Potential toxicity of cashew nut shell liquid (CNSL) on adult Bactrocera dorsalis (Hendel) (Tephritidae)

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a serious pest of papaya in all the regions. Cashew nut shell liquid (CNSL) Anacardium occidentale L. is a by-product of the shell of the nut after the kernel has been extracted to utilize as an insecticide to control fruit flies. This study investigated the insecticidal activity of topically applied dichloromethane and hexane extracts of CNSL against B. dorsalis. The LC50 values for the dichloromethane extract represented a 2.54-fold increase in toxicity from 6 h to 24 h (2.18 × 104 ppm) versus a 2.38-fold increase for hexane extracts after 24 h (2.94 × 104 ppm), which mortality was significantly impacted by time and concentration (p < 0.01). A GC-MS analysis of the two extracts showed the presence of higher phenol and 3-pentadecyl-. The CNSL was found to possess insecticidal activity against B. dorsalis, hence could be evaluated in the field to further assess their potential for B. dorsalis control.


Introduction
The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most serious pests of agricultural fruits [1].It attacks over 250 plants of economic importance, including a wide variety of commercial fruits and other agricultural crops [2].Its presence on commercial farms and orchards causes significant financial losses to farmers who lack a robust control measure.Affected fruits are subject to internal feeding by the larvae, which destroy the marketability of the fruits.Also, the slightest injury caused by female ovipositors reduces the market value of the fruits, hence, the infestation of B. dorsalis should remain below the economic injury level for a high-value crop [3].
The major challenge in fruit fly control is to prevent egg-laying by the female flies in the fruits and tender vegetative parts of the host plant.Larval development occurs after eggs hatch inside these tissues.These are areas beyond the reach of normal insecticide spray applications.Since papaya is a fruit that is usually eaten in its fresh form, total reliance on synthetic insecticides to control the pest becomes worrisome and causes a serious challenge [4].Furthermore, it has raised concerns among consumers because some insecticides are persistent and, therefore, need a long time to degrade.Thus, several control methods are integrated to reduce the damage done by the pest and, at the same time, prevent the likelihood of residual toxicity to consumers that may arise from consuming fruits sprayed with insecticides.
Studies show that adult fruit flies, after their emergence, need to regularly feed on carbohydrates and water to survive.For example, females require proteinaceous materials for the development of their gonads [5], hence mixing a killing agent such as the cashew-nut shell liquid with food is similar to the technique commonly referred to as insecticide bait spray.The flies are killed when they come in contact with, or feed on, the bait [6].
Many plant species possess compounds that have a multifaceted action against insects.They act as antifeedant, repellent, insecticidal or growth inhibitors to insects [7,8].For example, azadirachtin, which is isolated from Azadirachta indica A. Juss.(Meliaceae), has toxic and deterrent characteristics against a wide variety of insect species.Thus, it has created interest in plantderived insecticides due to the current concern from the public and policymakers regarding the problem of indiscriminate use and over-application of synthetic insecticides, causing pollution and the development of resistance in insects [9].
Interest in plant-derived compounds has further been boosted by the development of insecticide resistance in various Tephritid insect pests studied.For example, the oriental fruit flies B. dorsalis Hendel and B. cucurbitae Coquillett, field and laboratory breeds in the late 1950s and 1960s, from Hawaii, acquired resistance to DDT and methoxychlor, but not to malathion.However, 40 years later, in laboratory selection experiments with a B. dorsalis Taiwan population, resistant lines were produced not only for malathion and organophosphate but also for insecticides from methomyl and pyrethroid groups [10].Cross-and multi-resistance cases were also reported in the above investigation [11].
The Anacardium occidantale L. Cashew-Nut Shell (CNS) is released into the environment as an agricultural by-product and waste.However, inside the soft honeycomb of the shell, there is a valuable viscous liquid called cashew-nut shell liquid (CNSL) [12].CNSL is a unique source of naturally occurring long-chain hydrocarbon phenols and has been reported to exhibit entomotoxicity against insects [13].Ali et al. (8) [14] reported 65% mortality against the caterpillars of the cabbage white butterfly, Pieris brassicae, using a neem extract alone.This figure was higher than the combined effect of neem + insecticide (pyriproxyfen) though not statistically different.They also reported that mortality in a neem extract was higher than treatments with insecticides alone [15].Younus et al. ( 14), [15] in a study on the insecticidal activity of Euphorbia nivulia extract against the dusky cotton bug, Oxycarenus hyalinipennis, came up with a similar finding.They reported that the chloroform extract of E. nivulia recorded a higher mortality figure at 15% concentration than Oberon, a synthetic insecticide.
A common procedure used by investigators in determining the efficacy of newly-discovered compounds against insects is the contact action between insects and deposits of the pesticides being tested [16].However, the interaction between the insecticide and the insect can be quite complex.It is therefore difficult to ascertain whether the dose is a simple function of the rate of deposit [17].Thus, a more accurate procedure that can give the actual dose responsible for the mortality of the test insect is more reliable.Consequently, the topical application of tested compounds has been found to overcome this problem.
The topical application of insecticides on the dorsal region of the tested insect allows the investigator to make a reasonable estimate of the inherent toxicity of the insecticide by excluding all the other effects that are associated with insect behaviour, especially when the test insect is exposed to irritating and repellent insecticides [18].The outstanding advantage of the topical application method is the degree of precision and replicability of the procedure.Many tests can be done in a relatively short time with a small number of insects per replication.It is cheap in terms of equipment, and fewer toxins and reagents are required.Furthermore, the doses are independent of insect activity [16,19].
Bioassays aimed at evaluating the toxicity of plant extracts using a topical application have been performed by several investigators [20][21][22].This study aimed to evaluate the insecticidal potential of CNSL through the topical application of extracts on the dorsal surface of the thorax of B. dorsalis.

