Efficacy of Citrullus colocynthis seed extract on Earias vittella, Fabricius, (Lepidoptera: Noctuidae): environment sustainable approach

Earias vittella Fabricius, 1794 (Noctuidae: Lepidoptera) is deliberated to be one of the most destructive pests of cotton and okra vegetation in the world including Asia. The pest has established resistance to various synthetic insecticides. The use of bio-pesticide is one of the unconventional approaches to develop a vigorous ecosystem without harming non- target pests and beneficial natural insect fauna. In the present study, the toxicity levels of Citrullus colocynthis seed extract have been evaluated against the populations of E. vittella under standardized laboratory conditions. The toxic effects of C. colocynthis on development periods, protein contents and esterase activity of the life stages of E. vittella were also evaluated. The toxicity levels of methanol, ethanol, hexane, water and profenofos were evaluated on the 1 st instar larvae of E. vittella. LC 30 and LC 80 concentrations exhibited the effectiveness of methanol-based C. colocynthis seed extract against 1st instar larvae of E. vitella. The enhanced larval and pupal periods were revealed in treated samples during the comparison with untreated samples. The intrinsic rate of increase, net reproductive rate in the LC 30 and LC 80 concentrations exposed larvae remained less than the control treatment. Fecundity, the esterase activity and protein contents were declined in LC 30 and LC 80 treated samples as compared to the control. The present findings suggest that C. colosynthis extracts based botanical insecticides are beneficial, ecosystem sustainable and can be integrated with insect management programs from environment safety perspective.

Citrullus colocynthis is generally recognized as the colocynth, bitter apple, desert gourd and viny plant (Cucurbitaceae) intrinsic to the Asia and Mediterranean arena and well established in the temperate areas of Pakistan. Many active chemical compounds (cucurbitacin A, B, C, D, E, I, J, K, L, glucosides I, L and flavonoids) from C. colocynthis have been described in previous studies (Delazar et al., 2006;Sturm and Schneider, 2009). Isolated compounds exhibited diverse actions against insect pests especially the compound cucurbitacin B is responsible for reduced oviposition and anti-feeding activity (Seenivasan et al., 2004;Tallamy et al., 1997) and these are also exploited as an abortifacient (Madari and Jacobs, 2004). These chemical compounds can manage and control various insect pests (Ahmad, 2007). It's almost two millennia that plant-mediated pesticides are being used in agriculture (Thacker, 2002).
Many investigators explored the effects of different plant extracts on the life span, fertility, fecundity and physiology of insects (Baskar et al., 2015;Pavunraj et al., 2016). C. colocynthis leaves extract with ethyl acetate, benzene, petroleum ether and methanol was commenced very effectively in populations of mosquito, C. quinquefasiatus (Mullai and Jebanesan, 2007).
The present study investigated the lethal and sub-lethal effects of the methanol-based extracts of C. colocynthis various biological and physiological factors of E. vittella.

Insect culture and sampling
Laboratory culture of spotted bollworm (E. vittella) was established from the larvae collected from okra and cotton fields in the surrounding three districts (Rawalpindi, Taxila and Attock) of Punjab province, Pakistan for the continuous three years (2018)(2019)(2020). The infested pods from the fields were transferred into transparent plastic jars (20 cm length and 10 cm wide) and were retained within standardized conditions (27 ± 2 C, 60 ± 5% R.H., 16 hours light, 08 hours dark). Fresh Okra (Abelmoschus esculentus) fruits were utilized for sustaining the insect culture (Al-Mehmmady, 2000). Fresh okra fruits were thoroughly washed in clean water and desiccated before feeding to the neonates, and latterly these were cut into 0.5-1.0 cm pieces; 4-5 larvae were released per piece and placed in the plastic container. The food was replaced daily till pupation. The larvae were carefully removed from the okra pods and the excreta were cleaned. The pupae were shifted to another plastic jar (10 cm length and 5 cm width) until adult emergence. The emerged adults were shifted to adult cages (length x height x width: 38 x 34 x 30 cm) and nourished with 10% sugar solution. Nappy strips were provided in cages for oviposition and these strips were replaced regularly after observation of eggs.

