Secondary plant products against Culex pipiens (Linn.), with reference to some changes detected by scanning electron microscope

To find environment-friendly and low pest resistance natural mosquitocide, the bio-effects of smokes and oils obtained from leaves of Eucalyptus globulus and Ocimum basilicum against Culex pipiens (Linn.) were investigated. An influence of smokes and oils was tested on the loss ratio of eggs, larvae, pupae, and adult mortality of 1st and 2nd generations of Culex pipiens. Morphological changes of the 2nd generation, including antennae and mouthparts of larvae and adults, were examined by using the scanning electron microscopy (SEM). The smokes of O. basilicum and E. globulus reduced the ratio value of the insect, especially the eggs’ number and adults significantly within 2 generations that lasted 82 days. The impact of O. basilicum smokes on the larval molting and mortality rate was 83.15 and 95.05%, respectively, compared to 29.85 and 82.43 % for E. globulus. The lethal effect of O. basilicum smoke on adults had a loss rate of 100% in both generations than 75.86% by E. globulus. Essential oils from O. basilicum and E. globulus showed a severe and rapid killing effect of C. pipiens within 8 days with a loss ratio of 99.47 and 81.38% of larvae from eggs, respectively. Scanning electron microscopy showed that both smokes could cause prominent malformation to the larval antennae, siphon, and adult mouthparts. In conclusion, either E. globulus or O. basilicum smokes in the long term or either of their oils in the short term could be used as natural repellent agents against Culex pipiens.


Background
Plant natural products have been historically used in many parts of the world to repel and kill mosquitoes. Many plant species had been screened for their mosquito repellent and insecticide property (Shaalan et al., 2005;Ghosh et al., 2012;Ravindran et al., 2012). Repellent activities and their effects on the mosquitoes were reported for Eucalyptus maculate against Anopheles gambiae and Anopheles funestus (Trigg, 1996); Ocicum selloi against Anopheles braziliensis (Paula et al., 2003); neem plant extract against Aedes, Culex, Anopheles (Sharma et al., 1993;Dua and Sharma, 1995); and O. mericanum against Anopheles dirus, Aedes aegypti, and Culex quinquefasciatus (Tawatsin et al., 2001). The action of smoke from smoldering mosquito coils against Aedes aegypti, Anopheles stephensi, and Culex pipiens fatigans were studied by Chadwick (1975). Mosquito coil prepared from Mesue ferra leaf powder was investigated for its smoke effect against Cx. quiquefasciatus adults (Singha et al., 2011). They concluded that the smoke from M. ferra could play a crucial role in the pause of transmission of many diseases, whereas the mosquitoes act as vector at the individual level. Smoke toxicity test of Ocimum canum against Cx. quinquefasciatus, Anopheles stephensi, and Ae. aegypti was tested by Ramkumar et al. (2015) who found that smoke of the leaf powder could be used as an efficient toxicity agent against many species of mosquitoes. Murugan et al. (2016) reported that O. canum leaf extract and powder have toxicity effects on Cx. quinquefasciatus, Ae. aegypti, and An. stephinsi adults. Furthermore, Asiamah and Botchey (2019) stated that there were repellent and mosquitocidal properties of smoke from oil and the non-polar extracts of O. viride leaf against Ae. aegyptii.
The present study aimed to evaluate the effect of the smokes and oils from E. globulus and O. basilicum on morphological malformation and biological behavioral activities of the mosquito species, Cx. pipiens.

Insects rearing
Culex pipiens egg batches were obtained in year 2019 from the laboratory of Zoology Department, Faculty of Science, South Valley University (SVU), Egypt. The insects were reared in glass cages (50 mm in diameter and 100 mm high) with a little bit of bread crumbs and covered with cloth. Every day, water and bread crumbs of all jars were removed in order to maintain a population of young larvae (1st, 2nd, 3rd, and 4th instars) ready to be used in the experiments. The treatments were kept under the laboratory conditions of 25 ± 5°C, 25 ± 10% (RH), and a photoperiod of 16 h of light and 8 h of darkness.