Toxicity by topical application
The topical application method with some modification by using a handheld micropipette (Eppendorf Research plus) was used to apply the various concentrations of the CNSL as described by earlier investigators [16,[22][23][24].Insects were picked individually, and a volume of 0.5 µL of extracts was applied to the dorsal surface of the thorax of each insect with the aid of the micropipette.

Field trapping of adult male B. dorsalis
The source of fruit flies for the experiment was collected from an agricultural farm on mainland Penang.The adult flies were trapped using modified Steiner traps [25,26].Cotton wool soaked in methyl eugenol (ME) (3,3, dimethoxy (l) 2 propenyl benzene) was attached to a hook inside the traps as an attractant.Eleven traps were randomly hung on papaya plants 1.5 metres above the ground in a one-hectare farm.The traps were inspected after three hours.The insects were removed using a battery-powered handheld aspirator and transferred into 30×30×30 cm cages lined with a muslin cloth.The insects were allowed to acclimatize for 48 h in the laboratory before carrying out the experiment.Dead or abnormal individuals were removed from the cages and replaced with healthy individuals before the assays were done.

Maintenance of the Bactrocera dorsalis culture in the laboratory
Colonies of B. dorsalis were maintained in a 30×30× 30 cm cage in the Medical Entomology Laboratory, Vector Control Research Unit, School of Biological Sciences, Universiti Sains Malaysia (5 0 21 21.913 N, 100 0 18 4.575 E) in a room temperature of 28± 2 °C and relative humidity (RH) of 70±10%.The insects were maintained and fed on an artificial diet that consisted of two bananas, six egg yolks, 40 ml of honey, two tablets of vitamin B-complex (which was estimated to be 10 ml of syrup), 10 cm 3 of yeast and 120 gm of sugar.The insect was left to feed adlib [27][28][29].The cages were inspected daily, and dead insects were removed from them.The feeding devices were cleaned every two days to avoid contamination from fungi and bacteria.

Preparation of plant extracts
The cashew nut shell has a porous, spongy pith (honeycomb)-like structure that contains a caustic fluid referred to as cashew nut shell liquid.The liquid protects the nut from insect attack during growth [30].The dried nuts were cracked by hand to remove the kernel.The shells were ground into powder using a commercial electric blender (Panasonic: MX-899TM).The powder was then transferred into a 500 ml bottle and stored in a refrigerator at 4 °C until the extraction process.

Soxhlet extraction
Natural products, such as plant extracts, provide an enormous opportunity for new compound discoveries [31].A soxhlet extractor was used to extract the CNSL from the nuts using two different solvents, dichloromethane and hexane.For each extraction procedure, 40 grams of the smoothly ground cashew nut shell powder was put in a paper thimble (Favorit cellulose extraction thimbles: size 43×123 mm) and mounted on the Soxhlet apparatus, which was set to the appropriate boiling point of the different solvents 39.6 °C for dichloromethane and 69 °C for hexane.For each procedure, 2 litres of the solvent was used; the apparatus was allowed to run for 3 h until the colour of the solvent in the Soxhlet chamber was clear.The extract was concentrated using a Buchi 461 water bath with a Buchi 011 Rotavapor set at 100 rpm to remove the excess solvent.The water temperature was set to the boiling point of the different solvents (39.6 °C for dichloromethane and 69 °C for hexane).The dark brown viscous concentrate was placed in an Ecocell oven set at 40 °C and dried to constant weight.

