Introduction

Culex quinquefasciatus mosquito is a medical and veterinary insect globally because it is widespread in the world as it affects the health of humans and animals through the mechanical transfer of many pathogens to humans and animals such as dengue. It has a role in the transmission of Wuchereria bancrofti nematodes that cause Filaria vector, which currently affects about 120 million people in different parts of the world Wrchermesingh and Mendis1980), encephalitis virus, West Nile virus (Rutledge, Clarke, Curtis, & Sackett, 2003) and Vires Zika (Al-Zaidy, Parisi, Abed, & Salim, 2019; Vorou, 2016). There have been many methods of insect control for a long time. (Shono & Scott, 2003). Perhaps the most important reason that many interested in demanding a return to the use of pesticides of plant origin is their desirable qualities such as rapid decomposition and toxicity to humans and animals is very low and unlike chemical pesticides characterized by slow decomposition and high toxicity of mammals (Fraenkel, 1969). Plants Datura innoxia Mill belong to Solanaceae family which is an important plant because it contains many active compounds Hyoscyamine and Hyoscine Due to the lack of studies on the effect of the above plant on some life aspects of C. quinquefasciatus mosquitoes and their use as an alternative to chemical pesticides, we conducted the present research.

Materials and Methods of Work

Collect and diagnose plant samples

Collected Samples of leaves D. innoxia plants in November 2018 from private nurseries in the Qadisiyah governorate belongs to the Solanaceae family. The leaves of the plants were taken above and cleaned from the dust and washed and dried naturally in the shade and at room temperature. The leaves were then grinded by an electric grinder and kept in the dark plastic containers until use.

Preparation of the permanent farm Cx. qinquefasciatus

Collected immature instar (eggs and larvae) from one of the places of drainage water was placed in plastic basins filled with chlorine-free water and added rats' diet. For the purpose of obtaining a pure permanent farm, the pupa was transported in a plastic cage with a tulle clip and plastic plates containing cotton saturated with a 10% sugar solution To feed the modern insect and to obtain eggs boats followed the method (Mehdi & Mohsen, 1989), The female mosquitoes were fed three days after dawning on the blood of their pigeons. Then the boats were transferred to the eggs of new water contain food larvae were followed up until the emergence of full and Taking into account replacement of water every three days

Preparation of plant extracts

Preparation of organic solvent extracts for leaves of D. innoxia

Three different polar organic solvents were selected to obtain the organic extracts hexane, ethyl acetate and ethyl alcohol. Use (Harborne, 1984) for Prepare of organic solvent extracts 10 g of leaves plant powder were taken and placed Soxholet Extractor for 24 hours per organic solvent at 40-45 ° C The extracted material was then concentrated using a rotary evaporator at a temperature of 40-45 C, then the sample was transferred to glass plates and placed in the electric oven and at a temperature of 45 C to evaporate the solvent. The sample was taken and stored in the refrigerator until use.

Preparation of organic solvent extract

For the purpose of determining the bio-effectiveness of organic solvent extracts, a stock solution was prepared. 1 g of dry extract was dissolved by 5ml of the solvent and the volume was completed to 100ml distilled water. From this solution, the concentrations were prepared, which were (2000,1500,1000,500,250,100) ppm.

Determination of the biological activity of Organic Solvent Extracts (Hexane, Ethyl Acetate, Ethyl Alcohol) for leaves of D. innoxia of in Non - Cumulative mortality of Immature instar of Cx. quinquefasciatus

Determination of biological activity against eggs

To find out the effect of organic solvent extracts (hexane, ethyl acetate, ethyl alcohol) for D.innoxia leaves, eggs were taken 24 hours old with five replicates per concentration and one egg boat was placed in a 300ml plastic container with 100ml of each of the concentrations ppm (100250, 500, 1000, 1500, 2000) for the three extracts and spray the eggs Container surface and in a quantity of 2ml per repeater by hand sprayer with the same concentration that was put in it and after hatching the eggs were calculated.

Determination of biological activity against larvae

40 larvae were taken for each repetition from each of the four immature larval instar. 5 plastics containers containing 100ml of each of the concentrations of the extracts mentioned in the previous paragraph. ( 0.5) g of rats diet were added to each containers. Where the instar were prepared, they were followed up until they alienation to the later instar, and the mortality were recorded at each concentration after 24 hours.

Determination of biological activity against pupa

Isolated pupa from the permanent insect farm by 40 larvae per repeater and placed in a 300ml container of 5 replicates and followed the same test method as in the previous paragraphs except for the addition of bush.

Determination of the biological activity of organic solvent extracts (hexane, ethyl acetate, ethyl alcohol) for leaves of D. innoxia in the Cumulative mortality of immature instar of Cx. quinquefasciatus

To determine the effect of plant organic solvent extracts on the cumulative mortality of immature instar of Cx. quinquefasciatus Eggs were treated with ppm (100, 500,250, 1000, 1500, 2000) from each extract separately by inserting them into each concentration and spraying them superficially with a hand sprayer in addition to the control treatment, except that the extract was not added in the control treatment. They were distributed as in the previous paragraphs of larval number, number of replicates and experiment conditions. The extract was replaced every five days to avoid rot and dead insects were removed daily from the treatments.

