Introduction {#Sec1}
============

Mosquitoes are reported to cause nuisance to humans and transmit several viral and protozoan diseases of public health concern worldwide.  These are female mosquitoes which make a bite during their search for blood meal before oviposition which thus, increases their tendency to transmit several diseases including malaria, filariasis, dengue fever, japanese encephalitis, chikungunya, zika virus and yellow fever.  These diseases make life at risk of millions of people particularly in subtropical/tropical world^[@CR1],[@CR2]^. Of various mosquito species, *Cx.  quinquefasciatus* transmits various diseases i.e., West Nile virus, Japanese encephalitis, filariasis, bancroftian filariasis (*Wuchereria bancrofti*), St. Louis encephalitis, and avian malaria^[@CR3]^. In southern United States, St. Louis virus and West Nile virus (WNV) were transmitted by *Cx.  quinquefasciatus*^[@CR4],[@CR5]^. Almost 120 million people are affected annually only by lymphatic filariasis, whereas 1.3 billion are at risk resulting in nearly \$1.3 billion loss of productivity per year^[@CR6]^. Similarly, three billion individuals are at risk of being infected by Japanese encephalitis with 30,000--50,000 reported cases every year in disease endemic areas^[@CR7]^. Besides disease transmission in humans; *Cx.  quinquefasciatus* is also responsible for transmitting several diseases to livestock and companion animals viz.  Rift Valley fever, canine dirofilariasis (dog heartworm), avian malaria, avian pox, and West Nile encephalitis which lead to high mortalities or decreased productivity^[@CR8]^.

To avoid proliferation of mosquito borne diseases; mosquito control is necessary which is essentially performed through using chemical insecticides.  Many synthetic agents such as organochlorine and organophosphate compounds have been developed and employed with a considerable success despite of therein including toxicity to non-targeted organisms and fostered sometimes severe environmental and human health concerns^[@CR9]^. The continuous use of synthetic insecticide results in the development of resistance in mosquitoes.  Furthermore non-degradable nature of synthetic insecticide causes biomagnification which makes the situation overall more worst^[@CR10]--[@CR12]^. Collectively, the current status situation urges to find out environment friendly, cost-effective, biodegradable and target specific insecticides against mosquitoes.  Although, an eco-friendly alternative approach such as biological control became the central focus to exploiting certain natural enemies including predatory and parasitic species; however, mosquito control always remained a very serious issue. 

Plant extracts have been reported for the control of mosquitoes^[@CR13],[@CR14]^ and recently, weed plant extracts are being investigated^[@CR15]^. Nevertheless, the plant extracts are biodegradable, non-hazardous and have been found active against a number of insect pests^[@CR16]^. Previously, it was also focused on the commercial use of plant extracts as potent insect-control agents^[@CR17],[@CR18]^. Subsequently, plants derived secondary metabolites are responsible for defense to survive against selection pressure of herbivore predators and different environmental factors.  Numerous phytochemical groups like alkaloids, terpenoids, steroids, phenolics and essential oils extracted from various plants are reported as potent insecticides^[@CR16],[@CR19]^. For instance, *Salvia ballotiflora* contains 37 different compounds with β-caryophyllene and caryophyllene oxide as main components and resulted in 80% larval mortality of *Cx.  quinquefasciatus*^[@CR20]^. The phytochemical analysis showed the presence of aromadendrene, naphthalene, α-humulene, caryophyllene oxide, caryophyllene, phenol, 4-(3,7-dimethyl-3-ethenylocta-1,6- dienyl) and methyl hexadecanoate compounds in *Psoralea corylifolia* and caused DNA damage in *Cx.  quinquefasciatus*^[@CR21]^. Though, first report of genomic alterations using RAPD-PCR fingerprinting was reported by Lalrotluanga & Gurusubramanian^[@CR22]^ in mosquito larvae treated with various plant extracts.  Variations in DNA band were observed when *P. ferulacea* essential oil-treated larvae of *E. kuehniella* and were subjected to DNA damage analysis by RAPD assay^[@CR23]^. A significant DNA damage has been reported in *Aedes aegypti* due to *A. aspera*^[@CR24]^. Similarly, DNA damage was also shown in *Cx.  quinquefasciatus* larvae treated with *Curcuma longa* and *Melia azedarach* plant extracts^[@CR21]^.

It has also been described that the enzymatic profiles are modulated in response to natural oils from plants^[@CR25]^. Esterases, a major detoxifying enzyme in insects, are involved in detoxification of insecticides^[@CR26]^. Plant extracts are described as AChE inhibitors^[@CR10],[@CR27]^. Subsequent changes in Phosphatases enzyme activity are also reported in insects^[@CR25],[@CR26]^. Additionally, the discovery of highly toxic bacterial strains against dipteran larvae such as *Bacillus thuringiensis israelensis* (*Bti*) and *Bacillus sphaericus* Neide (*Bs*) provided an option to incorporate in mosquito control programs as a potent biolarvicides around the world^[@CR28]--[@CR30]^.