Experimental design
The extracts were transferred into a vial and used as a stock solution to prepare the various concentrations through a two-fold serial dilution.Since the two extracts were obtained by using dichloromethane and hexane as extracting solvents, acetone was used as a diluent to prepare the various concentrations (0.625, 1.25, 2.5, 5.0, 10.0, and 20.0% v/v).The same set was prepared using acetone alone as a control treatment.
A volume of 0.5 µL of prepared concentrations of dichloromethane and hexane extracts was deposited on the dorsal surface of the thorax of each insect with a micro-pipette (Eppendorf Research plus) separately.The control flies were treated with acetone alone following the same procedure of depositing 0.5 µL on the dorsal surface of the thorax of each insect.Treated insects were transferred individually into 115 cm 3 plastic cups with a 2 × 2 cm opening on the top lid for ventilation.A piece of mosquito netting was glued over the entire opening for ventilation.The insects were observed after 6, 12 and 24 h.Insects were assumed dead if they remained immobile and did not respond or move after probing with a blunt probe.The Completely Randomized Design (CRD) arranged the plastic cups in a tray.Each treatment was replicated four times.
Before applying the dosages, all the insects, including those used in the control treatment, were taken in batches of ten individuals and chilled for three minutes in a Hisense chest freezer at a temperature of −10 0 C before being placed on a chill plate to reduce their activity to ease the topical application procedure of the treatments.The immobilized insects were then picked singly with a pair of forceps to be treated using the dose stated above.For the control set, only acetone was used.A set of four batches of ten insects (n = 40) was used for each dose of concentration.

GC-MS analysis
The GC-MS analysis method adopted the Ravi et al. (31) [32] procedure with changes in solvent dilutions.This analysis of the crude extracts, A. occidentalis, was performed using a GC-MS-QP2010 Ultra (Shimadzu), BPX5 capillary column (30 m × 0.25 film thickness, maximum temperature 370°C).Helium was used as a carrier gas at a constant flow rate of 1.6 ml/min.The oven temperature was programmed from 150°C (hold for 2 min) to 280°C at a rate of 10°C/min.The CNSL extracts were diluted with hexane for the hexane extract and acetone for the dichloromethane extract.A 1000 ppm solution was prepared by adding 0.01gm of extract in a 10 ml solvent and filtering into headspace vials.The particle-free diluted extracts were taken using a syringe and injected into the injector.The percentage composition of the extract constituents was expressed as a percentage by the peak area.The identification and characterization of chemical compounds in various extracts were based on the GC retention time.The mass spectra were computer matched with those of the standards available in the NIST 08 mass spectrum libraries.

Statistical analysis
The data were analysed using the SPSS (Statistical Package of Social Sciences) version 20.The data were subjected to log-probit analysis for calculating LC 50 and LC 95 values.Mortality data were tested for normality and assumptions of multiple regression prior to analysis.A multiple regression analysis was done to determine the relationship between the dependent variable (mortality) and the two independent variables (time and concentration).