Collection of seed and solvent extraction
C. colosynthis plant seeds were collected from various crop fields throughout the temperate regions of the Punjab

Introduction
Spotted bollworm, Earias vittella Fabricius, 1794 (Noctuidae: Lepidoptera) is a global, polyphagous and most damaging pest of many economically important crops with wide host range which includes cotton, okra, hollyhock, China rose, shoe flower, sonchal and various other malvaceous plants (Syed et al., 2011). Spotted bollworm caused direct damage to fruiting bodies of cotton, Gossypium hirsutum and okra, Abelmoschus esculentus (Ahmad and Arif, 2009;Rahman et al., 2013). The larvae not only attack bolls and fruits though also damage the shoots, buds and flowers (Aziz et al., 2012). This notorious pest remained active throughout the year and at least 6 to 8 generations are observed each year. This pest causes up to 40% and 41.6% losses in seed cotton and okra, respectively whereas loss in cotton yield may reach up to 50% during severe infestation and larvae alone causes a damage between 50 to 70% including deformed fruits and low quality (Hassan and Ansari, 2010;Tanni et al., 2019).
Synthetic pesticides are the primary tools to manage insect pests of agriculture and veterinary. This sole dependence on chemicals, particularly in vegetables is not desirable due to health hazards. Furthermore, the injudicious exercise of chemicals has twisted numerous problems that jeopardize their efficiency further causing environmental pollution and intimidation to all biological and beneficial fauna (Hashmi, 1994). Recent years the indiscriminate use of insecticides leads to the development of resistance a big challenge for chemical control in insects (Ahmad et al., 2019;Bisset et al., 1997;Gulzar and Wright, 2015;Liu and Yue, 2000). It has been reported that many insect species have developed resistance against various insecticides (Whalon, 2008). The increasing concern on the development of resistance and restricted usage of pesticides has encouraged the application of bio-rational and botanical insecticides (Akhtar et al., 2012;Tome et al., 2013).
Botanicals or bio-pesticides can be an effective and advantageous replacement to commercial pesticides leading towards integrated pest approaches due to targetspecific, eco-friendly, low number of applications and higher acceptable properties. Phytochemicals extracted from the plant parts by using different solutions have acted as toxicants, repellents and growth regulators (Ahmad et al., 2021;Amer and Mehlhorn, 2006;Choudhury et al., 2021). Synthesized chemical compounds produced by plants have the potentials to way forward to integrate insect pest control methods (Tabashnik, 1994). Plants have the aptitude to produce various bioactive substances and tributary metabolites such as alkaloids, glycosides, carbohydrates, terpenoids, tannins, phenols, flavonoids and sitosterols (Georges et al., 2008;Mann, 1978). These chemical components of plants are necessary for the actual value of mythological cures of entomological fauna. These components are extensively utilized in agriculture, veterinary scientific researches and numerous diversified arenas (Vasu et al., 2009). This plant-based stuff derived from stems, leaves, twigs, flowers, roots and fruiting portions has developed the part pesticides since ancient times (Cragg and Newman, 2001). province, Pakistan. Impurities from the collected seeds were removed manually and kept for drying under room temperature (Memon et al., 2003). The desiccated seeds were crushed in the shape of elusive powder with an automatic grinder. Fifty grams of the ground seeds were drenched in 100 ml of methanol, ethyl acetate, hexane and ethanol discretely for 24 h for each solvent extract separately. The mixture was stirred for 1 h and kept below 4°C in the refrigerator for 2 days. These refrigerated mixtures were stirred again for 1 h. These mixtures were filtered twice through filter paper and finally dissolved in 10 ml of their particular solvent to be conserved as a stock solution.

Phytochemical analysis
Different solvent extracts (methanol, ethanol and hexane) were utilized to carry out the preliminary phytochemical analysis of C. colocynthis with already standardized methodologies to recognize the constituents (Harbone, 1973).

