Water Extracts From Trees Negatively Affect Oviposition Behavior and Fitness of Southern House Mosquito, Culex Quinquefasciatus Say (Diptera: Culicidae): A Simple, Easily Adoptable and Cost-Effective Approach to Control Mosquitoes

Background: The Culex quinquefasciatus Say is an important vector of many diseases of public health and veterinary importance. Environmental pollution, resistance development and health hazards associated with the use of synthetic insecticides have led to explore eco-friendly and cost effective management methods for mosquitoes. Methods: In our study, simple water extracts from leaves and bark of three tree species; Eucalyptus camaldulensis, Azadirachta indica (Neem), and Moringa oleifera, were tested to explore their potential negative effects on the oviposition behavior and tness of Cx. quinquefasciatus. Results: Oviposition bioassays showed a signicant delay in onset of 1 st oviposition in the all treatments as compred to control. Number of egg rafts and fecundity were signicantly lower in all treatments compared to control. Larval emergence was signicantly less in the all treatments as compared with the control except Moringa bark. Highest pupation was observed in control as compared to all other treatments. Importantly, no adult emerged in Eucalyptus leaf, bark and in Neem bark extracts. In case of toxicity bioassays, median lethal time (LT 50 ) was lower at higher concentrations and higher at lower concentrations. At highest tested concentration of every botanical extract, LT 50 was highly age dependent that is higher for elder larvae and vice versa. Conclusions: Therefore, it could be concluded that simple water extrcats, made locally, from trees may have a great potential to be incorporated in the mosquito control programs.

(References?). These major concerns have diverted the attention of researchers towards natural chemicals, such as plant extracts or oils, which are cost effective, safe, target speci c and easily biodegradable in the environment (Borah et al. 2010; Hafeez et al. 2011).
It has been shown by several studies that plant extracts or their non-volatile and volatile constituents have a great potential to be used as insecticides and insect repellents, respectively (Cetin et al. 2004; Lucantoni et al. 2006). Previously, different parts of medicinal plants such as leaves, seeds, peel, and succulent branches have shown great mosquitocidal properties (Ashfaq and Ashfaq 2012; Shaalan et al. 2005). In the current study, we have observed the female oviposition behavior and development period from hatching till adult emergence. This is the rst study where trees extracts have been tested while previously mostly small plant extracts have been tested on mosquitoes. Moreover, mosquitoes might be more familiar with small vegetation like grasses and shrubs to hide during the day, while large trees like Eucalyptu, Neem and Moringa are something new and may induce behaviroal and physiological changes more effectively than other plants. For this purpose, we have used leaves and bark extracts of three tree species; Moringa oleifera, Azadirachta indica and Eucalyptus camaldulensis to explore their potential negative effects on the oviposition behavior and tness as well as toxicity on immature stages of Cx. quinquefasciatus.

Insects
Pupae as well as larvae of Cx. quinquefasciatus were collected from the sewage line of student hostel area of Bahauddin Zakariya University, Multan (30°5′11N, 71°39′15E), Punjab, Pakistan. The pupae and larvae were kept in plastic containers (2 L), containing the same sewerage water (1L) and covered at open end with a muslin cloth in laboratory till emergence of adults. After emergence, male and female adults were kept together in the Plexiglas cages (1×1×1 ft) for mating purpose and provided with 10% sugar solution for feeding provided on an ad libitum hydrated cotton pad. After 3-4 days, only females were given a pigeon with exposed keel for a period of 12 h (overnight) for blood feeding in dark conditions. After blood-feeding, blood-fed females were kept for next 48 h post-blood feeding prior to use in further experiments. All the experiments were conducted under standard laboratory conditions of temperature at 27±2 ºC, light: dark (12: 12h) photoperiod, and relative humidity 70±5% (Shah et al. 2017).

Preparation of plant water extracts
Water based plant extracts were prepared by following the methods used by Wakil et al. (2014) and Arunpandiyan (2011) with some modi cations. Comprehensively, 500 g of fresh leaves as well as stembark of three different plant species (Moringa oleifera, Azadirachta indica and Eucalyptus camaldulensis) were collected separately in polythene bags. After collection, bark and leaves were grinded separately in mortar and pestle as well as in electric grinder (Black & Decker Company, New Britain, Connecticut, United States) by adding 500-800 mL of distilled water to make a uniform paste. Distilled water was added in the paste to make an amount of 2 L containing 500 gm of bark or leaves. Liquid mixtures of each botanical were poured in separate sterilized steel containers (5 L). These mixtures were stirred with a dried wooden stick continuously at an interval of 10-15 min for next three hours. After 3 h, mixtures were further extracted by using ne muslin cloth. After ltering the liquid, the remaining bark or leaf matter was pressed as much as possible to take out water from it. Furthermore, a standard solution (1000 mL) of 10% (w/v) for each type of extract was prepared by taking the required amount of ltrate and adding distilled water to prepare the solution of desired molarity. All the solutions were kept and maintained at 4°C in the refrigerator.

