Accepted Manuscript

Title: Novel and environmental friendly approach; Impact of

Neem (Azadirachta indica) gum nano formulation (NGNF) on
Helicoverpa armigera (Hub.) and Spodoptera litura (Fab.)

Authors: Chinnaperumal Kamaraj, Pachiyappan Rajiv Gandhi,

Gandhi Elango, Sengodan Karthi, Ill-Min Chung,
Govindsamy Rajakumar

PII: S0141-8130(17)32107-4
DOI: http://dx.doi.org/10.1016/j.ijbiomac.2017.08.145
Reference: BIOMAC 8138

To appear in: International Journal of Biological Macromolecules

Received date: 12-6-2017

Revised date: 14-8-2017
Accepted date: 27-8-2017

Please cite this article as: Chinnaperumal Kamaraj, Pachiyappan Rajiv

Gandhi, Gandhi Elango, Sengodan Karthi, Ill-Min Chung, Govindsamy
Rajakumar, Novel and environmental friendly approach; Impact of Neem
(Azadirachta indica) gum nano formulation (NGNF) on Helicoverpa armigera
(Hub.) and Spodoptera litura (Fab.), International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.145

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Novel and environmental friendly approach; Impact of Neem (Azadirachta indica) gum

nano formulation (NGNF) on Helicoverpa armigera (Hub.) and Spodoptera litura (Fab.)

Chinnaperumal Kamaraj1, Pachiyappan Rajiv Gandhi2, Gandhi Elango3, Sengodan Karthi4,

Ill-Min Chung5, Govindsamy Rajakumar5*

1
Department of Biotechnology, School of Biosciences, Periyar University, Periyar Palkalai

Nagar, Salem - 636 011, Tamil Nadu, India

2
Post Graduate and Research Department of Zoology, Auxilium College (Autonomous),

(Affiliated to Thiruvalluvar University), Gandhi Nagar - 632 006, Vellore, Tamil Nadu, India
3
Department of Zoology, Government Thirumagal Mills College, Gudiyattam-632602 Tamil

Nadu, India
4
Department of Biochemistry, Centre for Biological Sciences, K.S Rangasamy College of

Arts and Science (Autonomous), Tiruchengode - 637 215, Namakkal, Tamil Nadu, India
5
Department of Applied Bioscience, College of Life and Environmental Science,

Konkuk University, South Korea.

_______________

* Corresponding author.

E-mail address: kamaraj84@hotmail.com (Dr. C. Kamaraj),

govindr@konkuk.ac.kr (Dr. G. Rajakumar).

1
Highlights

 Neem gum mediated nano formulation (NGNF) as a bio-pesticide agent against

Helicoverpa armigera and Spodoptera litura.

 NGNF exhibited potential antifeedant, larvicidal and pupicidal activities against

Helicoverpa armigera and Spodoptera litura.

 Environmental toxicity was assessed by earthworm (Eudrilus eugeniae) there is no

significant toxicity observed in NGNF treatments.

ABSTRACT

The future of this study was to prepare a natural pesticide which will not harm the

environment and yet control pests. Neem gum nano formulation (NGNF), a novel

biopesticide prepared from the Neem gum extract (Azadirachta indica) (NGE) was evaluated

for its antifeedant, larvicidal and pupicidal activities against Helicoverpa armigera (Hub.)

and Spodoptera litura (Fab.) at 100ppm. The NGNF showed significant (100 %) antifeedant,

larvicidal and pupicidal activities against H. armigera and S. litura. The LC50 values of

10.20, 12.49 and LC90 values of 32.68, 36.68 ppm on H. armigera and S. litura, respectively

at 100ppm. The NGNF treatments showed differences in the activities of detoxifying

enzymes, carboxylesterases, glucosidases and glutathione S-transferases in the larval gut.

Earthworm toxicity illustrated that 6.25 ppm of chemical insecticides (cypermethrin) varied

widely in their contact toxicities compared to 100 ppm of NGNF and control in both contact

filter paper and artificial soil test. The NGNF were characterized and confirmed by FTIR,

XRD, SEM and EDX analysis. Ten compounds were identified from the Neem gum extract

(NGE) by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The major

compounds were fatty acids like Hexadecanoic acid, oleic acid, and ricinoleic acid. NGNF

could be used as an agent to prepare novel bio-pesticides formulations.

