorded at 30 min and the value for dinotefuran alone was 2.66-fold, and in combination with AMZ and CDM were 3.62- and 3.10-fold, respectively. Statistically significant synergistic effects were observed with dinotefuran alone at 3 hrs as well.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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Trehalase mRNA exp., (X control)
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(Fig. 6E). In contrast to the effects of the neonicotinoids, treatment with fenitrothion alone or in combination with AMZ and CDM did not affect free glucose levels (Fig. 6F). The effect of AMZ (Fig. 7A) at varying concentrations on total glucose level after 12 hrs is shown along with a parallel test with CDM after 6 hrs of treatment (Fig. 7B). The results obtained from Ae. aegypti larvae showed that the maximum level of stimulation by both formamidine was reached at the concentration range between 10 and 50 μg/ml. The maximum level of free glucose achieved by AMZ was higher compared to CDM.
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Time Fig. 5. Effectiveness of amitraz (AMZ) and chlordimeform (CDM) in synergizing the action of (A) deltamethrin and (B) dinotefuran: time-dependent effect of in vivo treatment of deltamethrin (0.29 ng/ml) and dinotefuran (131 ng/ml) and in combination with AMZ and CDM on trehalase mRNA expression level in 4th instar of Ae. aegypti. Statistical symbol and other conditions are as shown in Fig. 4.
Interestingly, when the same test was applied with deltamethrin alone on trehalase mRNA expression (Fig. 5A), it was found that this insecticide caused a steady increase in trehalase mRNA expression over the test period. A significant increase of trehalase mRNA by deltamethrin compared to the matched controls was found after 1 hr peaking at 6 hrs after treatment. When AMZ and CDM were co-treated with deltamethrin, it was found that both induced significant synergistic actions at 1 hr and 3 hrs. In the case of dinotefuran tested alone (Fig. 5B), it caused significant stimulation as in the case of deltamethrin. The highest increase of trehalase expression was observed after 6 hrs of treatment. A significant synergistic action of dinotefuran co-treatment with AMZ and CDM on trehalase mRNA expression was found after 1 hr. Treatment with deltamethrin alone caused a significant increase of free glucose levels at 3 hrs compared to the corresponding control (Fig. 6A). Furthermore, the action of deltamethrin on free glucose levels was significantly synergized by formamidines. Particularly noticeable was the synergistic effect of AMZ, which was significant at all points in time tested peaking at 3 hrs (12-fold) and declining to 4-fold after 24 hrs of treatment. In contrast, treatment with permethrin did not lead to significant changes on free glucose levels even in the presence of either formamidine (Fig. 6B). The effects of 3 neonicotinoid insecticides, dinotefuran (Fig. 6C), imidacloprid (Fig. 6D), and thiamethoxam (Fig. 6E) on the expression of free glucose levels were examined next. Dinotefuran, when tested alone, showed modest stimulatory effects on glucose levels at 12 hrs (Fig. 6C); however, co-treatment with AMZ and CDM synergized the action of dinotefuran significantly. Treatment with imidacloprid caused a maximum elevated level of free glucose at 12 hrs (Fig. 6D). The synergistic actions of both formamidines AMZ and CDM were significant after 6 hrs and 12 hrs of treatment. A similar trend was observed after treatment with thiamethoxam in co-treatment with AMZ
Formamidines have attracted the interest of many research groups for their biological activity and pharmacological potential [14]. To date, however, few studies have been conducted in dipterous insects, and, furthermore, practically no data exist on the possible biochemical and molecular mechanism of formamidines on Ae. aegypti. In our study, formamidines showed significant reduction of larval development of Ae. aegypti and Drosophila. In agreement with our observation of Drosophila larvae, Gruntenko et al. [15] showed that an increased level of octopamine was related to a decreased number of vitellogenic (stages 8–10) and mature (stage 14) oocytes in Drosophila virilis as well as a decreased fecundity of D. melanogaster and D. virilis. Our observation that exposure to formamidine suppresses insect feeding is supported by a large body of evidence. For instance, Lund et al. [5] found that CDM causes significant anorexia in M. sexta larvae through its action on the central nervous system, which was supported by the work of Hitoshi and Fukami [16] on other lepidopteran species. In the case of the American cockroach, Periplaneta americana, it is clear that increased hemolymph glucose levels are involved, since the neurotoxic symptoms in this species develop only at CDM doses approximately 1000-fold higher. Ismail and Matsumura [6] observed an increase in the level of both glucose and trehalose in the hemolymph of American cockroaches, following injection of CDM or octopamine itself. The increase in free glucose levels could be explained by the ability of these agents to