Bioassays
Through the diet emersion method, bioassays were performed and toxicity was measured for 1 st larval instar larvae of spotted bollworm. Different concentrations of C. colosynthis were prepared using different solvents (methanol, ethanol, hexane). Fresh okra fruits were dipped separately in each concentration for 10 seconds and then desiccated for 10 minutes at room temperature. Five okra fruits were used in one replication. Two 1 st instar larvae were released on each okra pod in all replications treated with different concentrations. Four replications were performed for each treatment. The control treatment was served with purified distilled water. The entire treated larvae were kept under standardized lab conditions (25 ± 2 °C; RH 65 ± 5%, 16 hours light, 08 hours dark) and datasets were recorded regarding larval mortality at 72 hours.

Sub-lethal effects of plant seed extracts on biological on biological parameters of Earias vittella
Age-stage, two-sex life table theory was considered to estimate the stuff of sub-lethal concentrations on the biological factors of E. vittella. For life table study, 210 eggs were used, which were collected after 24 hours of deposition by females of the laboratory population. Three treatments (control, LC 30 and LC 80 ) of methanolic extract were prepared for this experiment. Seventy eggs were treated with each treatment. Each egg in an individual Petri dish was reflected as a single replicate (Zhang et al., 2015;Chi and Yang, 2003). All petri dishes were kept under standardised conditions. The egg hatching data were recorded daily and the neonates from control, LC 30, LC 50 and LC 80 were shifted on the okra pods. The larval development was observed daily and fresh okra pods were provided after each instar. Pupae were removed and placed in new petri dishes until adult emergence. Then, the newly emerged adults were paired (one male and one female) and transferred to an individual plastic container for oviposition. The adults were patterned for oviposition and transported to fresh containers for egg-laying daily.
The fecundity (%) and survival rate of the adults were assessed until the death of the adults.

Assays for esterase activity and protein content
The general esterase activity was determined through the method proposed by Van-Asperen (1962). In this experiment, 10 mM of α-naphthyl-acetate (αNA) and β-naphthyl-acetate (βNA) were treated as substrates. The frozen supernatant from the above treatments was transported to a fresh micro tube and phosphate buffer was added for making dilution. 1mM Fast Blue RR salt was also supplemented to this solution tube. The calculation regarding the absorbance was retained at 630 nm. Bovine Serum Albumin was utilized as a standard for the determination of the total protein contents (Bradford, 1976).

Data analysis
The LC values were calculated based on mortality data using R Statistical Software version 2.9.0 (R Development Core Team, 2018) The datasets regarding different developmental stage periods, survival rate and fecundity were scrutinized with the help of model age-stage, twosex life table, theory and software (Chi, 1988(Chi, , 2021. The means of the biological parameters were compared by using 100,000 bootstrap techniques to achieve stable standard error estimates (Huang and Chi, 2012). The curves for age-specific survival rate, life expectancy, fecundity and reproductive values were generated by using Sigma Plot 14.0. The net reproductive rate was calculated using the following Formula 1: The intrinsic rate of increase (r) was calculated by using the iterative bisection method through Formula 2: With age indexed from zero (Goodman, 1982) the mean generation time (T) was calculated by the Formula 3: The Gross reproductive rate (GRR) was calculated by the Formula 4 as follow: The age-specific survival rate (l x ) and age-specific fecundity (m x ) were given as (Formula 5 and 6):

