Insecticidal and Genotoxic effects of some indigenous plant extracts in Culex quinquefasciatus Say Mosquitoes.

Five different weed plants viz. Convulvulus arvensis, Chenopodium murale, Tribulus terrestris, Trianthema portulacastrum, and Achyranthes aspera were investigated for their entomocidal and genotoxic effects against Culex quinquefasciatus mosquitoes. High mortality was observed at 72 hours in a dose dependent manner. Among all the tested plants, A. aspera was found highly significant which showed 100% mortality at 250 ppm after 72 hours with LC50 of 87.46, 39.08 and 9.22 ppm at 24, 48, respectively. In combination with Bacillus thuringiensis israelensis (Bti); A. aspera also caused 100% mortality at 250 ppm concentration after 72 hours (LC50 8.29 ppm). Phytochemical analysis of all the tested weed plants showed the presence of flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids. Random Amplification of Polymorphic DNA-Polymerase chain reaction (RAPD-PCR) and comet assay were performed to assess the genotoxic effect of A. aspera but no change in DNA profile was observed. Furthermore, FTIR showed the presence of phenolic compounds in A. aspera extract. It is suggested that certain phenolic compounds such as flavonoids modulate the enzymatic activity and, hence, cause the death of larvae of Cx. quinquefasciatus. Altogether, current study would serve as an initial step towards replacement of synthetic insecticides to plant-microbe based biopesticide against Culex mosquitoes in future.

Mortality bioassay test. Different concentrations of each weed plant extract from 10 ppm to 250 ppm were used to perform the bioassay test following WHO protocol 32 . Twenty larvae of Cx. quinquefasciatus were treated with different concentrations of each extract individually and in combination with microbes (1:1 volume); P. aeruginosa and Bacillus thuringiensis israelensis (Bti) along with control group and group treated with different concentrations of permethrin (10,20,30,50, 100, 150 and 250 ppm) in water. Three replicates were performed for each test and the mortality data was recorded at 24, 48 and 72 hours of post treatment 16 . Immovable larvae were considered as dead and removed to prevent decomposition which might cause the mortality of other intact alive larvae. The dead larvae were stored in ethanol in 1.5 ml eppendorf tubes to further determine the DNA damage by RAPD PCR and comet assay.
Esterases and phosphatases enzyme assay. The larvae of Cx. quinquefasciatus mosquitoes were thoroughly washed with distilled water and adhering water was removed using blotting paper. The larvae were homogenized using ice-cold Sodium Phosphate buffer (20 mM. pH 7.0) with the help of Teflon hand homogenizer. The homogenate was centrifuged at 8000 × g and 4 °C for 20 minutes using centrifuge machine, SIGMA, Germany. The supernatant was used for the estimation of Esterases and Phosphatases (AChE = acetylcholinesterase, AcP = acid phosphatases, AkP = alkaline phosphatases, α-Carboxyl = α-Carboxylesterases and β-Carboxyl = β-Carboxylesterases). All the solutions and glassware used for homogenization were kept at 4 °C prior to use and the homogenates were held on ice until used for assays. The protocols for enzymatic assays as already described Younes et al. 33 and Sultana et al. 15 were followed.