increase trehalase activity. Ismail and Matsumura [6] also demonstrated that octopamine causes elevation of trehalase in vivo and in vitro in several tissues of the American cockroach including its thoracic muscles. To elucidate the biochemical and molecular mechanism of the action of formamidines on Ae. aegypti larvae, we analyzed the mRNA expression of the OR and trehalase, as well as levels of the free glucose released. Both AMZ and CDM are capable of synergizing the action of a number of pyrethroids and neonicotinoids which is associated with their lethal actions as shown earlier [10]. Interesting to note in this regard is the missing synergistic action of formamidines in co-treatment with permethrin or fenitrothion. Although, in this particular case the conclusion of functional activation of OR is supported by induction of OR expression and formamidineinduced levels of free glucose, in addition to the in vivo observation of the exogenously added octopamine on the mRNA expression of OR and trehalase. The results may provide a molecular and biological mechanism for the correlation between the selective synergistic actions of AMZ and CDM in combination with active pyrethroids and neonicotinoids on Ae. aegypti larvae. Perhaps the most significant finding of the current study is the selective effect to increased glucose levels of deltamethrin-fenvalerate type pyrethroids and dinotefuran-thiamethoxam type neonicotinoids, but not by fenitrothion or permethrin, which was significantly amplified when Ae. aegypti larvae were co-treated with formamidines. Such findings provide a logical explanation for the basic synergistic action mechanism of formamidines to act as OR agonists to cause depletion of trehalose/glucose storage that eventually leads to the exhaustion of energy reserves in the affected insects. Indeed, those
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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6
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Fig. 6. Effectiveness of amitraz (AMZ) and chlordimeform (CDM) in synergizing the action of (A) deltamethrin (Delta), (B) permethrin (=Perm), (C) dinotefuran (DNF), (D) imidacloprid (=Imida), (E) thiamethoxam (=thiam), and (F) fenitrothion (=Fenit): time-dependent effects of in vivo treatment of deltamethrin (0.29 ng/ml), permethrin (0.45 ng/ ml), dinotefuran (131 ng/ml), of imidacloprid (360 ng/ml), thiamethoxam (130 ng/ml), and fenitrothion (3.50 ng/ml) and in combination with AMZ and CDM on glucose level in 4th instar larvae. Control value of glucose concentration was 0.002 μg/μl. Statistical symbol and other conditions are as shown in Fig. 4.
pyrethroids and neonicotinoids, which show significant synergistic actions with formamidines in terms of their lethal actions, are the ones that are regarded as strong neuro-excitants, meaning that they are expected to increase the release of free glucose, which consequently leads to the exhaustion of the insect. An interesting finding is the lack of such an action by permethrin or fenitrothion, despite their known action on the insect nervous system: to cause dysfunction of the sodium channel and to inhibit cholinesterase, respectively. Particularly intriguing in this regard is the case of permethrin, which has been regarded as the prototype of Type II pyrethroids such as cypermethrin. Perhaps, at least in the case of Ae. aegypti larvae, the action mechanism of permethrin might be very different from that of deltamethrin-fenvalerate type pyrethroids. In conclusion, the current study provides evidence that formamidines increase trehalase activity in vivo, which results in elevated levels of free glucose through their primary action of ac-
tivating the OR. Since increases in blood glucose has been suggested to cause anorexia in other animals [6,17,18], it is likely that such an action of formamidines to increase the glucose level serves as one of the major causes of anorexia as well as depletion of energy leading to the eventual exhaustion of the larvae of Ae. aegypti.
Acknowledgments Supported by the Hatch Fund, from the California Agricultural Experiment Station, College of Agricultural and Environmental Sciences, University of California Davis, and by a scholarship support for the senior author from the Oversea Doctoral Scholarship Fund, Ministry of Higher Education, the Government of Egypt grant number GM 670. We thank Dr. Frank Zalom, Department of Entomology and Nematology for reading this manuscript.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008
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(A) 0.016
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Fig. 7. Concentration-dependent effect of (A) amitraz (AMZ) treatment on the levels of free glucose on 4th instar of Ae. aegypti larvae in vivo as assessed after 12 hours of treatment and (B) the effects of chlordimeform (CDM) after 6 hours of treatment. Note that control value of glucose concentration in this series of tests was higher than that is shown in the previous testes (Fig. 6) because of the use of a higher number of larvae. *Statistically different from respective control with P ≤ 0.05.