Lethal and sub-lethal effect of methanolic extract of C. colosynthis fruit on life tables of E. vittella
The larval development period was recorded from 1 st instar to pupation stage of E. vittella treated with LC 80 and LC 30 as lethal and sub-lethal concentrations. The data of treated insects showed significant variation when compared with the untreated ( The average pupal weight of the insects exposed to lethal and sub-lethal concentrations was lower than the control treatment (Table 3, p˂0.001, df=2, F=204.1). The percent pupation after LC 80 treatment (52.48) was less than LC 30 (58.52) and control (91.75) treatments (Table 3, p˂0.001, df=2, F=11.93). The lethal and sub-lethal concentrations were recorded to reduce the percentage of adult emergence significantly with comparison to the control treatment (Table 3, p˂0.001, df=2, F=21.49).
The application of a methanol-based extract of C. colosynthis at lethal, sub-lethal and control concentrations (67, 41 and 89) resulted in the reduction of the mating pair's success (Table 3, p˂0.001, df=2, F=20.05). A significant decrease in the fecundity egg/female was perceived laid by each female in the lethal and sub-lethal treated population with comparison to control (Table 2, p˂0.001, df=2, F=62.93). The hatchability percentage of eggs (viability) was also affected and reduced significantly in the lethal and sublethal treated populations when compared to the control treatment (Table 3, p˂0.001, df=2, F=266.81).
C. colosynthis plant extracts significantly altered the population parameters of the E. vittella (Table 4). To estimate the population parameters, the bootstrap method with 100,000 replicate was applied. The increased intrinsic rate was decreased by treating with both the concentrations of C. colosynthis plant extract (0.128 d -1 and 0.140 d -1 for LC 80 and LC 30 respectively) as compared with the untreated larvae (0.166 d -1 ). Similarly, the trend was exhibited in the finite rate of increase (λ), as the highest value for λ was observed in control larvae (1.181 d -1 ), which gradually decreases with an increase in concentration from LC 30 to LC 80 as 1.150 d -1 and 1.137 d -1 respectively. A significant decrease was also observed in the net reproductive rate (Ro) after treatment from being highest in control (94.542) to 76.714 and 61.228 offsprings/individual for LC 30 and LC 80 treated larvae, respectively. The highest Gross reproductive rate (GRR) was recorded for control larvae (183.42), while the lowest for LC 80 treated larvae (136.77) progeny/individual which was both statistically different to each other. The mean generation time was prolonged in the treated larvae in comparison with the larvae of control treatment. Minimum mean generation time was taken by the control larvae (27.255 d -1 ), followed by LC 30 treated larvae (30.967 d -1 ). The maximum days were recorded on LC 80 treated larvae (31.917 d -1 ). The S xj represents the age-stage survival rate, it shows the chances of afresh laid eggs to survive till (age x and stage j) as shown in (Figure 1). Significant variations were observed in the growth rate of the various entities in the population (Figure 1). The lethal and sub-lethal treatments show overlapping curves of the developmental stages as compared to control. Each developmental stage showed a different pattern of the peak curves for lethal and sub-lethal of C. colosynthis plant extract and control. The curve line for the male adults ends earlier (Figure 1) as compared to female adult lines. Survival rate (l x ), agespecific fecundity (m x ) and age-specific maternity (l x m x ) were calculated for lethal and sub-lethal concentrations for E. vittella (Figure 2). A significant decline of l x curves was observed in the lethal and sub-lethal treated population much earlier than that in control. Higher peaks for f x curves were noted in control, while much lower f x curves were noted in LC 80 and LC 30 treated populations. The e xj (life Table 3. Mean biological parameters of E. vittella treated with lethal and sub-lethal concentrations of methanolic extract of C. colosynthis.

Treatment
No expectancy) curves varied significantly among the treated (LC 80 and LC 30 ) and untreated for E. vittella (Figure 3). The minimum life expectancy for eggs was observed on the lethal, followed by sub-lethal, while the maximum value of life expectancy for eggs was observed in the control. Reproductive rate (vxj) is defined as the measure of dedication to newly coming offspring in the future from age x to stage j (Figure 4). The contribution of males in the population to the next generation was not well defined; therefore the curve for males was not included. A decline

Total protein content and esterase activity of E. vittella treated with plant extract
Total protein content in E. vittella treated with lethal and sub-lethal deliberations of C. colosynthis plant extract   were shortened to a significant level as compared to the untreated treatment (Table 5, p˂0.001, df=2, F=349.5). The average esterase enzyme activity in E. vittella treated with lethal and sub-lethal concentrations was significantly reduced when compared with the untreated control populations (Table 5, p˂0.001, df=2, F=1704.2).