Results
This study showed that both the dichloromethane and hexane extracts had a toxic effect against B. dorsalis as shown by their LC 50 and LC 95 values.The LC 50 values for the dichloromethane extract changed from 5.54×10 4 ppm after 6 h of exposure to 2.18×10 4 ppm after 24 h, representing a 2.54-fold increase in toxicity.A similar trend was observed for the LC 95 values, which showed  1).
On the other hand, the LC 50 values of the hexane extract showed a lower level of toxicity against B. dorsalis between the 6 and 24 h exposure periods recording a 2.38-fold increase in toxicity from 7.00×10 4 ppm to 2.94×10 4 ppm (Table 1).Further, scrutiny of the dichloromethane and hexane extracts within the same exposure periods of 6, 12 and 24 h indicated that except for the 12-hour exposure, period, there was an overlap in the 95% CI for LC 50 values, which suggests no significant difference.
The LC 95 value of the hexane extract recorded a 2.10 fold increase in toxicity from the first 6 h of exposure to 24 h (Table 1).It was observed that the 95% CI for the LC 95 values had no overlaps for both a 12 and 24hexposure, indicating a difference in toxicity between the two extracts against B. dorsalis.
The dichloromethane extract recorded an LC 95 value of 8.08×10 4 ppm, and the hexane extracts a value of 19.77×10 4 ppm after 12-hour exposure.However, after 24 h of exposure, the LC 95 values of 5.59×10 4 ppm and 15×10 4 ppm for dichloromethane and hexane, respectively, were recorded (Table 1).
Results from the multiple linear regression after the dependent variable (mortality) was regressed on the predicting variables (time and concentration) significantly predicted mortality for dichloromethane extract F = 137.80;df 2, 81; p < 0.01 (Table 2) and for the hexane extract F = 56.16;df 2, 81; p < 0.01 (Table 3).This indicates that for both the dichloromethane and hexane extracts, time and concentration had a significant impact on mortality.Furthermore, an R 2 value of 0.773 was recorded for dichloromethane and 0.581 for hexane extract (Figure 1B).
Hence, for the CNSL extracted using dichloromethane, for every unit increase in concentration, the   1A), whereas faster reaction can be seen for the cashew nut shell hexane extract.In the hexane extract, for every unit increase in concentration, mortality was increased by 0.411 units.Meanwhile, a unit increase in hours, mortality increased by 1.482 units.Hexane extracts exhibited a fitted regression model equation of y = 3.25 +0.411 concentration + 1.482 h (Figure 1B).The GC-MS analysis of the two extracts showed the CNSL dichloromethane extract contained 15 compounds and had four peaks of phenol and 3-pentadecylconstituting 95.55% of the total compounds detected (Table 4), whereas in the cashew nut shell liquid hexane extract, 14 compounds were detected, and three peaks of phenol, 3-pentadecyl-which constituted 46.75% of the total compounds were detected (Table 5).The constituents of the extracts shown by the peak area and the identification of chemical compounds in two extracts  were based on the GC retention time.The mass spectra were computer matched with standards available in the NIST 08 mass spectrum libraries and a comparison of the GC-MS data with those in the literature [33,34].compounds 4, 5, 6 and 8 in the dichloromethane extract and compounds 1, 3 and 9 in the hexane extract were identified as phenol, 3-pentadecyl-.