Weeds are undesired flora which compete with crop plants for food space and nutrients and provide the alternative food for pests.  In attempting to eradicate these weeds; why not this is appropriate to use them for insect control and to develop an environmentally safe insect-control agent in future^[@CR15]^. Therefore, the current study was designed to evaluate the entomocidal impact of different weed plant extracts individually and in combination with microbial strains along with the genotoxic effect of weed plant extracts against *Cx.  quinquefasciatus* mosquitoes. 

Materials and methods {#Sec2}
=====================

Collection and Rearing of *Culex quinquefasciatus* {#Sec3}
--------------------------------------------------

The adults and larvae of *Cx.  quinquefasciatus* were collected from main drain of Government College University, Faisalabad.  The mosquitoes were then reared in plastic and enamel tray with tap water under standard conditions (26 ± 1 °C, 60 ± 10% RH with 12 hours day/night cycle) in Lab, Department of Zoology in Government College University Faisalabad.  The newly emerged larvae were fed on grounded Fish Food and 5--8 days old larvae were fed on two tablets of Purina Cat Food daily.  The grounded Cat Food was added after 8 days.  Tray was kept in insect cage after the formation of pupae.  After 2 days, adults were emerged from the pupae.  A beaker having cotton soaked in 10% sugar solution was kept to provide sugar contents to adult mosquitoes.  For blood feeding to female *Cx.  quinquefasciatus* mosquitoes, an albino rat in a small cage was left overnight in the rearing cage^[@CR31]^.

Collection of microbes {#Sec4}
----------------------

The *Psuedomonas aeruginosa* isolate was procured from Department of Microbiology whereas *Bacillus thuringiensis israelensis* (*Bti*) was purchased from Summit^®^, USA. * P. aeruginosa* was isolated from burn wound samples using *Psuedomonas* agar (oxoid, UK).  The bacterial count was adjusted to 1 × 10^8^ colony forming unit/ml (CFU/ml).  This stock solution was used to prepare different ppm solutions.  Similarly, 100% solution was prepared by dissolving 10% dunk of *Bti* in 10 ml distilled water.  Then 1000 ppm stock solution was prepared by dissolving 10 µl of this *Bti* solution in 90 µl distilled water.  This stock solution was used for further dilutions. 

Collection and identification of weed plants {#Sec5}
--------------------------------------------

Weed plants were collected from different areas of District Faisalabad and identified from Department of Botany, Government College University Faisalabad and Department of Botany, University of Agriculture Faisalabad, Pakistan.  The used weed plants for extraction are listed in Table [1](#Tab1){ref-type="table"}.Table 1List of weed plants used for oil extraction. Sr \#Common nameScientific NameCollection AreaGPS Coordinates1Lilly*Convulvulus arvensis*Sammundri31.0691°N, 72.9361°E2Krund*Chenopodium murale*Sammundri31.0691°N, 72.9361°E3Bhakhra*Tribulus terrestris*Satyana31.2047°N, 73.1711°E4Itsit*Trianthema portulacastrum*Satyana31.2047°N, 73.1711°E5Puthkanda*Achyrnathes aspera*Tandlianwala31.0368°N, 73.1379°E

### Petroleum ether extraction of weed plants {#Sec6}

Weed plants were cleaned by washing with clean water and shade-dried in Lab for a week.  Dried plants were crushed in small pieces for further grinding.  Crushed dried plants were again oven dried at 60 °C for 20 minutes and grinded in electrical grinder to obtain powder form.  Extracts were obtained from 15 g of powder in 150 ml of petroleum ether of each weed plant using Soxhlet apparatus after several rotations in 8 hours.  The extracts were then stored in clean and air tight bottles at 4 °C^[@CR15]^.

Mortality bioassay test {#Sec7}
-----------------------

Different concentrations of each weed plant extract from 10 ppm to 250 ppm were used to perform the bioassay test following WHO protocol^[@CR32]^. 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^[@CR16]^. 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 {#Sec8}
---------------------------------------

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*.^[@CR33]^ and Sultana *et al*.^[@CR15]^ were followed. 

Phytochemical analysis of weeds extracts {#Sec9}
----------------------------------------

Phytochemical analysis of five tested weed plant extracts were performed in order to detect the chemical constituents as described by Harborne^[@CR34]^, Trease and Evans^[@CR35]^ and Sofowara^[@CR36]^.

Fourier transform infrared spectroscopy (FTIR) analysis {#Sec10}
-------------------------------------------------------

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^[@CR37]^.

DNA extraction {#Sec11}
--------------

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^[@CR38]--[@CR40]^.

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:$$\documentclass[12pt]{minimal}
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                \begin{document}$${\rm{DNA}}\,{\rm{Conc}}.\,\mu {\rm{g}}/\mu {\rm{l}}={\rm{Dilution}}\,{\rm{fold}}\times {\rm{absorbance}}\,{\rm{at}}\,260\,{\rm{nm}}$$\end{document}$$

RAPD-PCR amplification {#Sec12}
----------------------

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*.^[@CR39]^ and Zahoor *et al*.^[@CR41]^.

Haemocyte collection {#Sec13}
--------------------

*Cx.  quinquefasciatus* haemocytes were collected according to Irving *et al*.^[@CR42]^. 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 {#Sec14}
-----------

The comet assay was performed according to Singh *et al*.^[@CR43]^ 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^[@CR21],[@CR44],[@CR45]^.

