Insect growth regulatory and larvicidal activity of chalcones against Aedes albopictus
Abstract
Widespread use of chemical insecticides has resulted in development of insect resistance and natural products with biological activities could become an attractive alternative to control insect pests. In order to find more effective insecticides for controlling mosquito, various mosquitocidal compounds are studied. Recently, juvenile hormone antagonists (JHANs) have been found to be to safe and effective insecticides for control of mosquito. In order to identify novel insecticidal compounds with JHAN activity, several chalcones were surveyed on their JHAN activities and larvicidal activities against Aedes albopictus larvae. Among them, 2′-hydroxychalcone and cardamonin showed high levels of JHAN and mosquito larvicidal activities. These results suggested that chalcones with JHAN activity could be useful for control of mosquito larvae.
Introduction
The Asian tiger mosquito, Aedes albopictus, is a medically important insect pest that transmits various diseases, including dengue fever (Sang et al. 2015), chikungunya (Reiter et al. 2006), and zika virus (Grard et al. 2014). The global health problem is becoming serious because they have spread to at least 28 other countries from Asia (Benedict et al. 2007). Due to its rapid expansion and vectorial capacity, effective control of A. albopictus is required for public health (Bonizzoni et al. 2013). Chemical insecticides such as DEET (N,N-diethyl-m-toluamide), pyrethroids, and temephos have been commonly used to control mosquitoes (George et al. 2015). However, because of their toxicity to the environment and non-targets, and also the development of resistance, there is an urgent need to develop novel effective insecticides (Vontas et al. 2012).
Insect growth regulators (IGRs) could become an effective alternative to control mosquitoes and other vectors. Important advantages of IGRs are relatively low toxic to environment and selectivity (Pener & Dhadialla 2012). Synthetic pyrethroids are reported to be highly toxic to aquatic organisms, especially fish, while there is a growing interest in the use of IGRs such as methoprene, pyriproxyfen, novaluron, and diflubenzuron for mosquito control (Chavasse et al. 1997; Raghavendra et al. 2011). Methoprene and pyriproxyfen, particularly, belong to the juvenile hormone (JH) agonists (JHAs) that are one of three classes of IGR insecticides effective against mosquitoes both as larvicides and inhibitors of adult emergence (Ansari et al. 2005). However, according to recent studies, mosquitoes and other pests also have developed resistance to methoprene and pyriproxyfen (Paul et al. 2005; Silva & Mendes 2007; Dennehy et al. 2010). Hence, there is an urgent need to develop novel IGR insecticides.
Chalcones are aromatic ketones with two phenyl rings which are abundant in a variety of plant species. They show various biological activities such as antidiabetic, antineoplastic, antihypertensive, antibacterial, antifungal, antiprotozoal, antiviral, anti-inflammatory, antihistaminic, anti-oxidant, and antimalarial activities (Chen et al. 1997; Elias et al. 1999; Mishra et al. 2001; Israf et al. 2007; Detsi et al. 2009; Kantevari et al. 2011; Romagnolo & Selmin 2012; Mahapatra et al. 2015). Recently, it has been reported that one of the chalcone derivatives, kanakugiol, acts as a juvenile hormone antagonist (JHAN) by disrupting the juvenile hormone receptor complex (Lee et al. 2015). JHANs could be effective for control of insect pests because they fatally affect the JH-regulated phyisiological processes such as molting, metamorphosis, reproduction, polyphenism, caste differentiation, and various physiological functions in insects (Riddiford 1994; Nijhout 1998; Raikhel et al. 2005; Hartfelder & Emlen 2012). In this study, we investigated several chalcones for their JHAN activity and mosquito larvicidal activity against A. albopictus.
Materials and methods
Insect
The A. albopictus was provided by the Korea National Institute of Health (Cheongwon, Korea). The mosquitoes were reared at 28°C and 70 % relative humidity with a 12 h dark/12 h light cycles in aged tap water. Larvae were fed on a diet of Tetramin fish flakes, and adults were reared using 10 % sucrose solution.
Chemicals
(E)-1,3-diphenylprop-2-en-1-one (3), (E)-1-(3-chloro-2-hydroxyphenyl)-3-phenylprop-2-en-1-one (5), (E)-1-(2-hydroxy-3,4-dimethoxyphenyl)-3-phenylprop-2-en-1-one (6), 2-hydroxybenzoic acid (1), and (E)-3-phenylprop-2-enoic acid (2) were purchased from Sigma-aldrich (USA). (E)-1-(6-hydroxy-2,3,4-trimethoxyphenyl)-3-phenylprop-2-en-1-one (8) and (E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (4) were purchased from HANCHEM (Korea). (E)-1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylprop-2-en-1-one (7) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Chemical structures of chalcones used in this study are shown in Fig. 1.