Phytochemical analysis of weeds extracts. Phytochemical analysis of five tested weed plant extracts
were performed in order to detect the chemical constituents as described by Harborne 34 , Trease and Evans 35 and Sofowara 36 .
Fourier transform infrared spectroscopy (FTIR) analysis. The functional groups of active components in the extracts of weed plants were identified using FTIR spectrometer (Bruker Tensor II) on the basis of vibrational frequencies between atomic bonds. The extracts were chilled at −80 °C followed by lyophilization to obtain the IR spectrum of lyophilized extract (Alpha, Bruker, California, USA). FTIR spectra were measured in the frequency ranges from 400-4000 cm −1 sample by scanning the sample. The samples were run in triplet form 37 . DNA extraction. The stored samples of mosquito larvae were homogenized in 300 µl lysis buffer (0.4 M NaCl, 2 mM EDTA, and 10 mM Tris-HCL pH 8.0), 100 µl Proteinase K (100 mg/µl) of BIOSHOP, Canada and 20% sodium dodecyl sulphate (SDS). The homogenate was incubated at 55 °C for one hour, then, 300 µl of 5 M NaCl was added and vortexed for few seconds. The mixture was centrifuged at 13,000 rpm for 10 minutes. The DNA from supernatant was precipitated by adding ice cold ethanol in equal volume, and kept at −20 °C for 1 hour and afterwards recovered by centrifugation. The DNA pellet was air dried and resuspended in D 3 H 2 O [38][39][40] .
The optical absorbance of each sample was calculated by measuring the absorption at 260 nm wavelength of UV light using spectrophotometer of HITACHI, Japan. DNA Concentration was calculated as: = × µ µ DNA Conc g/ l Dilution fold absorbance at 260 nm RAPD-PCR amplification. A total of five RAPD primers (GenLink: A-03, A-04, A-06, A-18 and C-04; Supplementary Table) were selected to amplify the mosquitoes genomic DNA following the PCR conditions as described by Bibi et al. 39 and Zahoor et al. 41 .
Haemocyte collection. Cx. quinquefasciatus haemocytes were collected according to Irving et al. 42 . The collected larvae from trail beaker were first washed with distilled water, sterilized in 5% bleach and dried. The cuticle was removed with two fine forceps. The haemolymph and haemocytes were taken in microcentrifuge tubes. The pooled haemolymph was centrifuged at 300 × g and 4 °C for 10 minutes, the supernatant was discarded and the pellet was resuspended in 20 µL of cold PBS.
Comet assay. The comet assay was performed according to Singh et al. 43 with minor modifications. The cell samples were carefully suspended in 140 µL of 0.75% LMA (Low melting agar) and then layered onto microscope slides coated with 150 µL of 1% NMA (Normal melting agar) and dried at room temperature. The two gels on each slide were mounted, covered with a coverslip and put at 4 °C for10 minutes to let solidify the gel. The coverslip was immediately removed after agarose solidification. The slides were immersed in a cold fresh lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris, 1% Triton X-100 and 5% DMSO) for 2 hours at 4 °C in a dark chamber. The slides were placed in a horizontal gel electrophoresis tank filled with cold electrophoretic buffer (1 mM Na 2 EDTA and 300 mM NaOH, pH 13) for 25 minutes for DNA unwinding. The electrophoresis was performed in the same buffer for 20 min at 25 V and 300 mA (0.73 V/cm). After electrophoresis, the slide was washed twice with 0.4 mM Tris (pH 7.5) for 5 minutes to neutralize the slides. The slides were stained with 20 µL of DAPI (1 µg/mL) per gel and examined at 400× magnification with Komet 5.5 Image Analysis System fitted to an Olympus BX50 fluorescence microscope equipped with 590 nm barrier filter and 480-550 nm wide band excitation filter. One hundred randomly selected cells (50 cells per two replicate slides) per treatment were analyzed 21,44,45 .
Data analysis. Probit Analysis program (version 1.5) was used to determine the LC 50 between concentration and percent mortality at various concentrations of plant extract and bacteria 46 . Abbot's formula was used to analyze the data of mortality obtained through bioassay tests. The corrected mortality data was subjected to ANOVA using Statistica 13.0 for Windows 15 . The means were separated using Tuckey's HSD (Honest Significant Difference) test at a significance level of 0.05. A value of p < 0.05 was considered statistically significant 14,47,48 . PCR products were analyzed using gel electrophoresis and the genetic data were analyzed using POPGENE software 40 . For comet assay, the DNA damage in cells of Cx. quinquefasciatus larvae was assessed by two distinct types of DNA damage measurements: the length of DNA comet tail and the percentage of fragmented DNA present in the tail after electrophoresis 21 .

Results
Mortality assay of culex quinquefasciatus using weed plant extracts at various concentrations and different time intervals.

Mortality Assay of Culex quinquefasciatus mosquitoes using Achyrathes aspera in-combination with microbes at various concentration and different time intervals.
A. aspera showed significant results for mortality and thus, it was selected for combinatorial trials with Bti and P. aeruginosa. The mean mortality induced by Bti and Pseudomonas individually and in combinations with A. aspera extract at different concentrations and time intervals is shown in Table 4. It was observed that mortality increased with increase in concentration and exposure time. High mortality was observed at 250 ppm concentration after 72 hours, whereas, highest mortality (100%) was shown by of A. aspera with Bti followed by combination of Pseudomonas and A. aspera (72.79%) at 250ppm concentration as compared to control treatment (Table 4). www.nature.com/scientificreports www.nature.com/scientificreports/ fourier transform infrared spectroscopy (ftiR) analysis of Achyranthes aspera extract. The Infra-red spectrum of A. aspera extract revealed peak at 3338. 23 cm −1 which corresponds to -OH group for phenols. Moreover, peak at around 1317.85 cm −1 and 1377.38 cm −1 was assigned to C=H group and 1077.88 cm −1 for C-O Stretching. The peak at 779.00 cm −1 shows the stretching vibration of plane C-H bending. In addition, the peak at 669.12 cm −1 corresponds to aromatic ring having phosphate group (Fig. 1). Hence, the FTIR results showed the presence of phenolic compound in extract of A. aspera.