Discussions
With restrictions on the use of synthetic insecticides and increasing public demand for organic and pest-free food products, there is a need to develop environmentfriendly pest management technologies such as the use of plant-derived insecticides.Our study demonstrated that both the dichloromethane and hexane extract of CNSL showed insecticidal activity against B. dorsalis, with the dichloromethane extract causing a 100% mortality after 24 h of exposure, while the hexane extract caused an 82.5% mortality on the tested population after a 24 h exposure.The results from this study suggest that both extracts could be used against B. dorsalis.
We attribute the difference in toxicity between the two extracts against B. dorsalis result of the difference in the Cumulatively, the dichloromethane extract registered a higher quantity of this compound than the hexane extract.
Natural CNSL is composed of phenolic compounds with a side chain of fifteen carbon atoms which contain one, two, and three unsaturated bonds [35].The CNSL comprises three main compounds, namely anacardic acid, cardol, and cardanol.These compounds have been found to inhibit the Acetylcholinesterase (AChE) enzyme activity [36].
The inhibition of the AChE causes the accumulation of acetylcholine in the central nervous system and the death of the affected pest [37].The process of inhibiting the enzyme, acetylcholinesterase, is reported to be the mode of action of many insecticides [38], hence the presence of the phenolic compound phenol, 3pentadecyl-in our CNSL extracts may also control B. dorsalis through this mechanism.
Gerolt (38) [39], investigating the most vulnerable point for topically applied insecticides, reported that the exceedingly susceptible areas are the head and prothorax regions.The areas of application further away from these points are less effective.He went on to point out that the slowest response was observed with an application to the anal region.This viewpoint was supported by Sugiura et al. (39), [40,41] who stated that the speed of action was thought to depend on the distance between the point of application and the site of action, that is the central nervous system.The authors further reported that the knockdown time for pyrethroids following direct aerosol spraying on the German cockroach, Blattella germanica, was fastest when the insecticide was applied to the mesothoracic spiracle of the insect.This procedure was adopted in our study, in which the extracts were deposited on the dorsal surface of the mesothoracic region.This might have been why a 100% mortality was achieved with the dichloromethane extract, and 82.5% was recorded for the hexane extract after a 24-hour exposure.
The topical application of plant-derived extracts has shown high toxicity against insects, just like synthetic insecticides.Using the same procedure, Srivastava et al. (41) [42] obtained results with myristicin from Piper mullesua against the 4th instar larvae of Spilarctia obliqua and after a 24-hour exposure, the LD 50 was 104 mg/larva.
The dichloromethane extracts outperforming the hexane extracts by recording higher mortality on test insects have been reported by Pung and Srimongkolchai (42) [43].Crude extracts of Lantana camara flowers from these solvents were topically applied on the third thoracic segment of the second instar larvae of Spodoptera litura.The dichloromethane extract inflicted a 56% mortality, whereas the hexane extract caused 34% deaths in 7 days.The findings of our current study on B. dorsalis gave a better result in that topically applied dichloromethane, and hexane extracts caused higher mortality after a 24-hour exposure.
The GC-MS analysis of the two CNSL extracts showed the presence of phenol, 3-pentadecyl-, which is at times referred to as cardanol, tetrahydroanacardol, or hydro cardanol.The toxicity of this compound is often associated with the presence of one or two functional groups.Lomonaco et al. (34) [35] reported that cardanol's bioactivity might be due to the presence of the double bonds in its side chain.
Poonsri et al. ( 43) [44] reported on the median lethal dose (LD 50 ) of various Bauhinia scandens extracts against second instars larvae of Plutella xylostella.They tested four extracting solvents (hexane, dichloromethane, ethyl acetate, and ethanol) and in descending order of their toxicity, dichloromethane was found to be the most toxic extract (dichloromethane > hexane > ethyl acetate > ethanol).This is similar to our findings, in that the dichloromethane extract exhibited a more toxic effect against B. dorsalis than the hexane extract.
Information about CNSL extracts as a botanical insecticide against B. dorsalis is limited.Thus the study, adds to the existing data and supports the findings of other investigators who have worked on the insecticidal activity of CNSL extracts [35][36][37][38][39][40][41][42][43][44][45][46].It has demonstrated its suitability as a possible candidate that could be used as a botanical insecticide against B. dorsalis, a pest in many regions of the world where papaya is grown.

Conclusion
The results from this study confirm the insecticidal properties of CNSL against B. dorsalis pests.It has also indicated that the dichloromethane extract was more toxic than the hexane extract against B. dorsalis because of the lower LC 50 values recorded and the percentage mortality achieved in 24 h.While a 100% mortality of the population after 24 h was observed for the dichloromethane extract, the hexane extract, on the other hand, required either a higher concentration or a prolonged period of exposure to achieve the same level of mortality as was obtained with the dichloromethane extract.However, since the topical application method allows a reasonable estimation of the inherent toxicity of the extracts against the test insects, the results could be used to determine the required concentration to be applied.Furthermore, in the event that there is an interest to commercialize the product, the findings of this study could be a guide in formulating ecological and environmentally safe plant-based insecticides.

Figure 1 .
Figure 1.Multiple linear regression model to explain the mortality of Bactrocera dorsalis adults after the topical application of (A) dichloromethane and (B) hexane extracts of Cashew Nut Shell Liquid (CNSL) at 6, 12 and 24 h after application.

Table 1 .
LC 50 and LC 95 values of the dichloromethane and hexane extracts of Cashew Nut Shell Liquid 6, 12 and 24 h after their topical application on Bactrocera dorsalis male adults.

Table 2 .
Multiple linear regression between factors (concentration and hours) and mortality of Bactrocera dorsalis by dichloromethane extract of cashew nut shell liquid.

Table 3 .
Multiple linear regression between factors (concentration and hours) and mortality of Bactrocera dorsalis by hexane extract of cashew nut shell liquid.
df = degree of freedom, SS = sum of squares, MS = mean square, F = f-value, p = p-value * * * Significant at p < 0.001 mortality was increased by 0.878 units.If a unit increase in hours, mortality was increased by 1.589 units.The fitted regression model for dichloromethane was a y = 3.25 +0.878 concentration + 1.589 h (Figure

Table 4 .
GC-MS result Cashew Nut Shell Liquid dichloromethane extract.

Table 5 .
GC-MS analysis of Cashew Nut Shell Liquid hexane extract.