Oviposition bioassay
For oviposition bioassays, 10 mL of 10% extract was added into a plastic cup (120 mL) containing 90 mL distilled water to make 1% (w/v) solution (i.e. 1 gm leaf weight in 99 mL water). Plastic cups containing 100 mL of different extracts (1%) i.e. Neem leaf (Neem L), Neem bark (Neem B), Eucalyptus leaf (Eucalyptus L), Eucalyptus bark (Eucalyptus B), Moringa leaf (Moringa L), and Moringa bark (Moringa B) were placed singly in adult cages as oviposition substrates and 10 blood-fed females were released in each cage including control containing only distilled water and 5 replications were completed for each and every extract type and control. Oviposition was observed on daily basis till death of last female in each treatment. Ovipositional substrates (botanical solution or distilled water in case of control) were replaced with the fresh ones at an interval of 48 h to avoid any fungal development. Number of egg rafts and number of eggs in each egg raft were counted (under a compound microscope having a magni cation of 4X) on daily basis throughout the life span of females. Female longevity was also noted by observing the female mortality on daily basis till the last female died.

Larval development
Effects of tested plant extracts on the larval development of Cx. quinquefasciatus were also examined. An egg raft was placed in plastic cup containing 100 mL of 1% plant extract or the only distilled water (control). Larval emergence, larval mortality, pupae formation, and adult emergence were observed on daily basis (24 h) till all the larvae became pupae and adults. An egg raft was considered as a replicate and each treatment was replicated ve times. Rearing mediums (extract or distilled water) were refreshed after every 48 h.

Larvicide bioassays
The larval toxicity of tested botanicals (mentioned above) was evaluated against the 24h, 48h and 72h old larvae of Cx. quinquefasciatus following the methodology described by World Health Organization (1996) with some modi cations. Four concentrations of each botanical were prepared ranging from 100 ppm to 100,000 ppm. Each concentration was replicated ve times. Ten (for each age group) larvae were used in each replicate, i.e., 50 larvae per concentration and 250 larvae in one bioassay including control. The control was consisted of only water. The data was recorded at 24h, 48h, 72h and 96h intervals. All those larvae which were unable to move were considered dead.

Data analysis
The Shapiro-Wilk test was applied to test the normality of the data and P-values of less than 0.05 were considered as signi cant (data set was taken as non-normal). Therefore, non-parametric test, Mann-Whitney U-test was used for pairwise comparison of different parameters of each treatment including delay in 1 st oviposition, female fecundity, larval emergence, larval development, pupation, adult emergence, larval survival, adult survival and longevity compared to that of control. For signi cance level, α was set at 5% and P-values of less than 0.05 were considered as signi cant. All the data were analyzed by using IBM SPSS Statistics v 21. The concentration response data was analyzed by probit analysis (Finney 1971) to determine the median lethal time (LT 50 ) values, their 95% con dence intervals (CI), slope ± standard error (SE) and chi square (χ 2 ). LT 50 values were considered signi cantly different when their 95% CI did not overlap (Litch eld and Wilcoxon 1949; Robertson and Preisler 1992). Pearson correlation was applied to determine the coe cient of correlation and signi cance of correlation (P<0.05) between the LT 50 values and different age groups of exposed larvae at highest tested concentration of different botanicals. Moringa bark (U=1, P=0.08) did not differ signi cantly than that of control ( Figure 2). Females exposed to botanical extracts of Eucalyptus bark (U=0.0, P=0.05), Eucalyptus leaf (U=0, P=0.05) and Neem bark (U=0, P=0.05) laid similar number of egg rafts signi cantly lower as compared with that of control. Moringa leaf (100,000 PPM), Eucalyptus leaf (100,000 PPM) were similar to each other based on the overlapping 95%CI and were the least time taking among all tested concentrations to kill the 50% population of the exposed larvae (Table 1). While, Neem leaf (100 PPM), Moringa leaf (100 PPM) and