### Data analysis {#Sec15}

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^[@CR46]^. 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^[@CR15]^. 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^[@CR14],[@CR47],[@CR48]^. PCR products were analyzed using gel electrophoresis and the genetic data were analyzed using POPGENE software^[@CR40]^. 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^[@CR21]^.

Results {#Sec16}
=======

Mortality assay of *culex quinquefasciatus* using weed plant extracts at various concentrations and different time intervals {#Sec17}
----------------------------------------------------------------------------------------------------------------------------

The comparison of insecticidal activity of five different weed plant extracts against larvae of *Cx.  quinquefasciatus* using different concentrations at various exposure intervals is shown in Table [2](#Tab2){ref-type="table"}.  High mortality was obtained by all the weed plant extracts after 72 hours exposure time compared to *Azadirachta indica* (Neem) extract at 250 ppm concentration.  But as compare to synthetic pesticide (Permethrin) only *T. terrestris* and *C. murale* showed the low mortality among all weed plants extracts.  The highest mean mortality was shown by *Achyrathes aspera* (42.76, 61.28, 66.93, 78.11, 88.22, 93.43 and 100%) followed by *C. arvensis* (34.34, 42.76, 44.67, 49.50, 62.96, 71.85 and 88.22%) and *T. portulacastrum* (32.66, 44.44, 49.86, 62.96, 74.75, 79.06% and 88.22%) at 10, 20, 30, 50, 100, 150 and 250 ppm concentrations, respectively. * T. terrestris* showed low mean mortality (15.82, 29.29, 31.78, 34.34, 52.86, 59.32 and 73.06%) whereas, lowest mortality among all the five extracts was observed with *C. murale* (15.82, 20.88, 22.68, 27.61, 42.76, 49.82 and 64.65%) at 10, 20, 30, 50, 100, 150 and 250 ppm concentrations, respectively.  Permethrin *s*howed (13.51, 18.01, 39.28, 43.36, 51.95, 67.14 and 76.46%) mortality at the same concentrations, respectively. * A. indica* (Neem) extract showed lowest mortality when compared to all the tested plants and Permethrin (Table [2](#Tab2){ref-type="table"}).  The overall results indicated that mortality was increased with increased extract concentrations. Table 2Mean mortality of *Culex quinquefasciatus* by different weed extracts at various concentration and different time intervals. TreatmentConc. F\
ValuedfP\
valueMean mortality with different time intervals24 hours48 hours72 hoursZ110 ppm41.912\<0.056.67 ± 1.67c16.67 ± 1.67b34.34 ± 2.92a20 ppm88.232\<0.0511.67 ± 1.67c23.33 ± 1.67b42.76 ± 1.68a30 ppm68.492\<0.0515.68 ± 1.68c28.34 ± 1.68b44.67 ± 1.67a50 ppm59.052\<0.0520.00 ± 2.89c36.67 ± 1.67b49.50 ± 0.00a100 ppm78.872\<0.0525.00 ± 2.89c48.33 ± 1.67b62.96 ± 1.68a150 ppm92.582\<0.0529.88 ± 2.88c55.67 ± 1.67b71.85 ± 1.68a250 ppm134.032\<0.0538.33 ± 1.67c65.00 ± 2.89b88.22 ± 1.68a10 ppm14.132\<0.053.33 ± 1.67b8.33 ± 1.67b15.82 ± 1.68a20 ppm8.612\<0.0511.67 ± 1.67b13.33 ± 1.67b20.88 ± 1.68a30 ppm7.792\<0.0512.38 ± 1.67b15.67 ± 1.67b22.68 ± 1.68aZ250 ppm27.272\<0.0513.33 ± 1.67c20.00 ± 0.00b27.61 ± 1.68a100 ppm61.982\<0.0516.67 ± 1.67c26.67 ± 1.67b42.76 ± 1.68a150 ppm89.462\<0.0517.58 ± 1.67c29.78 ± 1.67b49.82 ± 1.68a250 ppm146.342\<0.0518.33 ± 1.67c35.00 ± 0.00b64.65 ± 2.92a10 ppm7.522\<0.056.67 ± 1.67b11.67 ± 1.67ab15.82 ± 1.68a20 ppm13.882\<0.0513.33 ± 1.67b23.33 ± 1.67a29.29 ± 2.92a30 ppm28.952\<0.0514.86 ± 1.67b24.33 ± 1.67a31.78 ± 1.68aZ350 ppm34.622\<0.0518.33 ± 1.67c26.67 ± 1.67b34.34 ± 0.00a100 ppm116.092\<0.0518.33 ± 1.67c26.67 ± 1.67b52.86 ± 1.68a150 ppm189.232\<0.0520.67 ± 1.67c28.33 ± 1.67b59.32 ± 1.68a250 ppm247.442\<0.0523.33 ± 1.67c33.33 ± 1.67b73.06 ± 1.68a10 ppm39.432\<0.0511.67 ± 1.67c21.67 ± 1.67b32.66 ± 1.68a20 ppm93.722\<0.0518.33 ± 1.67c28.33 ± 1.67b44.44 ± 0.00a30 ppm67.432\<0.0522.88 ± 1.67c32.67 ± 1.67b49.86 ± 1.68aZ450 ppm89.452\<0.0531.67 ± 1.67c43.33 ± 1.67b62.96 ± 1.68a100 ppm42.372\<0.0541.67 ± 1.67c55.00 ± 2.89b74.75 ± 2.92a150 ppm89.752\<0.0543.33 ± 1.67c59.67 ± 1.67b79.06 ± 1.68a250 ppm109.472\<0.0553.33 ± 1.67c68.33 ± 1.67b88.22 ± 1.68a10 ppm53.492\<0.0518.33 ± 1.67c31.67 ± 1.67b42.76 ± 1.68a20 ppm39.002\<0.0531.67 ± 1.67c43.33 ± 1.67b61.28 ± 1.68a30 ppm63.062\<0.0534.67 ± 1.67c57.78 ± 1.67b66.93 ± 1.68aZ550 ppm108.822\<0.0543.33 ± 1.67c58.33 ± 1.67b78.11 ± 1.68a100 ppm35.342\<0.0568.33 ± 1.67c78.33 ± 1.67b88.22 ± 1.68a150 ppm83.492\<0.0572.86 ± 1.82c82.96 ± 1.87b93.43 ± 1.68a250 ppm45.502\<0.0581.67 ± 1.67c91.67 ± 1.67b100.00 ± 0.00a10 ppm12.432\<0.052.34 ± 1.67b3.02 ± 1.67a3.89 ± 1.67a20 ppm16.022\<0.052.89 ± 1.67b3.98 ± 1.67a4.25 ± 1.67a*Azadirachta indica*30 ppm17.872\<0.053.56 ± 1.67b4.08 ± 1.67a4.78 ± 1.67a50 ppm8.982\<0.056.06 ± 1.67b8.21 ± 1.67a9.32 ± 1.68a100 ppm44.212\<0.058.23 ± 1.67b8.95 ± 1.67b11.63 ± 1.67a150 ppm62.80.2\<0.059.78 ± 1.67b10.67 ± 1.67b13.84 ± 1.68a250 ppm78.272\<0.0512.36 ± 1.67c14.01 ± 1.67b16.17 ± 1.68aPermethrin10 ppm7.562\<0.052.32 ± 1.78c5.67 ± 1.29b13.51 ± 1.57a20 ppm16.672\<0.053.85 ± 1.25c8.92 ± 2.18b18.01 ± 1.92a30 ppm23.022\<0.059.43 ± 3.62c22.46 ± 2.57b39.28 ± 1.98a50 ppm28.772\<0.0512 ± 2.48c29.84 ± 1.89b43.36 ± 0.2.16a100 ppm37.532\<0.0519.27 ± 2.38c35.17 ± 2.61b51.95 ± 2.58a150 ppm127.052\<0.0523.63 ± 1.58c41.21 ± 1.68b67.14 ± 2.18a250 ppm273.382\<0.0529.32 ± 1.68c52.89 ± 1.49b76.46 ± 2.47a^\*^*Convulvulus arvensis* (Z1), *Chenopodium murale* (Z2), *Tribulus terrestris* (Z3), *Trianthema portulacastrum* (Z4), *Achyranthes aspera* (Z5), *Azadirachta indica* (Neem) and Permethrin. 