2
Key words

Helicoverpa armigera; Spodoptera litura; NGNF; Larvicidal; Detoxifying enzymes; GC-MS

1. Introduction

The extensive use of synthetic insecticides during the last five decades has sire

environmental pollution and also in the development of physiological resistance in major

Lepidopteran pests species in addition to the increased costs of insecticides. The synthetic

pesticides persist in the aquatic environment and accumulate in lipids, facilitating their bio-

magnification in aquatic food chains and toxicity to predators, and represent a risk for human

health (Schlechtriem et al., 2012). Botanical insecticides have been instrumental in the

discovery and development of synthetic chemical products. Examining botanical insecticide

chemistry provides a solid foundation built upon evolutionary selection for chemistries that

may be more specific to a pest or pathogen, while having faster degradation, lower

environmental persistence, and usually do not bioaccumulate in the environment. Thus

continued discovery and development of botanical-based products exemplify the direction for

designing new, better pesticides (Kamaraj et al., 2008a, b; Senthil-Nathan, 2015).

Nanotechnology will be a bottom-up technology, dwelling upward from the molecular

scale. It will make a hit with an action in cro-magnon man abilities relish that brought by

production industries. Synthesis and formulation of patrician metal oxide nanoparticles, in

diverse, titanium dioxide nano-formulations, using fabricate gum has become a major focus

3
for researchers guerdon to their purity of procedures, stability, and their weight applications

in chemical sensing, biological imaging, gene silencing, and drug delivery (Wei and Qian,

2008). They have versatile properties such as self-assembly, specificity, encapsulation,

stability, and biocompatibility. Among the available nanomaterials, titanium dioxide

nanoparticles form a major part of the medical and agricultural industry due to their

possession of anticancer, antimicrobial, antileishmanial, antiviral, antiparasitic and larvicidal

properties (Rajakumar et al., 2014; Xu et al., 2015; Nakayama et al., 2016; Tahir et al.,2016;

Abamor and Allahverdiyev,2016; Kamaraj et al., 2017).

The cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a

worldwide polyphagous pest inflicting crop damage in India to the sum of one billion dollars

annually and it attacks over 200 crop species belonging to 45 families (Choudhury et

al.,2010). H. armigera is a major pest in many economically important crops including

cotton, pigeonpea, chickpea, tomato, okra, and blackgram. The tobacco armyworm

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is an economically important

polyphagous pest in India, China, and Japan, causing enormous economic ceasing to exist to

manifold vegetable and employment crops. This vermin attacks greater than 112 line of

cultivated crops and causes severe loss varies between 10% and 30% for major crops (Baskar

et al., 2011; Ferry et al., 2004). These pests are well justified in their polyphagy on all

economically important crops and the hurdles in its management. This necessitates the seek

for in a superior way potent insecticides which are safer to the user and consumer.

Earthworms are soil invertebrate it is best bio-indicators and decomposers associated with

soil fertility and fauna diversity. They are the essential producer of the vermicomposting

process and increase the soil surface area for microbes by fragmentation of the ingested

material through muscular action (Lazcano et al., 2008; Ravindran et al., 2015). Eudrilus

eugeniae (Kinberg) inhabits the surface layer (epigeic) of moist soils with a rich organic

4
matter which utilized in organic, industrial, and biotechnology for vermicomposting

(Nattudurai et al., 2014). Changes in earthworm behavior depend in large part upon the

degree of exposure to contaminants in the soil (Langdon et al., 2005). In terrestrial

ecotoxicology, earthworms are sensitive indicators of soil quality (Gao et al., 2015). Neem

gum is exudates of naturally occurring water-soluble, complex polysaccharides derived from

the bark of Azadirachta indica (family Meliaceae). The Neem gum is clear, bright and brown

colored which was used in many industries such as cosmetics, paper, textile, and

pharmaceuticals (Srivastava and Rai, 1963; Ogunjimi and Alebiowu, 2013).The plant-based

gum is a heteropolysaccharide of natural gum, and its morphological, compositional,

physicochemical, thermal, rheological, and emulsifying properties have been well studied

(Srivastava and Rai, 1963; Aspinall et al., 1965; Jefferies et al., 1977).

The biopolymer of gum is a rich-arabinose, acidic protein mixed calcium, magnesium,

potassium and sodium salts (Tischer et al., 2002). Such interesting features of the neem gum

encouraged us to use this biopolymer as a template for the nanoformulation and stabilization

of NGNF due to its (i) water solubility and easy availability; (ii) non-toxic and natural

materials; (iii) abundance of hydroxyl, acetyl, carbonyl and carboxylic functional groups; and

(iv) metal-biosorption properties. Therefore, in the present study, we have reported for the

first time the detailed investigation of the nano formulation procedure for the production of

NGNF using the aqueous extract of neem gum. The synthesis was carried out in an aqueous

suspension of the gum extract by autoclaving, without the addition of any external chemical

reducing agent. The present study aims to (a) determination of major active compounds

present in Neem gum extract (NGE) using GC-MS technique; (b) detection of behavioural

changes in H. armigera and S. litura treating with different concentrations of NGNF; (c) the,

nano-formulated NGNF characterized and confirmed by FTIR, XRD, EDX, and SEM; (e)

evaluation of toxicity of NGNF to earthworm E. eugeniae compared to chemical pesticides.