Number of eggs/female
Eucalyptus leaf (100 PPM) were longest time taking concentrations among all the tested concentrations against 24h old larvae to kill the 50% population of the exposed larvae. Neem bark (100 PPM) and Moringa bark (100 PPM) did not kill any of the exposed population.
When 48h larvae were exposed to different botanical concentrations, Neem leaf (100,000 PPM), Neem bark (100,000 PPM), Eucalyptus Leaf (100,000 PPM) and Eucalyptus bark were the concentrations having similar LT 50 based on overlapping 95% CI and the shortest time taking (Table 2). Moreover, the neem leaf (100 PPM) was the longest time taking concentration among all tested concentrations to kill fty percent of the population. However, Moringa bark (100 PPM) did not kill any larvae exposed to it.  Our results suggest that onset of 1 st oviposition was delayed as compared with control, when females were exposed to each of tested botanical extracts. Previously, Rajkumar  plant extracts such as Aegle marmelos, Andrographis lineate, Andrographis paniculata, Cocculus hirsutus, Eclipta prostrata, and Tagetes erecta have oviposition-deterrent, ovicidal, and repellent activity against An. subpictus. As oviposition was signi cantly delayed in exposed females in all treatments, oviposition deterrent activity of plant extracts might induce physiological changes leading to behavioral avoidance in female adult mosquitoes. It might also be due to volatile chemicals released from respective plant water extracts, which were added in their oviposition substrates. Tawatsin et al. (2006) has also reported that the relatively high oviposition deterrent activity was induced by essential oils of Curcuma longa, Zingiber o cinale, Vitex trifolia, Melaleuca cajuputi, Manglietia garrettii, and Houttuynia cordata. The delay in oviposition leads to lower female fecundity and thus it is obvious that with vast biodiversity, many bioactive compounds could be isolated and utilized for mosquito control especially Cx. quinquefasciatus. Moreover, adding simple water extracts of potential botanicals in the standing waters in ditches, ponds or in sewerage lines, which are the main breeding sites of Cx. quinquefasciatus, may help to deter gravid females or delaying the oviposition leading to less female fecundity and ultimately less population of Cx. quinquefasciatus. In the present study, no pupation occurred in Eucalyptus leaf and in Eucalyptus bark, while very few pupae were formed in Neem leaf and Neem bark. These results are similar to NJOM et al. (2011) where higher concentration of Moringa seed extracts did not allow any pupation. Previously, hexane based leaves extract of Eucalyptus has shown potential to suppress the pupal formation in from larvae of different exposed mosquito species including Cx. quinquefasciatus (Singh et al. 2007). This also indicates that plant extracts can kill the larvae as well as pupae of mosquitoes.
In the present study, no adult emergence in Eucalyptus leaf, Eucalyptus bark and Neem bark while was highly signi cantly lower in Neem leaf. Elimam et al. (2009) reported that aqueous extracts from Ricinus communis inhibit adult emergence and also showed larvicidal as well as oviposition deterrent effect against Cx. quinquefasciatus and An. arabiensis.

Conclusions
The water based botanical extracts of bark and leaves of the all tested species showed a variable range of larvicidal activity against the larvae of the Cx. quinquefasciatus but have potential to be used as the traditional control agents. Among the tested botanicals, mortality of the larvae was highly dose dependent. Highest mortality at higher doses and lowest or no mortality was observed at lowest doses of the tested botanicals in our study. Previously, Govindarajan et al. (2011b) reported the similar dose dependent response of the Cx. quinquefasciatus to botanicals. Cetin et al. (2006) reported a similar dose dependent response of Cx. pipens exposed to plant extracts. Therefore, application, keeping in view the dose response of the target species, of these easily made botanical extracts shall be considered at least at small scale level, e.g., homes and poultry farms.

Declarations
Ethics approval and consent to participate: No human beings were subject of experiments; therefore, no consent to participate was required.    Figure 1 Effects of different plant water extracts in delaying 1st oviposition in Culex quinquefasciatus. *, ** and *** indicate the signi cant delay in those treatments compared to the control at P<0.05, P <0.005 and, P <0.0001 respectively. "L" and "B" denote leaf and bark, respectively. The error bars represent the standard error of the mean.

Figure 2
Effect of different botanicals on the production of egg rafts in Culex quinquefasciatus. *, ** and *** indicate the signi cant delay in those treatments compared to the control at P<0.05, P <0.005 and, P <0.0001, respectively. The error bars represent the standard error of the mean.

Figure 4
Effects of different plant water extracts on the larval emergence of the Culex quinquefasciatus. *, ** and *** indicate the signi cant delay in those treatments compared to the control at P<0.05, P <0.005 and, P <0.0001, respectively. The error bars represent the standard error of the mean.

Figure 5
Effects of different plant water extracts on the pupation (%) of the Culex quinquefasciatus. *, ** and *** indicate the signi cant delay in those treatments compared to the control at P<0.05, P <0.005 and, P <0.0001 respectively. The error bars represent the standard error of the mean. Figure 6