Statistical analysis

The results of the present study were analyzed statistically according to the method. The percentages of mortality were corrected according to the (Abbott, 1925). LC50 and LC90 were calculated using Probit analysis, and K-squared, P-value and regression equation for each extract or compound were determined

Results and Discussion

Effect of organic solvent extracts of leaves of D. innoxia on the non-cumulative mortality of immature instar of Cx quinquefasciatus The Effect in the mortality of eggs

The effect of the concentrations of organic solvent extracts (hexane, ethyl acetate, ethyl alcohol) of D. innoxia leaves on the mortality percentage of Cx. quinquefasciatus eggs is shown in Figure 1 Where we observe the most effective of the hexane extract over the extracts of ethyl acetate and ethyl alcohol as well as we note a direct relationship between the concentration of the extract and the percentage of non-cumulative mortality of eggs, where the proportion of mortality increases with the concentration of the extract as the concentration of ppm 2000 gave the highest mortality rate for all extracts using D. innoxia which reached (60, 55,48)%of (hexane, ethyl acetate, ethyl alcohol). Table 1 shows that LC50 for hexane extract of D. innoxia ppm (1495.8) and LC90 were in the concentrations of ppm (42281.2). Ethyl alcohol was LC50 ppm (2199.9) and LC90 was ppm (65392). Increased mortality in hexane extract indicates that the most effective compounds found in the leaves D. innoxia have been extracted with this solvent and the most effective compounds are non-polar and insoluble in water (Halify & Zubaidi, 1989).

In mortality percentage of eggs. Cx. quinquefasciatus

(Traboulsi et al., 2005) explained that the hexane extract of leaves Myrtus communis had an effect on the mortality of C.pipiens mosquito eggs where the lethal concentration of LC50 was 16 mg/ml and shown seed S.nigrum plant led to 100% mortality of An.stephensi eggs at 10 mg / mL and shown Almrmdh(2014). The hexane extract of R.sanctus was less efficient than that of ethyl acetate and ethyl alcohol extract in M. domestica eggs.

The effect in the mortality of Larval instar

The effect of the concentrations of organic solvent extracts (hexane, ethyl acetate, ethyl alcohol) on the leaves of D. innoxia on the percentage of larval mortality of Cx. quinquefasciatus is shown in Figure 2. We note the hexane extract was the most most effective from ethyl acetate and ethyl alcohol as well as a direct correlation between the concentration of the extract and the ratio of non-cumulative mortality as the percentage of mortality increases with the concentration of the extract as the concentration of ppm 2000 gave the highest rate mortality for all extracts. The values ​​of hexane extract were reached (90, 82, 72, 62 )%The ethyl acetate extract was (80 70, 65 and 60)%. In ethyl alcohol extract, the percentage of larval instar was ( 77, 68, 60 and 55)%. From Table 2, the relationship between the values ​​of LC50 and the exposure periods of the extracts is reduced. Affected by the extracts where the resistance increases as the instar of life increasesTable 4.

From Table 2, hexane extract was superior to that of ethyl acetate and ethyl alcohol extract using D. innoxia leaf. Table 2 above also showed the relationship between LC50 values ​​and exposure periods for extract.

As the value of LC50 decreases as the exposure period of the extracts increases, we also note that the first larval insta is the most sensitive instar with the highest values ​​of mortality ratios and all the solvents used compared with the fourth larva linstar, which was less affected by the extracts where the resistance increases as the life of the instar increases. The cause of the first instar mortality may be explained by a higher percentage than the rest of the instar in all treatments due to several reasons, including the thinning of the cuticle layer surrounding the larvae in the first larval instar. Extracted with food into the gut leading to poisoning or may be caused by the compounds of these plants are anti. The results show that the LC50 of hexane extract of Leaves D.innoxia ppm (245.8, 301.1, 418.4, 1128.6). The value of LC90 was ppm (411.81, 773.7, 47936.8,19410), and the ethyl acetate was LC50 ppm (246.9, 375.3, 622.6, 1188.2) and LC90 was ppm (3757.1, 49537,30420,14234) respectively. LC50 for ethyl alcohol extract was( 33 2.4, 568., 1004.7, 5 1446.6) The LC90 (11685.2, 14673.7, 38915.1 and 67538.4) were for the four larval instar respectively. The results are consistent with that of (Choochote et al., 1999) in that the hexane extract of A.galagal was more effective than the methanolic extract of the same plant in killing the larvae of the fourth stage Cx. Quinquefasciatus, Singh, et al., in 2006, found that the hexane extract of Leaves M.chranatia was effective in the Cx. quinquefasciatus (LC50) was 1.29% and (Misvar & Aneesh, 2014) found that the hexane extract of C. citrinus leaves most effect from extracts (methanol, acetone, ethyl acetate) against the fourth larvae instar of Cx. quinquefasciatus with a value of LC50 511.9 (ppm). Kadhim et al., 2019 show The hexane extract, ethyl acetate and ethyl alcohol of Musa acuminate L Mortality of (100%, 92%, 100% and 90%) was achieved in the four larval instar of C. molestus, respectively