Yeast two-hybrid β-galactosidase assays
The yeast two-hybrid binding test using quantitative β-galactosidase assay was carried out using Y-187 yeast cells transformed with A. aegypti Met-FISC as previously described (Lee et al. 2015). The transformed Y187 cells were incubated at 30°C in DDO (SD -Leu/−Trp) media until OD600 values reached 0.3–0.4. After harvest, the cells were suspended in the fresh media at a concentration of 2.0 × 106 cells / mL and 100 μL of the cells was distributed in 96-well plates. To determine JHAN activity, 100 μL of yeast cells (2.0 × 106 cells / mL) distributed in 96-well plates was treated with 0.033 ppm of pyriproxyfen and corresponding concentrations (0.1, 1, or 10 ppm) of compound. A negative control treated with 0.033 ppm of pyriproxyfen and control solvent (dimethyl sulfoxide, DMSO) was placed in each tested plate. The treated cells were incubated for further 3 h and subjected to the β-galactosidase assays using the Yeast β-galactosidase Assay Kit (Thermo Scientific, Waltham, MA, USA). The obtained OD420 values were converted to an arbitrary unit representing JHAN activity (Lee et al. 2015).
Larval toxicity tests
Twenty third-instar larvae of A. albopictus in 5 mL tap water with food mixtures were treated with 5 ppm of each compound. The number of dead larvae was counted at 24 h intervals after treatment for 3 days. The toxicity assays were performed in triplicates.
Statistical analysis
Differences among treatments were analyzed by one-way analysis of variance (ANOVA). Homogeneity of variances was determined using Levenes's test, and determined significance between treatments using Tukey HSD and Dunnett's T3 statistic (P < 0.05). All statistical analyses were conducted using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA).
Results and discussion
The basic chemical structure of chalones is the enone which connects two aromatic rings, ring A and B. The ring A is linked to the ketone and the ring B is linked to the alkene of the enone. It has been reported that variety of biological activities of chalcones are closely associated with the substitution patterns on the two aromatic rings (Begum et al. 2011). When the (1) corresponding to ring A moiety of chalcones and (2) corresponding to ring B moiety were subjected to in vitro yeast two-hybrid β-galactosidase assays, (2) interfered with the pyriproxyen-mediated binding of A. aegypti Met-FISC, demonstrating that this compound has JHAN activity (Fig. 2A). In contrast, (1) did not affect the pyriproxyfen-mediated binding of A. aegypti Met-FISC. To determine their ability to interfere with pyriproxyfen-mediated binding of A. aegypti Met-FISC, another six chalcones were tested using in vitro yeast two-hybrid β-galactosidase assays (Fig. 2B). All chalcones tested were found to highly interfere with the binding in the concentration-dependent manner. These results suggested that the aromatic ring B linked to the alkene of chalcones is crucial for JHAN activity. The addition of chalcones with high JHAN activity to yeast cells transformed with A. aegypti Met-FISC resulted in normal growth of yeast cells, suggesting that their JHAN activities are not false signals originated from their anti-yeast activity and they directly disrupt the formation of JH receptor complex (Fig. 3).
To investigate larvicidal activities of the chalcones with JHAN activity, third instar larvae of A. albopictus were treated with a 5 ppm of each compound (Fig. 4). Among these chemicals, (4) and (7) showed the highest larvicidal activity with mortalities over 80 %. (3) and (5) also caused larval mortalities over 50 %. These result were consistent with the previous report that insecticidal activities of (3) increased when they incorporated a hydroxyl substituent at 2′-position or electron donating group on ring A (Begum et al. 2011). However, mosquito larvicidal activities of chalcones were dramatically reduced when their 2-hydroxylated ring A incorporated a methoxy group as in (6) and (8). In addition, (2) corresponding to aromatic ring B moiety of chalcones showed very low larvicidal activities against A. albopictus, suggesting that both ring A and B are necessary for mosquito larvicidal activity and that other biological activities of chalcones beside JHAN activity might contribute to their mosquito larvicidal activities.
In this study, chalcones with high level of JHAN activity were identified using in vitro yeast two-hybrid screening assay. Among them, (3), (4), (5) and (7) showed high level of mosquito larvicidal activity against of A. albopictus. These results suggested that chalcones with JHAN activity could be useful for the development of environmentally benign IGR insecticide to control mosquitoes.
Acknowledgments
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agro-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 316030-3). Lee SH, Lee BR, Fang Y, Kim JH, Park DH, Park MG, and Woo RM were supported by the Brain Korea 21 plus project, Seoul National University, Republic of Korea.