LC 50 of Achyrathes aspera extract in-combination with microbes against
Random amplified polymerase DNA polymerase chain reaction (RAPD-PCR) analysis. The extracted DNA from larvae of Cx. quiquefasciatus (treated with Achyrathes aspera and its combination with Bti) was amplified using RAPD-PCR. The banding profile showed that no DNA damage had occurred due to application of A. aspera individually and in combination with Bti compared to control (Fig. 2).
Comet assay. The comet assay was performed to detect the DNA damage in cells of treated larvae of Cx. quiquefasciatus with significant plant extract A. aspera and its combination with Bti. It was found that no DNA damage had occurred in treatment group compared to control (Fig. 3).

Discussions
During the present study, weed plant extracts were exploited for their insecticidal potential in Cx. quiquefasciatus. Five different weed plants extracts C. arvensis, C. murale, T. terrestris, T. portulacastrum and A. aspera were employed against larvae of Cx. quiquefasciatus. The extracts of three weed plants viz. A. aspera, T. portulacastrum and C. arvensis showed significant results as compare to synthetic pesticide Permethrin, and in comparison with A. indica (neem) extract all plants showed high mortality. A. indica extract had already been reported by Sagheer et al. 14 which showed 16.11% mortality at 15% concentration. Recently, Sultana 49 and Sultana et al. 15 described that weed plant extracts have more insecticidal activity as compared to A. indica extracts. Subsequently, A. aspera revealed LC 50 value 9.22 ppm with 100% mortality which is lower than that of Clausena dentate (LC 50 28.60 ppm) against Cx. quinquefasciatus larvae as described by Sakthivadivel et al. 50 . Similarly, it was also revealed that A. aspera had more entomocidal potential when compared the LC 50 89.03 ppm of Croton rhamnifolioides as reported by Santos et al. 51 .
In current study, all the plant extracts showed high mortality at 72 hours compared to 24 and 48 hours. And no LC 50 was found more than 250 ppm (LC 50 < 250 ppm) at 72 hours exposure time. Thus, the insecticidal activity of weed plant extracts is time dependent 15 . It was thus found that the mortality had increased with increased concentrations of weed plant extracts. Hence, the percent mortality and toxicity data is in accordance to the previous findings of Odeyemi and Ashamo 52 , Sultana et al. 15 and Sagheer et al. 14   www.nature.com/scientificreports www.nature.com/scientificreports/ (LC 50 = 1189.30 ppm) and Citrullus vulgaris (LC50 = 1636.04 ppm) and found higher LC 50 than 1000 ppm (maximum concentration) 53 .
Plant extracts have also been used in combination with certain microbes. Kumar et al. 54 described that B. thuringiensis in combination with Solanum xanthocarpum showed higher larval mortality 54 . Recently, B. thuringiensis was reported as very effective causing high mortality when used in combination with weed plant extracts 49 . In addition, Kupferschmied et al. 28 extensively reviewed the root-associated Pseudomonas with their insecticidal activities against various insect pests. Consistently, the current findings showed that A. aspera in combination with B. thuringiensis had caused 100% mortality against larvae of Cx. quinquefasciatus.
Phytochemical analysis of the weed plant extracts used in present study indicated the presence of flavonoids. It has been reported by Gautam et al. 55 that flavonoid extracted from aerial parts of Androgrpahis paniculata was inactive at 600 ppm against Ae. aegypti larvae, but it caused 70% mortality against An. stephensi at 200 ppm concentration. However, flavonoid extracted from flower-buds showed 100% mortality for Ae. aegypti and An. stephensi at 600 & 200 ppm concentration, respectively. A. aspera weed plant contains flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids. These compounds are also present in other tested plants, except steroids and cardiac glycosides which were found absent in C. arvensis and T. terrestris. Similarly, anthrequinones and terpenoids were also found absent in C. muale. Low mortality was shown by C. muale in the current study which might be due to the absence of anthrequinones and terpenoids, and absence of steroids in case of T. terrestris.    Table 6. Percent inhibition of enzyme activity in Culex quinquefasciatus larvae using different concentrations of Bti at 30% concentrations. Convulvulus arvensis (Z1), Chenopodium murale (Z2), Tribulus terrestris (Z3), Trianthema portulacastrum (Z4) and Achyranthes aspera (Z5). AChE = acetylcholinesterase, AcP = acid phosphatases, AkP = alkaline phosphatases, α-Carboxyl = α-Carboxylesterases and β-Carboxyl = β-Carboxylesterases. Means sharing the same letter within each treatment is not statistically different.