Bioassay tests Effect of Eucalyptus globulus and Ocimum basilicum smokes on Cx. pipiens
This experiment was conducted to determine the dosedependent toxicity smokes of Eucalyptus globulus and Ocimum basilicum against different stages of Cx. pipiens. Exposing such stages to the tested smokes was conducted daily by burning 10 dried grams of the leaves from each plant. Smoking process was continued for about 15 min for each plant in 4 rooms (length 5.40 m, width 2.15 m, and height 3 m). After treatments, daily monitoring was carried out for 82 days (two generations of the insect) for each dose of smokes, to estimate the mortality rate of Cx. pipiens larvae and adults by picking and separating the dead individuals.
Effect of the essential oils of O. basilicum and E. globulus on Cx. pipiens O. basilicum and E. globulus essential oils were purchased from the Captain Company accredited and registered to the Ministry of Health No. 2978/2002. For the treatments' application, the total volume used in each spray was 1 ml, applied on glass flasks (50 mm in diameter and 100 mm high) layered with a sterilized filter paper, to be sure that the treatments were retained, at 40 kPa. Three doses (6, 12, and 24 l/Petri dish) of O. basilicum and E. globulus were sprayed and 3 replicates were done. A treatment without essential oils was used as a control. After the application of treatments, daily monitoring, by separating and counting the dead larvae individuals, was carried out during the following 8 days after the application of each dose of essential oil.
Scanning electron microscope (SEM) Chaudhary and Gupta (2004) protocol for SEM studies, was followed. Egg batches were collected from water drains by taking surface water included egg batches with a small bucket. The developed larvae out of the collected eggs were stored remaining in 70% ethanol, besides, some continued to be reared in the laboratory. The specific identification was made from the reared adult mosquitoes. For SEM studies, both larvae and adults were passed through the essential dry point and placed on SEM stubs with only a thin double-sided adhesive tape strip (Kirti and Kaur, 2011). Under the smokes of E. globulus and O. basilicum, larvae and adults of Cx. pipiens were scanned by JSM-6100 SEM. The effects on the larval antennae and siphons and adult antennae and mouthparts were investigated.

Statistical analysis
Data were analyzed by SPSS program using one-way analysis of variance (ANOVA). Mean differences were compared using multiple comparison range test by Excel program for three replicates

Results and discussion
The effect of the smoke was continued for 2 generations (82 days), intervals of 15 min smoke every day. Extracted oils from E. globulus and O. basilicum had been investigated for their effect on the biological behavioral activities of Cx. pipiens. The data recorded about the eggs, alive larvae, larval molting, larval mortality, alive pupae, alive adults, and adult mortality of Cx. pipiens under the effect of E. globulus smoke and in control treatment conditions are presented in Table 1. It was found that in the 1st generation, the Cx. pipiens under normal conditions showed very low of loss rates in all stages, while in the 2nd generation, it was found only one adult had been lost represented (9.47%). It showed also the lowest loss rate (3.46%) in eggs, and it ranged between 8.40 and 8.65% in alive larvae, larval molting, larval mortality, alive pupae, and alive adults.
Effect of E. globulus and O. bacilicum smokes on Cx. pipiens These findings are in agreement with the other researches that showed that the smoke possesses toxicity to various pests. As plant products contain multiple chemical constituents, smoke burning from it also contains some of those active principles. Several plant smokes showed a toxic effect against mosquitoes. For example, smoke from Albizza amara reported to have a more toxic effect on Ae. aegypti than O. bascilicum smoke (Murugan et al., 2007). It was reported that smokes of Ficus krishnae had a toxic efficacy against Anopheles stephensi Liston and Cx. vishnui group of mosquitoes (Haldar et al., 2014). The smoke obtained from various solvent extracts from Lantana camara leaves showed different levels of repellence against female anopheles mosquitos (Akumu et al., 2014). Volatiles found in the smoke resulted either from burning or from fresh leaves of Corymbia citriodora and Ocimum suave and showed remarkable repellent activities against host-seeking Ae. aegypti and An. arabiensis mosquitoes (Dube et al., 2011).