References [1] P.D. Evans, J.D. Gee, Action of formamidine pesticides on octopamine receptors, Nature (London) 287 (1980) 60–62. [2] R.M. Hollingworth, L.L. Murdock, Formamidine pesticides: octopamine-like actions in a firefly, Science 208 (1980) 74–76.
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[3] J.A. Nathanson, E.J. Hunnicutt, N-desmethyl-chlordimeform: a potent partial agonist of octopamine-sensitive adenylate cyclase, Mol. Pharmacol. 20 (1981) 68–75. [4] R.W. Beeman, F. Matsumura, Anorectic effect of chlordimeform in the American cockroach, J. Econ. Entomol. 71 (1978) 859–861. [5] A.E. Lund, R.M. Hollingworth, D.L. Shankland, Chlordimeform: plant protection by a sub lethal, noncrystalline action on the central nervous system, Pestic. Biochem. Physiol. 11 (1979) 117–128. [6] S.M.M. Ismail, F. Matsumura, Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the American cockroach P. americana, Pestic. Biochem. Physiol. 39 (1991) 219–231. [7] M.F. Treacy, J.H. Benedict, K.M. Schmidt, R.M. Anderson, T.L. Wagner, Behavior and spatial distribution patterns of tobacco budworm (Lepidoptera: Noctuidae) larvae on chlordimeform treated cotton plants, J. Econ. Entomol. 80 (1987) 1149–1151. [8] T.C. Sparks, B.R. Leonard, J.B. Graves. Formamidine metabolism and pyrethroid interaction in pyrethroid resistant tobacco budworms. 1989 Proceedings of the Beltwide Cotton Production and Research Conferences. National Cotton Council of America, Memphis, Tenn., 1989, pp. 330–333. [9] A. Paul, L.C. Harrington, J.G. Scott, Evaluation of novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae), J. Med. Entomol. 43 (2006) 55–60. [10] M.A.I. Ahmed, F. Matsumura, Synergistic actions of formamidine insecticides on the activity of pyrethroids and neonicotinoids against Aedes aegypti (Diptera: Culicidae), J. Med. Entomol. 49 (2012) 1405–1410. [11] C.F. Vogel, E. Sciullo, F. Matsumura, Activation of inflammatory mediators and potential role of ah-receptor ligands in foam cell formation, Cardiovasc. Toxicol. 4 (2004) 363–373. [12] E.M. Sciullo, C.F. Vogel, D. Wu, A. Murakami, H. Ohigashi, F. Matsumura, Effects of selected food phytochemicals in reducing the toxic actions of TCDD and p,p’-DDT in U937 macrophages, Arch. Toxicol. 84 (2010) 957–966. [13] S. Rozen, H.J. Skaletsky, Primer3 on the www for general users and for biologist programmers, in: S. Krawetz, S. Misener (Eds.), Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totowa, NJ, 2003, pp. 365–386. [14] D.D. Diaz, M.G. Finn, Formamidine urea as tunable electrophoresis, J. Eur. Chem. 10 (2004) 303–309. [15] N.E. Gruntenko, E.K. Karpova, A.A. Alekseev, N.A. Chentsova, E.V. Bogomolova, M. Bownes, et al., Effects of octopamine on reproduction, juvenile hormone metabolism, dopamine, and 20-Hydroxyecdysone contents in Drosophila, Arch. Insect Biochem. 65 (2007) 85–94. [16] W. Hitoshi, J. Fukami, Stimulating action of chlordimeform and desmethylchlordimeform on motor discharges of Armyworm, Leucania separata Walker (Lepidoptera: Noctuidae), J. Pestic. Sci. 2 (1977) 297–302. [17] E.D. Hensley, L.H. Conch, D.J. Tassel, E.S. Horton, R.E. Musty, Behavioral and metabolic effects of sucrose-supplemented feeding in hyperactive rats, Am. J. Physiol. 253 (1987) 434–444. [18] S.M.M. Ismail, F. Matsumura, Studies on the biochemical mechanisms of anorexia caused by formamidine pesticides in the tobacco hornworm Mandela septa, Insect Biochem. Mol. Biol. 22 (1992) 713–720.
Please cite this article in press as: Mohamed Ahmed Ibrahim Ahmed, Christoph F.A. Vogel, Fumio Matsumura, Unique biochemical and molecular biological mechanism of synergistic actions of formamidine compounds on selected pyrethroid and neonicotinoid insecticides on the fourth instar larvae of Aedes aegypti (Diptera: Culicidae), Pesticide Biochemistry and Physiology (2015), doi: 10.1016/j.pestbp.2015.01.008