5
2. Materials and methods

2.1. Insect culture

H. armigera and S. litura cultures were maintained at the laboratory. Post-hatching larvae

were reared on castor leaves (Ricinus communis) to the prepupal stage. Pre-pupae were

detached and provided with vermiculture clay as pupation sites. Emerging adult moths were

transferred to cages and fed on a 10% (w/v) sucrose solution. Moths were transferred at a

ratio of 1 male: 2 females to oviposition cages containing castor leaves and covered with

sterilized muslin cloth for oviposition. The muslin cloth containing eggs were moistened to

enhance hatching. After hatching the larvae were reared on new growth castor leaves. These

laboratory reared larvae were used for bioassay. All experiments and culture were carried out

at 27 ± 2 ºC, 85% relative humidity, with a 14 h light: 10 h dark cycle (Kamaraj et al., 2008 a,

b; Srinivasan et al., 2016).

2.2. Collection and preparation Neem gum suspension

Crude Neem gum was collected from the incised bark of A. indica trees in the forest area

of Amman Nagar, India. The gum was dried under direct sunlight for 2 weeks to completely

remove the moisture. A fine powder was obtained from the dried gum using a kitchen

blender. The gum powder was sieved to separate a powder form of the particles using a 25 m

sieve. A homogenous 0.5% (w/v) gum stock solution was prepared by adding the fine powder

of sieved gum to a brown bottle containing Milli-Q water with constant stirring on a magnetic

stirrer at 60 ºC for 2 h. Then this solution was centrifuged to revoke the insoluble materials,

and the supernatant was used for all the experiments.

2.3. Synthesis of NGNF

6
 Titanium tetrachloride (TiCl4) (99.9% pure) analytical grade were purchased from Sigma-

Aldrich, India. A. indica gum as a stabilizing agent. For the synthesis of NGNF, the

Erlenmeyer flask containing 100 mL of 5 mM TiCl4 was stirred for 2 h. Diff erent

concentrations of neem gum aqueous suspension were prepared and interacted with the TiCl4

solution mixing ratio of 5:95, 10:90, 15:85, 20:80 and 25:75 mL, separately. 20 mL of gum

aqueous suspension was added to 80 mL of TiCl4 solution for the optimization of NGNF

synthesis. The pure TiCl4 solution and the aqueous gum suspension did not show any color

change. Whereas aqueous gum suspension with TiCl4 showed the change of color to light

brown. Diff erent reaction parameters (concentrations of aqueous gum suspension, substrate

concentrations, pH, temperature and reaction time) were optimized to synthesize NGNF with

controlled properties.

2.4. Characterization of synthesized NGNF

The NGNF solution thus obtained was purified by repeated centrifugation at 12,000 rpm

for 20 min followed by re-suspension of the pellet in Milli-Q water and filtered through

Millipore filter (0.45 μm). In X-ray diffraction studies (XRD), dried nanoparticles were

coated on an XRD grid, and the spectra were recorded by using a Philips PW 1830 X-ray

generator operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation.

Fourier transform infrared (FT-IR) spectroscopic analysis of the samples was measured using

a PerkinElmer Spectrum one instrument in the diffuse reflectance mode at a resolution of 4

cm−1 in KBr pellets. Powder samples for the FTIR were prepared similarly to powder

diffraction measurements. FTIR spectra of the synthesized NGNF display the possible

functional groups and the formation of nanoformulation. For the scanning electron

microscopic studies, 25 μL of sample was sputter-coated on the copper stub. Morphological

7
and topographical analysis of the nanoformulations were investigated by scanning electron

microscopy (SEM; JEOL, model JFC-1600) equipped with EDX.