LC~50~ of weed plant extracts against *Culex quinquefasciatus* mosquitoes {#Sec18}
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*A. aspera* showed lowest LC~50~ (87, 39 and 9 ppm; p \< 0.05) at 24, 48 and 72 hours, Similarly, low LC~50~ (205, 123.65 and 34.64; p \< 0.05) was found with *T. portulacas* at 24, 48 and 72 hours, respectively; followed by *C. arvensis* (LC~50~ 304, 151, and 57; p \< 0.05). * C. murale* showed highest LC~50~ (\>250 ppm) at 24, 48 hours among all five extracts.  High LC~50~ was observed with *T. terrestris* (\>250 ppm) at 24, 48 hours, while it showed low LC~50~ (126 ppm; p \< 0.05) but higher than LC~50~ (98 ppm; p \< 0.05) of Permethrin at 72 hours.  The LC~50~ of Permethrin was LC~50~ \> 250 ppm and 247 ppm at 24 and 48 hours, respectively.  Overall, it was found that all the weeds extracts showed significant results at 72 hours (p \< 0.05) as compare to *A. indica* (Neem) extract, which showed LC~50~ (\>\>250 ppm) after all exposure time (p \> 0.05; Table [3](#Tab3){ref-type="table"}).Table 3Toxicity of weed plant extracts against *Culex quinquefasciatus*.TreatmentObservationNLC50 (ppm)\
(Upper + lower values)Slope ± SEX2 ± dfSEPZ124100LC50 \> 250 ppm0.0039830 ± 0.00069825.296516.820.0048100151.0515 (125.8312 ± 185.6088)0.0049704 ± 0.00067378.383514.520.007210057.1057 (37.0145 ± 75.4342)0.0063345 ± 0.00077911.31959.500.00Z224100LC50 \>\> 250 ppm0.0018983 ± 0.00077776.1745246.230.3248100LC50 \> 250 ppm0.0018983 ± 0.00077776.174553.400.1072100164.9892 (140.0672 ± 199.3816)0.0053894 ± 0.00067893.451514.470.05Z324100LC50 \>\> 250 ppm0.0020102 ± 0.00073824.3555184.870.2248100LC50 \>\> 250 ppm0.0019029 ± 0.00068026.0975133.470.1072100126.5443 (106.1374 ± 151.5917)0.0058605 ± 0.00069528.582511.220.03Z424100205.0356 (169.5248 ± 262.854)0.0043157 ± 0.000667812.09522.030.0348100123.6593 (99.0462 154.7968)0.0046799 ± 0.0006749.695513.550.007210034.6413 (13.5864 ± 51.9514)0.0069505 ± 0.000846611.7459.450.00Z52410087.4652 (70.6095 ± 105.3030)0.0069648 ± 0.000752317.6058.640.014810039.0875 (21.3783 ± 54.3348)0.0078991 ± 0.000907310.6358.16870.00721009.2235 (6.3036 ± 19.7265)0.014552 ± 0.0021145.60856.24590.0024100616.028(429.372 ± 1279.82)0.0029602 ± 0.00085612.5775147.950.75*A. indica* Neem48100600.278(420.494 ± 1220.25)0.0028745 ± 0.00081602.547135141.5350.7672100546.163(394.025 ± 1005.21)0.0029899 ± 0.00077713.863395116.0250.56Permethrin24100LC50 \> 250 ppm0.03432162 ± 0.006576214.2512615.72620.0348100247.0248 (179.6136 ± 298.7321)0.0042325 ± 0.000716978.1245613.230.027210098.2859 (86.7189 ± 145.4137)0.0068465 ± 0.000824185.8267.490.01^\*^*Convulvulus arvensis* (Z1), *Chenopodium murale* (Z2), *Tribulus terrestris* (Z3), *Trianthema portulacastrum* (Z4), *Achyranthes aspera* (Z5), *Azadirachta indica* (Neem) and Permethrin. 