The effect in the mortality of pupa instar

The effect of concentrations of organic solvent extracts (hexane, ethyl acetate, ethyl alcohol) on leaves of D. innoxia leaves on the percentage of pupa mortality of mosquitoes is shown in Figure 3. Cx. quinquefasciatus. We note the hexane extract most effect from ethyl acetate and ethyl alcohol extract. We also note that there is a direct correlation between the concentration of the extract and the percentage of the cumulative mortality as the mortality rate increases with the concentration of the extract as the concentration of ppm 2000 gave the highest mortality rate (48, 45, 37)%As shown in Table 3. As shown in Table 3, LC50 for hexane extract ppm (1146.5), LC90 was (32966.3), for ethyl acetate extract was LC50 ppm (1179.6) and LC90 was ppm (81091.8). (1249.6) and LC90 was ppm (172705.6) ethyl alcohol extract. The preference of hexane over ethyl acetate and ethyl alcohol solvents in mortality pupa is due to the incompatibility of the compounds present in the extract with the action of the endocrine system, leading to defect growth and increased insect mortality (Halify et al., 1989). The death of pupa and the non-emergence of adults is the effect of extracts used as insect growth regulators and their effect on adult exit hormone (Makkar, Siddhuraju, & Becker, 2007).

In mortality percentage of a pupa. Cx. quinquefasciatus

(Sakthivadivel & Thilagavathy, 2003) noted that the hexane, acetone and petroleum ether extracts of Argemone Mexicana seeds inhibited the conversion of Ae.aegypti into adults and (Aarthi & Murugan, 2010) tested several plants for their medicinal effective by using several solvents including hexane solvent. Hexane for the leaves of Campanulata Spathodea most effect in the mortality of pupa An.stephensi. The mosquito mortality of Cx. quinquefasciatus was limited between (5-12%) by the effect of hexane extracts, petroleum ether and chloroform In concentrations between ppm (200-240). While (Ramar, Paulraj, & Ignacimuthu, 2015) concluded that hexane extract of leaves Corton sparciflorus was more effective than chloroform and ethyl acetate in the mortality of the pupa Table 5. The hexane extract recorded a 89% mortality after 24 hours of treatment. Mo, Nagmouchi, Al-K, and Benammar (2019), said that the ethyl alcohol extract of Azadirachta Indic and Cymbopogon citratu and Allium Sativa of Cx. quinquefasciatus achieved 100% mortality after 72 hours of treatment of the above-mentioned plants at a concentration of 40 mg/ml.

Cumulative Effect of Organic Solvent Extracts (Hexane, Ethyl Acetate, Ethyl Alcohol) of D. innoxia Leaves on the Percentage of Cumulative mortality of Immature instar Cx. quinquefasciatus

Figure 4 illustrates the effect of hexane extract of D. innoxia leaves on cumulative mortality ratios for immature instar of Cx. quinquefasciatus where hexane extract most effect from that ethyl acetate and ethyl alcohol All the larval instar mortality in the first phase of the hexane extract The ethyl acetate extract all the larval instar mortality in the second instar the ethyl acetate extract, where the mortality in the third instar of the ethyl alcohol extract of the D. innoxia plant

This extract has a negative impact on the stages of the development of the insect and the instar of adults and explains the reason for the presence of high poisoning and the accumulation of active substances of the above-mentioned plants in the gut. It is worth mentioning that the high mortality recorded by the extract of hexane indicates that the most effective compounds found in the plant have been extracted In this solvent are also indicated It also indicates that the active compounds were the most non-polar and insoluble in water and the superiority of the non-polar extracts over the polar extracts in the destruction of larvae. Cx quinquefasciatus (Abed, 2017; Ewaid & Abed, 2017; Mansour, Messeha, & Hamed, 1995). The results of the present study are consistent with the findings of Al-Zaidy et al. (2019) reported that exposure of Culex pipien larvae to the hexane extract of Azadirachta excels resulted in a 100% cumulative at a concentration of 1 mg/ml and Prevent adult exit (Arivoli, John, Raveen, & Tennyson, 2012; Ewaid & Al-Ansari, 2019; Ewaid, Abed, & Al-Ansari, 2019). Hexane extract of Hyptis suaveolen was the most effective in. Cx quinquefasciauts and show up. The hexane extract of the leaves of A. hispidum plant was at 69.82 ppm. The hexane extract of Musa acuminate recorded a 100% cumulative mortality at concentration 2mg /ml against Culex molestus .

Conclusions

The LC50 for ethyl alcohol extract (332.4, 568.5 1004.7, 1446.6) ppm. In instar pupa, the value of LC50 (1146.5, 1179.6, 1249.6) ppm for hexane extract, ethyl acetate and ethyl alcohol, respectively. As for the cumulative depreciation, the cumulative mortality rate of the immature stage reached 100% at the concentration of 2000ppm and for all organic solvents compared to the control, which was limited to (8-17) %.