As shown in
Obtained results showed that the complete mortality of adults of Cx. pipiens was observed by E. globulus smoke treatment under room conditions during the 2nd generation. Moreover, the current results showed the  severe mortality appeared only after 82 days of continuous smoking. Thus, it could be interpreted that we smoked the Cx. pipiens in few minutes and other researchers smoked very heavily in closed areas. Furthermore, the present results found that the toxicity efficacy between smokes of E. globulus and O. basilicum either for the 1st or 2nd generations varied greatly, as for example that there was an increase in the loss rate of larval molting and larval mortality under O. basilicum smoke than that of E. globules. However, the loss rate in the number of egg production and adult mortality caused by E. globulus was higher than that caused by smoke of O. basilicum and could be due to the nature of the chemicals present in each plant (Brooker and Kleinig, 2006). 1, 8-Cineole has been reported to be the most important chemical in the genus of Eucalyptus and is largely attributed to various pesticide characteristics (Duke, 2004). On the other hand, O. basilicum essential oil was mainly composed of aromatic ether estragol (74.0%) and the terpene with alcohol group linalool (17.8%), which is known to be a repellent and had toxic activities against insect species (Regnault-Roger et al., 1993).
The current results could be due to the layer thickness of oils covering the water surface. Meanwhile, these larvae breathe by their syphon of the terminal body end. Moreover, the number of released larvae of E. globulus was fewer than that was treated by O. basilicum, and this may be due to the thickness of the E. globulus oil that was more than that of the O. basilicum oil.
Effect of the essential oils on Cx. pipiens According to Table 2, the results showed an acute effect on hatched eggs for those treated by oils of E. globulus and O. bacilicum than the control across 8 days. In the case of control, the loss ratio of larvae resulted from hatched egg was 2.338% and those treated by oils of O. basilicum and E. globulus were 81.38% and 99.47%, respectively. Again, this support that both smokes from leaves of E. globulus and O. bacilicum had a toxic effect probably due to specific toxic chemicals against Cx. pipiens. It was found that E. globulus oil possesses a wide spectrum of biological activity including anti-insecticidal repellent (Batish et al., 2008). Previous researches showed that oils of many plants had various activities against eggs hatching, adults, developmental traits, and larva/pupa susceptibilities (Regnault-Roger and Hamraoui, 1994;Papachristos and Stamopoulos, 2002).

SEM observations
SEM of the larval antennae, siphons, adult antennae, and mouth parts of Cx. pipiens subjected to the E. globulus and O. basilicum smokes were found to be morphologically abnormal compared to control (Figs. 3 and 4). It was evident that the bending of Cx. pipiens under E. globulus smoke (Fig. 3b) was more than that O. basilicum smoke (Fig. 3c). Also, the number of sensory hairs on the larval antennae of non-exposed Cx. pipiens to smoke was more than those exposed to E. globulus and O. basilicum smokes. The variation in the number of sensory hairs is probably due to the oily layer formed from E. globulus and O. basilicum smokes and why larvae underwent abnormal growth with little bed sensory hairs. As shown in Fig. 3d, there are no active processes in the terminal ends of the siphon of the control sample, while complete active processes in the siphons were observed under smokes of E. globulus and O. basilicum (Fig. 3e, f). Also, it was observed that there was abnormal growth of the antenna as there was a cut in some segments under E. globulus smoke (Fig. 4b) and prominent curvature under the effect of both smokes of O. basilicum and E. globulus ( Fig. 4e). A curvature in the antennae may be a result from an increasing surface area provided with more sensory hairs and plates to have a benefit from any amount of oxygen that is contaminated by smokes. These results were confirmed by Sujatha et al. (1988) who estimated the effect of Acorus calamus extract on Cx. quinquefasciatus and to Cx. quinquefasciatus and found morphological malformations to the Culex spp.

Conclusion
Smokes and oils of E. globulus and O. basilicum leaves affected the life cycles of Cx. pipiens with varying degrees of mortality rate. Therefore, both smokes and oils of E. globulus and O. basilicum can be applied as natural products against mosquitoes.