Mortality Assay of *Culex quinquefasciatus* mosquitoes using *Achyrathes aspera* in-combination with microbes at various concentration and different time intervals {#Sec19}
-------------------------------------------------------------------------------------------------------------------------------------------------------------------

*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](#Tab4){ref-type="table"}.  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](#Tab4){ref-type="table"}).Table 4Mean mortality of *Culex quinquefasciatus* using *Achyranthes aspera* with microbes at various concentration and different time intervals. TreatmentConc. F\
ValuedfP\
valueMean mortality with different time intervals24 hours48 hours72 hours10 ppm27.812\<0.0512.46 ± 1.68c20.07 ± 1.70b30.27 ± 1.70a20 ppm16.902\<0.0522.56 ± 1.68c33.67 ± 2.95b43.88 ± 2.95a30 ppm30.672\<0.0524.86 ± 1.68c35.48 ± 1.70b45.63 ± 1.70a*Bti*50 ppm39.902\<0.0530.98 ± 1.68c42.18 ± 1.70b52.38 ± 1.70a100 ppm35.342\<0.0542.76 ± 1.68c50.68 ± 1.70b59.18 ± 0.00a150 ppm31.612\<0.0545.24 ± 1.68c53.67 ± 1.70b63.58 ± 1.70a250 ppm34.512\<0.0551.18 ± 1.68c60.88 ± 1.70b71.09 ± 1.70a*Bti* + Z510 ppm77.642\<0.0517.51 ± 1.68c30.27 ± 1.70b47.28 ± 1.70a20 ppm46.872\<0.0530.98 ± 1.68c43.88 ± 2.95b60.88 ± 1.70a30 ppm43.582\<0.0535.28 ± 1.68c48.33 ± 2.57b65.45 ± 1.70a50 ppm39.332\<0.0542.76 ± 1.68c55.78 ± 3.40b72.79 ± 1.70a100 ppm54.702\<0.0554.55 ± 2.92c69.39 ± 2.95b89.80 ± 0.00a150 ppm98.192\<0.0557.72 ± 1.68c74.68 ± 1.70b92.53 ± 1.70a250 ppm183.902\<0.0562.96 ± 1.68c86.39 ± 1.70b100.00 ± 0.00a*Pseudomonas*10 ppm7.252\<0.051.35 ± 1.35b4.76 ± 1.70ab9.86 ± 1.70a20 ppm41.062\<0.054.04 ± 0.00c11.56 ± 1.70b21.77 ± 1.70a30 ppm78.542\<0.054.89 ± 0.00c11.93 ± 1.70b23.91 ± 1.70a50 ppm52.232\<0.057.41 ± 1.68c13.27 ± 0.00b26.87 ± 1.70a100 ppm41.512\<0.0514.14 ± 0.00c21.77 ± 1.70b31.97 ± 1.70a150 ppm63.462\<0.0517.26 ± 1.68c23.45 ± 1.70b36.62 ± 1.70a250 ppm83.412\<0.0522.56 ± 1.68c30.27 ± 1.70b52.38 ± 1.70a*Pseudomonas* + *Bti*10 ppm15.502\<0.054.04 ± 0.00c9.86 ± 1.70b14.97 ± 1.70a20 ppm55.102\<0.057.41 ± 1.68c14.97 ± 1.70b31.97 ± 1.70a30 ppm86.982\<0.059.04 ± 1.68c16.63 ± 1.70b33.47 ± 1.70a50 ppm46.232\<0.0512.46 ± 1.68c21.77 ± 1.70b35.37 ± 1.70a100 ppm33.512\<0.0522.56 ± 1.68c31.97 ± 1.70b42.18 ± 1.70a150 ppm98.342\<0.0526.77 ± 1.68c36.52 ± 1.70b49.32 ± 1.70a250 ppm150.662\<0.0532.66 ± 1.68c43.88 ± 0.00b65.99 ± 1.70a*Pseudomonas* + Z510 ppm39.252\<0.052.69 ± 1.35c9.86 ± 1.70b18.37 ± 0.00a20 ppm46.042\<0.057.41 ± 1.68c16.67 ± 1.70b30.27 ± 1.70a30 ppm28.922\<0.0510.63 ± 1.68c21.34 ± 1.70b36.82 ± 1.70a50 ppm36.172\<0.0515.82 ± 1.68c28.57 ± 2.95b42.18 ± 1.70a100 ppm52.502\<0.0527.61 ± 1.68c40.48 ± 1.70b59.18 ± 2.95a150 ppm47.482\<0.0531.23 ± 1.68c46.59 ± 1.70b64.34 ± 2.95a250 ppm58.842\<0.0536.03 ± 1.68c52.38 ± 1.70b72.79 ± 3.40a^\*^*Achyranthes aspera* (Z5), *Psuedomonas*, *Bacillus thuringiens isisraeliensis* (*Bti*). 