Subsequent studies by Cárdenas-Ortega et al. 20 reported 37 different compounds with β-caryophyllene and caryophyllene oxide as main components in Salvia ballotiflora resulted in 80% larval mortality. Evans 56 reported that alkaloids cause the death of treated organisms due to their ability to bind DNA of organisms and affecting the replication process and synthesis of molecules. Alkaloids compound were identified in all of our used weed plants extracts but no DNA damage was observed in the treated larvae of Cx. quinquefasciatus mosquiotes. The enzymatic profiles are also modulated in response to natural oils from plants 25 . For instance, Esterases, a major detoxifying enzyme in insects and have been reported to involve in detoxification of insecticides 26 . Plant extracts have been reported as AChE inhibitors 27 . The death in insects due to treatment with plant extracts suggested that the molecules present therein possibly interfere at the cholinergic synapse and destroyed the communication network from one exonic end to another; thereby, blocking the nerve impulse transmission. Thus, the lethal effect may also be due to the accumulation of acetylcholine (ACh), a neurotransmitter, at synaptic junctions, which interrupts the coordination between the nervous and muscular junctions (neurotoxicity) 27 . Subsequent changes in enzyme activity are also reported for Phosphatases in insects. The hydrolysis of acid phosphatase (ACP) and alkaline phosphatase (ALP) phosphomonoesters under acid or alkaline conditions, respectively 25 . Alkaline phosphatase (ALP) is used as a membrane marker enzyme, active in intestinal epithelial cells, malpighian tubules and hemolymph of insects 25,57 . A decrease in ACP levels due to plant extract could be attributed to reduced phosphorous liberation for energy metabolism, decreased rate of metabolism as well as decreased rate of transport of metabolites 25,26 . The enzyme activity of AChE, AcP, AkP, α-Carboxyl and β-Carboxyl was inhibited with increase of concentration of all the plant extracts which is also in agreement to Santos et al. 51 who also reported that essential oil of Croton rhamnifolioides showed the inhibitory effect on a digestive enzyme (Trypsin) from larvae of Ae. aegypti.
The genotoxicity and carcinogenicity in the cell genome are caused by genotoxic agents having some lethal or sub-lethal effects which are induced by some xenobiotic substances 22,26 .
Thus, the DNA damage due to exposure of an organism to plant extracts 21,22 may result from the formation of covalently bound adducts between metabolites and DNA; and the faulty repair of these adducts often results in  www.nature.com/scientificreports www.nature.com/scientificreports/ mutations and sometimes cytogenetic changes. Recently A. aspera was found genotoxic against Ae. aegypti with significant changes in the RAPD profiles. These changes suggested that certain phytocomponents in A. aspera caused the probable DNA damage and mutations in the larval g-DNA which could be the possible reason of larval  www.nature.com/scientificreports www.nature.com/scientificreports/ mortality 24 . In contrast, no DNA damage was found in the current findings. Moreover, FTIR analysis of A. aspera indicated the presence of phytochemicals composed of hydrogen bonded -OH functional group. Mostly phenolic phytochemicals such as tannins and flavonoids are composed of -OH functional group 58 . FTIR spectrum of C. arvensis showed the presence of alkaloids. These all compounds are reported as toxic to insects and produced insecticidal activities. It has already been reported that phenolic compounds can be potentially used for the control of insect pests of various crops 59 .
Hence, it is suggested that the mortality in larvae of Cx. quinquefasciatus cannot be attributed due to genotoxicity. Rather, perhaps it is caused by the presence of certain phenolic phytochemicals such as flavonoids which modulate the enzymatic activity and thus, cause the death of larvae of Cx. quinquefasciatus. Thus, it is suggested that A. aspera weed plant can be further exploited for extraction and purification of phenolic compounds to use against mosquitoes. In addition, the current study which is the first one performed in Pakistan using weed plants extracts; also suggests that weed plants can be explored for their insecticidal activity against other insect pests.

conclusions
The petroleum ether extracts of five weed plants were used against the larvae of Cx. quinquefasciatus. A. aspera extract showed highest mortality. Thus, based on LC 50 values (p-values), Achyrathes aspera weed plant extract was used along with Bti and Pseudomonas bacteria for further trials. The highest mortality of C. quinquefasciatus was found using A. aspera with Bti. Enzyme inhibition activity of AChE, AcP, AkP, α-Carboxyl and β-Carboxyl was found in tested weed plant extracts. Phytochemical analysis showed the presence of flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids. Moreover, FTIR analysis showed that A. aspera contains phenolic compounds which have been reported to show insecticidal activity. Genotoxic activity was also observed using RAPD-PCR and comet assay. It was found that no DNA damage had been occurred due to either A. aspera extractor using the extract in combination with Bti. It is suggested that certain phenolic compounds such as flavonoids which modulate the enzymatic activity and, causes the death of larvae of Cx. quinquefasciatus. A. aspera plant is easily available in Pakistan and its extract could be very used to control Culex mosquitoes. In future, further studies are needed to extract and characterize the particular potential of phenolic compound found in A. aspera to use in mosquito control programs.