Abstract: Mosquito larvicidal efficacy of seven different synthesized imidacloprid compounds (IMD) with different substitutions (chlorine ion, methyl group, methoxy group, ester group, phenyl ring and pyridine ring) of Imidacloprid were studied on the IV stage larvae of mosquito, Culex quinquefasciatus. LC50 of each compound (IMD1-IMD-7) was determined using probit analysis. Effects of these compounds on biochemical toxicity were also determined by observing the changes in total protein, lipid and Acetylcholine esterase activity of the mosquito larvae. The results show that due to the action of these compounds, higher amount of protein production (p<=0.001) and AchE activity (p<=0.001) and significant decrease in total lipid activity (p<=0.001) were observed when compared to the control. Similar types of results were also reported on increase production of protein, lipid and acetylcholine activity due to the action of insecticides in other insects. Hence, it is concluded that, these seven synthesized compounds can be used as mosquito larvicides.
INTRODUCTION
The neonicotinoids are new insecticide class, which include the commercial products imidacloprid (IMD). Its chemical analogs are also being used as insecticides for many agricultural and public health pests (Yamamoto and Casida, 1999; Mason et al., 2000) which directly act on the nicotinic acetylcholine receptor (nAchr) (Tomizawa and Casida, 2001; Suchail et al., 2003).
Imidacloprid, 1(6-chloro-3-pyridinyl)methyl-N-nitro-2-imidazolidinimine, is a new neonicotinoid insecticide, has been widely used as pest control agent on many crops (Elbert et al., 1991). Imidacloprid causes blockage in neuronal pathway which leads to the accumulation of more amount of acetylcholine, resulting to paralysis and eventually leads to death of the insect (Casida, 1998; Caroline Cox, 2001; Suchail et al., 2003). Neurophysiological studies have also shown that imidacloprid has multiple agonist and antagonist effects on neuronal nicotinic acetylcholine receptor channels in clonal rat phaeochromocytoma cells (Nagata et al., 1998). However, these compounds are shown to have very less or no toxic effect against higher organisms including humans. This selective toxicity of imidacloprid is attributed to differences in the binding affinity at the nicotinic acetylcholine receptor (Chao and Casida, 1997) or due to structural differences in the neuronal nicotinic acetylcholine receptor binding sites of mammals and insects (Casida, 1998; Wang et al., 2007).
Mosquitoes (Diptera: Culicidae) pose the greatest threat to public health because of their ability to act as vectors of pathogens causing malaria, dengue, encephalitis and filariasis, etc. which affect many millions of people all over the world, particularly in South East Asian countries. Culex quinquefasciatus Say, is one of the potential vector of Wuchereria bancrofti, the causative agent of human lymphatic filariasis (Kabir et al., 2003). Recent estimates suggest that 120 million people infected with filariasis globally (Michael, 2000). It is reported that C. quinquefasciatus and Anopheles gambiae developed resistance to many effective insecticides (Guessan, 2003). So, there is urgent need to identify some effective insecticides to suppress the mosquito control.
MATERIALS AND METHODS
Seven different synthesized analogues of imidacloprid were produced from the
Department of Organic Chemistry, Indian Institute of Chemical Technology (IICT),
Hyderabad and used for the present study. The process of synthesis briefly is:
various substituted nicotinaldehydes were synthesized from enamides by Vilsmeier
reaction. These aldehydes were reduced by using Sodium borohydride to get alcohol.
Alcohols were then converted to chloride by using thonylchloride in the presence
of n-Heptane solvent and catalytic amount of Dimethylformamide (DMF). These
chlorides were again coupled with tetrahydro 2-nitro imine imidazole in presence
of Potassium carbonate, catalytic amount of Cesium chloride and solvent was
acetonitrile and yielded analogue of IMDs (substituted -2-pyridyl methyl tetrahydro-1H-2-imidazolyliden)-1-oxo-1-hydraziniumolate)
(Fig. 1). These products were well characterized by 1H
Nuclear Magnetic Resonance (1HNMR), Mass, IR and 13C NMR
and named as Imidacloprid (IMD 1-7).
| Fig. 1: | Step wise synthesized of imidacloprid analogues (IMD1-IMD7) |
Insect
Cultured stock mosquito, C. quinquefasciatus colonies from the insectary
of Biology division, IICT, Hyderabad was used for this experiment. The temperature
was maintained at 27±2°C and relative humidity (RH) at 60±5%
throughout the experiment. Ten percent yeast suspension was provided as food
source for the mosquito larvae.
Larvicidal Bioassay
The larval bioassay toxicity study was performed using standard protocol
as described by World Health Organization (WHO, 1963), on 4th instar mosquito,
C. quinquefasciatus larvae. For each group, 25 larvae were placed in
a disposable plastic cup containing 250 mL of tap water. To determine the toxicity
of the each analogue seven different concentrations, i.e., 1, 2, 3, 4, 5, 10
and 15 ppm were prepared by dissolving with acetone and were inoculated to respective
cup. All the cups were coded and kept separately. Simultaneously, positive control
(only IMD pure compound) and negative control (249 mL of tap water and 1 mL
of acetone) were kept separately. For all the experiments, 3 replications were
maintained. Larval mortality from each compound was recorded after 24 h of treatment
and LC50 and LC90 were determined using Probit Analysis
(Finney, 1971).