LC~50~ of *Achyrathes aspera* extract in-combination with microbes against *Culex quinquefasciatus* mosquitoes {#Sec20}
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The combination of *A. aspera* and *Bti* showed low LC~50~ (137, 49 and 8 ppm; p \< 0.05) at 24, 48 and 72 h, respectively.  However, the combination of *Pseudomonas* and *A. aspera* showed very high LC~50~ (\>250 ppm) at 24 h and, moderate to high LC~50~ (213 and 111 ppm; p \> 0.05) was found at 48 and 72 h, respectively.  For control treatment, the LC~50~ of *Bti* (213.45, 145.80 and 76.12 ppm; p \< 0.05) was found lower than *Pseudomonas* (\>250 ppm) at 24, 48 and 72 h, respectively.  The LC~50~ of combination of *Pseudomonas* and *Bti* is shown in Table [5](#Tab5){ref-type="table"}.Table 5Toxicity of *Achyranthes aspera* extract in combination with microbes against *Culex quinquefasciatus*.TreatmentObservationNLC50 (ppm)\
(Upper ± lower values)Slope ± SEX2 ± dfSEP value*Bti*24100213.4507 (172.9759 ± 284.6938)0.0038483 ± 0.000662911.840325.7890.0048100145.8052 (113.6218 ± 194.4482)0.0036579 ± 0.000657510.203318.8930.017210076.1231 (42.6566 ± 107.7533)0.0036931 ± 0.00067237.694315.5070.05*Bti* ± Z524100137.0498 (109.2724 ± 175.2752)0.0042117 ± 0.000664416.361315.8100.004810049.6853 (27.9094 ± 68.5992)0.0061320 ± 0.00077238.902310.0340.00721008.2924 (−7.7614 ± 18.9967)0.014400 ± 0.0021291.47536.40150.00*Pseudomonas*24100LC50 \> 250 ppm0.0044863 ± 0.00082785.935353.8150.1148100LC50 \> 250 ppm0.0034591 ± 0.00072675.432361.3880.1472100226.9449 (187.1139 ± 294.4419)0.0042459 ± 0.00067076.336325.1550.09*Pseudomonas* + *Bti*24100LC50 \> 250 ppm0.0044517 ± 0.00073797.302339.2600.0648100LC50 \> 250 ppm0.0039967 ± 0.00068065.565332.6510.1372100153.6023 (127.0986 ± 190.8331)0.0047483 ± 0.00067117.794315.3990.05*Pseudomonas* + Z524100LC50 \> 250 ppm0.0047247 ± 0.000725816.513332.2420.0048100213.0870 (177.4763 ± 270.5488)0.0044985 ± 0.000672211.693322.0670.0372100111.5831 (90.7470 ± 135.9624)0.0055370 ± 0.000693813.540311.1470.68^\*^*Achyranthes aspera* (Z5), *Psuedomonas*, *Bacillus thuringiens isisraeliensis* (*Bti*). 