Discussion
In the present study, the presentation of lethal and sublethal deliberations of C. colosynthis seed mines significantly influences the developmental time, pupal weight, pupal percentage, adult emergence and adult longevity. The methanolic extract of C. colosynthis prolonged larval development, pupal developmental time as compared with the control. Ramzi et al. (2015) also described related results, when the caterpillars of Apomyelois ceratoniae were pickled with C. crenata as compared with the populace of control treatment, this difference might be the metamorphosis interruption grounds.
The auspicious results of C. colosynthis to control E. vittella might be due to the presence of compound cucurbitacin B and responsible for the reduced hatchability antifeedant activity (Tallamy et al., 1997). The present study reported that C. colocynthis seed extract significantly affects the mating, fecundity and egg viability in E. vittella treated with lethal and sub-lethal concentrations. Such kind of results was also described by various researchers (D' Incao et al., 2012;Khani et al., 2013;Khosravi et al., 2011). Lethal and sub lethal effects along with reduced fecundity and fertility rates were reported in Spodoptra frugiperda when treated with saponins extract, obtained from Passiflora alata (D' Incao et al., 2012). The extract of Piper nigrum and Jatropha curcas reduced the egg viability and adult emergence of C. cephalonica with control comparison (Khani et al., 2013).
The intrinsic rate of natural increase (rm) provides an estimate of the growth probability of insect populations (Rabinovich, 1972) which can offer great perception into the population growth of species besides other life-history factors. Whereas it is also conceivable to estimate the net rate of reproduction (R o ) dependency of natural increase in the intrinsic rate principally based on productiveness, % hatching, growth and adult emergence (Khan et al., 2013;Saeed et al., 2010). Aiming the purpose, the above mentioned divergent life-history abilities might simulate the population increase rate of E. vittella. The intrinsic rate of natural increase (r m ) was significantly declined in lethal and sub-lethal concentrations as compared with control treatment. These findings are also described by Hafeez et al. (2019) in a way that the intrinsic rate of increase was significantly changed due to lethal and sub-lethal concentrations of lufenuron whereas present study tested plant extracts also showed the comparable results. Correspondingly, the average generation time was significantly protracted in the treated population equal to the mean generation time of the untreated population. The escalation was observed in the mean generation time of S. litura when treated with different doses of imidacloprid (Abbas et al., 2012). Our results exhibit that parameters of the life table of E. vittella were adversely transformed with the lethal and sub-lethal concentrations of C. colocynthis plant extracts. A significant change was found in the S xj , which decreased in the treated population compared to the untreated population. f xj and m x were also found to be decreased in the lethal treated population as compared to the control population. A measure of the influence of new individuals in population growth (e xj ) declined sharply in second larval instars in the lethal and sub-lethal treated population. The methanolic extract of C. colosynthis significantly reduced the esterase activities in larvae of E. vittella. The reduction in the esterase activity which is a vital detoxifying mechanism may indications to a substantial control of various insect pests (War et al., 2014). Equivalent results about the retarded esterase activity due to plant extract in insects were perceived (Khosravi et al., 2011). Furthermore, by applying the C. colocynthis extracts on 1 st larval instar of E. vittella reduction in total protein contents was observed. Similar reduction in the activity of α-amylase and deterioration of protease was observed when compared with the control in Glyphodes pyloalis (Khosravi et al., 2011). The decrease in protein content might be due to the interruption intention in ATP synthesis. These insecticidal activities might be due to individual efficacy or the synergistic action of biological compounds present in these plants.

Conclusion
The methanol extract of C. colosynthsis displayed toxic properties on the development and survival of E. vittella. The present results suggested that plant extract-based control measures may be integrated into the pest management programs with special emphasis on the environmental tactics for ecosystem safety.