Selection of Doses
By observing the LC50 dose of each compound, one lowest and one
highest concentration of each test compound were selected and tested for the
biochemical analysis.
Biochemical Assays
From each set of experiment, total protein content by Lowry et al.
(1951) method total lipids by Van Handel (1985) and Acetylcholine Esterase (AchE)
activity using Ellman et al. (1961) method were determined. The results
were subjected for statistical analysis (t-test).
RESULTS AND DISCUSSION
Larval Mortality
The LC50 values of Imidacloprid analogues were determined (IMD1,
2, 3, 4, 5, 6 and 7) were 7.15, 5.67, 6.18, 6.38, 8.86, 4.14 and 5.48 ppm, respectively.
Among all the compounds, IMD-2, 6 and 7 showed higher larval mortality when
compared to other 4 compounds on C. quinquefasciatus larvae (Table
1). This differential activity of these different analogues of IMD may be
due to the position of the active functional groups in the native ring. When
larvae were exposed to different groups of Imidcloprids analogs for 24 to 72
h post treatment of compounds, 100% of larval mortality was recorded from all
the test groups including in the positive control Imidacloprid. This increased
in larval mortality, may be due to the uptake of the active moiety of the compound,
which could be time dependent. There is also every possibility of production
of more toxic metabolites in the larval integument and alimentary canal leading
to death of the larvae (Kabir et al., 2003).
Biochemical Analysis
Effect on Total Protein
The total protein content from the positive control batch exposed to only
IMD was 30 μg mL-1 while in the negative control it was 23.2
μg mL-1. Higher amount of total protein were also recorded from
all the different metabolites of IMD such as 54.7, 58.6, 51.8, 47, 43.2, 54.7
and 64.3 μg mL-1 in IMD-2 (5 ppm), IMD-3 (3 ppm), IMD-4 (4 ppm),
IMD-5 (4 ppm), IMD-6 (2 and 3 ppm), IMD-7 (4 and 5 ppm), respectively (p≤0.001)
(Table 2). This higher amount of total proteins production
in presence of insecticides may be due to the over expression of the target
gene (Mouches et al., 1990). A very less amount of total protein were
found in IMD-3 (3 ppm)-19.2 μg mL-1 and IMD-5 (5 ppm)-16.3 μg
mL-1, which may be due to the more toxic nature of these metabolites
at specific concentration as per as the total amount of protein is concerned.
Effect on Total Lipid
Lipids are known to be the important constituent of the insect for the defending
action against various kinds of pathogens by blocking the passage inside the
body cavity. It is reported that various toxic chemicals interfere in the metabolism
of lipids in animals. However, the physiological changes in insects depend upon
the type of exposed toxic chemicals. In the present study, all the IMD analogues
including pure IMD caused massive damage in the total lipids in mosquito. In
control group, the total lipid was found to be 165.99 mg mL-1 while
in pure IMD it was 45mg mL-1 (p≤0.001). In all the 7 different
IMD compounds, drastic drop in the total lipid content was observed (p≤0.001)
(Table 2). This decrease in the total lipid may be due to
the increase in the lipid peroxidation in the plasma membrane with the exposure
of these compounds.
| Table 1: | Toxicity pattern of different IMD compounds on Culex quinquefasciatus larvae |
Data collected from three independent replicates | |
| Table 2: | Effect of different concentrations of IMD analogues on biochemical parameters in IV stage Culex quinquefasciatus larvae |
Data collected from three replicates; ±is Standard Error of Mean | |
Effect on AchE
High amount of AchE were obtained with many folds in the presence of different
metabolites of IMD when compared to control groups. In the negative control
group, AchE was recorded 2.52 μL min-1 mg-1 while
in the positive control (pure IMD), it was 7.075 μL min-1 mg-1.
High concentration of the AchE were obtained in all the different IMD compounds.
The amount of AchE was also increased with the increase in the concentration
of the IMD metabolites tested (p≤0.001) (Table 2). This
increase in the AchE in presence of the different metabolites of IMDs may be
due to the inhibitory action of the AchE activity in post-synaptic region of
the nerves.
CONCLUSIONS
Nicotinoids are neurotoxic in nature. These molecules bind to the receptor molecule in the nervous system that normally receives the molecule acetylcholine. Toxicological studies of Imidacloprid shows the rapid appearance of neurotoxicity symptoms in honey bee (Suchail et al., 2003). It is also reported that, IMD metabolites are more toxic than IMD (Caroline, 2001; Axel Decourtye et al., 2004). From this study it is observed that, the toxicity pattern of all the tested compounds do not differ much and on par with the toxicity of the (IMD). Among all the analogues tested, IMD-6, IMD-7 and IMD-2 (chlorine atom with different position and contain phenyl substitution), (ester group and substitution of pyridine ring) and methoxy group) respectively showed higher toxic effect on C.quinquefasciatus larvae when compared to other four compounds. To use these compounds as mosquito larvicides, further studies on non-target organisms are required.
ACKNOWLEDGMENT
Authors are thankful to the Director, Indian Institute of Chemical Technology for his encouragement and support.