Enzyme inhibitory effect of weed plant extracts in *Culex quinquefasciatus* mosquitoes {#Sec21}
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Maximum inhibition activity of AChE, AcP, AkP, α-Carboxyl and β-Carboxyl was found for *A. aspera* as 54.68, 34.28, 24.58, 71.08 and 59.96 at 250 ppm, respectively.  The mean value for inhibition activity for aforementioned mentioned enzymes by *C. arvensis* was found 52.94, 32.02, 21.01, 63.88 and 46.91%, respectively; followed by *C. murale* as 59.95, 29.98, 26.08, 45.04 and 50.01% at 250 ppm concentration.  Similalry, *T. terrestris* showed inhibition of AChE, AcP, AkP, α-Carboxyl and β-Carboxyl as 48.78, 28.16, 25.02, 47.13 and 47.04, respectively; likewise, *T. portulacastrum* showed as 51.14, 29.08, 24.98, 47.68 and 49.69, respectively at 250 ppm.  Overall, the enzyme assay showed that the inhibition of enzyme activity increased with increase of concentration (Table [6](#Tab6){ref-type="table"}).Table 6Percent inhibition of enzyme activity in *Culex quinquefasciatus* larvae using different concentrations of *Bti* at 30% concentrations. PlantsConcentrationAChEAcPAkPα-Carboxylβ-Carboxyl**Z1**50 ppm14.85 ± 2.83 a14.90 ± 1.75 a8.35 ± 1.08 a30.01 ± 2.02 a26.65 ± 2.23 a100 ppm31.14 ± 3.36 b21.07 ± 1.43 b12.95 ± 0.78 a52.80 ± 3.01 b43.87 ± 3.34 b250 ppm52.94 ± 2.43 c32.02 ± 1.14 c21.01 ± 1.57 b63.88 ± 1.67 c46.91 ± 3.08 bF, df and P value(F = 92.99; df = 2; P \< 0.05)(F = 32.03; df = 2; P \< 0.05)(F = 24.56 df = 2; P \< 0.05)(F = 78.92; df = 2; P \< 0.05)(F = 23.02; df = 2; P \< 0.05)**Z2**50 ppm17.83 ± 2.3 a15.77 ± 1.43 a8.87 ± 1.16 a24.08 ± 2.13 a26.13 ± 3.38 a100 ppm31.12 ± 3.32 b20.97 ± 1.03 b12.91 ± 0.93 b33.10 ± 4.11 a39.78 ± 3.03 b250 ppm59.95 ± 2.47 c29.98 ± 1.23 c26.08 ± 0.69 c45.04 ± 3.513b51.01 ± 2.57 bF, d.f and P value(F = 90.86; df = 2; P \< 0.05)(F = 38.79; df = 2; P \< 0.05)(F = 128.87; df = 2; P \< 0.05)(F = 13.05; df = 2; P \< 0.05)(F = 14.03; df = 2; P \< 0.05)**Z3**50 ppm11.93 ± 2.69 a19.61 ± 1.38 a9.49 ± 1.19 a22.93 ± 2.82 a16.18 ± 3.74 a100 ppm24.03 ± 2.59 b21.83 ± 1.68 a16.09 ± 1.39 b36.15 ± 3.08 b36.24 ± 3.49 b250 ppm48.78 ± 1.82 c28.16 ± 1.24 b25.02 ± 1.29 c47.13 ± 3.39 c47.04 ± 2.52 bF, d.f and P value(F = 59.57; df = 2; P \< 0.05)(F = 12.89; df = 2; P \< 0.05)(F = 39.94; df = 2; P \< 0.05)(F = 23.29; df = 2; P \< 0.05)(F = 23.31; df = 2; P \< 0.05)**Z4**50 ppm20.67 ± 2.87 a19.96 ± 1.49 a11.03 ± 1.49 a28.49 ± 2.59 a22.03 ± 4.87 a100 ppm27.79 ± 2.39 a23.21 ± 1.54 b13.48 ± 1.58 a45.08 ± 2.78 b35.03 ± 3.61 ab250 ppm51.14 ± 2.78 b29.08 ± 1.29 c24.98 ± 1.38 b47.68 ± 3.71 b49.69 ± 3.89 bF, d.f and P value(F = 45.89; df = 2; P \< 0.05)(F = 42.39; df = 2; P \< 0.05)(F = 33.54; df = 2; P \< 0.05)(F = 12.89; df = 2; P \< 0.05)(F = 11.23; df = 2; P \< 0.05)**Z5**50 ppm20.69 ± 2.03 a18.07 ± 1.08 a10.17 ± 0.74 a37.93 ± 2.11 a31.01 ± 2.93 a100 ppm38.76 ± 3.06 b23.29 ± 1.39 b15.34 ± 0.74 b58.17 ± 2.89 b48.16 ± 3.04 b250 ppm54.68 ± 1.35 c34.28 ± 1.13 c24.58 ± 1.14 c71.08 ± 3.03 c59.96 ± 1.68 cF, d.f and P value(F = 59.24; df = 2; P \< 0.05)(F = 47.87; df = 2; P \< 0.05)(F = 51.02; df = 2; P \< 0.05)(F = 52.23; df = 2; P \< 0.05)(F = 31.07; df = 2; P \< 0.05)*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. 

Phytochemical constituents in weed extracts {#Sec22}
-------------------------------------------

Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids in all the five weeds extracts used against *Cx.  quiquefasciatus* larvae (Table [7](#Tab7){ref-type="table"}).Table 7The chemical constituents present in five weeds extract. Sr.  No. Code\#Weed PlantsChemical ConstituentsFlSaTnStCGAlAnthTer1Z1*C. arvensis*+++−++++2Z2*C.muale*++++++−−3Z3*T. terrestris*++++−+++4Z4*T. portulacastrum*++++++++5Z5*A.aspera*++++++++^\*^*Convulvulus arvensis* (Z1), *Chenopodium murale* (Z2), *Tribulus terrestris* (Z3), *Trianthema portulacastrum* (Z4) and *Achyranthes aspera* (Z5).^\*^Fl = flavonoids, Sa = saponins, Tn= tannins, St = steroids, CG = Cardiac glycosides, Al= alkaloids, Anth= anthrequinones and Ter = terpenoids. 

Fourier transform infrared spectroscopy (FTIR) analysis of *Achyranthes aspera* extract {#Sec23}
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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](#Fig1){ref-type="fig"}).  Hence, the FTIR results showed the presence of phenolic compound in extract of *A. aspera*.Figure 1Full FTIR spectrum of *Achyranthes aspera*.  The spectrum shows a range of 4000 to 400 cm^−1^ wave number (along X-axis) the function of percent (%) transmittance (along Y-axis).  The peaks observed at 669.12 cm^−1^ correspond to aromatic ring having phosphate group, 779.00 cm^−1^ out of plane CH bending, 1077.88 cm^−1^ C-O Stretching, 1317.85 cm^−1^ and 1377.38 cm^−1^ C=H group, 3008.66 cm^−1^ alkyl C-H group stretching and 3338.23 cm^−1^--OH group for phenols. 

Random amplified polymerase DNA polymerase chain reaction (RAPD-PCR) analysis {#Sec24}
-----------------------------------------------------------------------------

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](#Fig2){ref-type="fig"}).Figure 2Comparison of RAPD profile for genotoxicity using C-04 primer. ( 1) Control group (Non-treated *Cx.  quiquefasciatus* larvae). ( 2) Treated larvae with *A. aspera* extract. ( 3) Treated larvae with *Bti*. ( 4) The combination of *A. aspera* with *Bti*. 

Comet assay {#Sec25}
-----------

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](#Fig3){ref-type="fig"}).Figure 3Comparison of Comet assay profile for genotoxicity in larvae of *Culex quiquefasciatus*. (** A**) Control. (** B**) *A. aspera*. (** C**) *Bti*. (** D**) *A. aspera* + *Bti*. 

Discussions {#Sec26}
===========

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*.^[@CR14]^ which showed 16.11% mortality at 15% concentration.  Recently, Sultana^[@CR49]^ and Sultana *et al*.^[@CR15]^ 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*.^[@CR50]^. 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*.^[@CR51]^.

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^[@CR15]^. 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^[@CR52]^, Sultana *et al*.^[@CR15]^ and Sagheer *et al*.^[@CR14]^ that the plant extracts become more toxic with increased dose and exposure time.  Interestingly, few LC~50~ values in the current findings were extrapolated which are in line with study of Prabakar and Jebanesan^[@CR53]^ who reported LC~50~ of *Benincasa cerifera* (LC~50~ = 1189.30 ppm) and *Citrullus vulgaris* (LC50 = 1636.04 ppm) and found higher LC~50~ than 1000 ppm (maximum concentration)^[@CR53]^.

Plant extracts have also been used in combination with certain microbes.  Kumar *et al*.^[@CR54]^ described that *B. thuringiensis* in combination with *Solanum xanthocarpum* showed higher larval mortality^[@CR54]^. Recently, *B. thuringiensis* was reported as very effective causing high mortality when used in combination with weed plant extracts^[@CR49]^. In addition, Kupferschmied *et al*.^[@CR28]^ 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*.^[@CR55]^ 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*. 

Subsequent studies by Cárdenas-Ortega *et al*.^[@CR20]^ reported 37 different compounds with β-caryophyllene and caryophyllene oxide as main components in *Salvia ballotiflora* resulted in 80% larval mortality.  Evans^[@CR56]^ 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^[@CR25]^. For instance, Esterases, a major detoxifying enzyme in insects and have been reported to involve in detoxification of insecticides^[@CR26]^. Plant extracts have been reported as AChE inhibitors^[@CR27]^. 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)^[@CR27]^. 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^[@CR25]^. Alkaline phosphatase (ALP) is used as a membrane marker enzyme, active in intestinal epithelial cells, malpighian tubules and hemolymph of insects^[@CR25],[@CR57]^. 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^[@CR25],[@CR26]^. 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*.^[@CR51]^ 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^[@CR22],[@CR26]^.

Thus, the DNA damage due to exposure of an organism to plant extracts^[@CR21],[@CR22]^ may result from the formation of covalently bound adducts between metabolites and DNA; and the faulty repair of these adducts often results in 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 mortality^[@CR24]^. 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^[@CR58]^. 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^[@CR59]^.

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 {#Sec27}
===========

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. 

Supplementary information
=========================

 {#Sec28}

Supplementary Dataset . 

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Supplementary information
=========================

is available for this paper at 10.1038/s41598-020-63815-w.

The facilities provided by Department of Zoology, Government College University Faisalabad (GCUF) are highly acknowledged to conduct this research work.  The authors are also thankful to Department of Microbiology for kind gift of bacteria.  The authors declare that there is no conflict of interest. 

M.K.Z. designed and supervised the work.  M.Z. performed experiments.  H.N.M., A.R. and K.R. did collection, extraction and helped in chemical as well as data analyses.  A.A., M.A.Z. and F.J. gave their input during write up.  All authors read and approved the final version of the manuscript. 

The authors declare no competing interests.