2.5. Larvicidal bioassay

Bioassays were performed with second, third and fourth instars of H. armigera and S.

litura were using at 6.25, 12.5, 25, 50 and 100 ppm concentrations of NGNF. Sterile castor

leaves (75-125cm2) were sprayed with different concentrations of NGNF and air dried for 10

min to remove the excess moisture content. Control leaves were treated with sterile distilled

water containing 0.05% of ethyl alcohol at the ratio of (1:1). The treated leaves were placed

in the bioassay chamber (9 X 5 X 4 cm3) checked with wetted cotton and tissue paper which

provide humidity and water supply for the leaves. The bioassay chamber was incubated at 28

± 1ºC with 95% humidity and 15:9 (L: D) photoperiod. A minimum of 20

larvae/concentration were used for all the treatments, and these treatments were replicated

five times (n = 100). The dried leaves were transferred every 24 h, and replaced with fresh

untreated castor leaves. The mortality was observed from the fourth-day post treatment to day

10. The percentage mortality was calculated by using the formula (1) and corrections for

natural mortality when necessary were done by using Abbott's formula (1925) (2) and

subjected to Probit analysis (Finney, 1971) to calculate the treatment concentrations for

biological studies.

Number of dead larvae

(1)
Percentage of mortality = X 100
Number of larvae introduced

n in T after treatment
X 100 (2)
Corrected percentage of mortality = 1 -
n in C after treatment

8
2.6. Antifeedant bioassay

Antifeedant bioassay of the NGNF was studied using leaf disc no choice method (Isman et

al., 1990). Fresh castor leaves discs of 4 cm in diameter were punched using cork borer and

were dipped in NGNF at 6.25, 12.5, 25, 50 and 100 ppm concentrations. The leaf discs

treated with water were used as negative control, and Azadirachtin (40.86% purity, obtained

from EID-Parry, India Ltd., Chennai) was used as positive control. In each petri dish (1.5 cm

X 9 cm) wet filter paper was placed to avoid the early drying of the leaf discs, and one third

instar larva was introduced into each petri dish. Progressive consumption of leaf area by the

treated and control larvae after 24 h was recorded utilizing leaf area meter. Leaf area, eaten

by larvae in treatment was corrected from the negative control. Five replicates were

maintained for each treatment. The antifeedant activity was calculated using the formula (3)

of Isman et al. (1990).

Leaf area consumed in control - treated leaf

Antifeedant activity= = X100 (3)
Leaf area consumed in control + treated leaf

2.7. Pupicidal bioassay

The method was described by Arasu et al. (2013) with minor modification; the larvae

which survived were continuously fed with a normal diet as specified in the larvicidal

activity until they became pupae and adults. Pupicidal activity was calculated by subtracting

the number of emerging adults from the total number of pupae.

2.8. Enzymatic analysis of H. armigera and S. litura

2.8.1. β-glucosidase (Glu) activity

Insect guts from NGE, NGNF treated and untreated larvae were homogenized in 0.1 M

potassium phosphate buffer (pH 7.0) and centrifuged to collect the supernatant for further

9
experiments. Gut extracts were stored at-20 ºC till use. Estimation of b-glucosidase was done

according to the Low et al. (1986) method. The reaction mixture containing 1 mL of 0.1 M

acetate buffer (pH 5), 0.5 mL of p-nitro phenyl glucopyranoside (PNPG-0.02M) and 20 µL of

enzyme solution extracted from the insect gut was incubated for 15 min at 37 ºC. To this 2

mL of 0.2 M, sodium carbonate was added to stop the reaction. Absorbance was read at 400

nm using water as a blank.

2.8.2. Carboxyl esterase (CaE) activity

Estimation of carboxyl esterase enzyme activity was done according to the method

described by Govindappa et al. (1987) with minor modifications. Larval gut extracts were

obtained by homogenizing the insect guts in 0.1 M potassium phosphate buffer (pH 7.0) and

centrifuging. The supernatant was collected and stored at -20 ºC till further use. Estimation of

carboxyl esterase was done by taking 150 µL of crude homogenate and 1.35 mL of 0.27 mM

of a-naphthyl acetate and incubated for 20 min at 30 0C. To this 500 lL of 1% fast blue salt

and 5%, sodium lauryl sulfate was added in 2:5 v/v ratios. Absorbance was read at 600 nm.

2.8.3. Glutathione-S-transferase (GST) activity

This enzymatic assay was carried according to Habig et al. (1974). Potassium phosphate

buffer (0.1 M, pH-7.0) was used to homogenize the larval insect guts. Estimation of enzyme

activity required reaction mixture of 1 mM substrate, CDNB (1-chloro- 2,4-dinitrobenzene),

1 mL of 1 mM Glutathione reduced (GSH), 1 mL of phosphate buffer (pH-7.0) along with

0.1 mL of crude enzyme extract obtained from the insect larval guts. Formation of S-(DNP)

GS was continuously monitored at 340 nm up to 5 min with 30 s interval.

10
2.9. Earthworm toxicity

2.9.1. Earthworm culture

The earthworm, E. eugeniae was used as stock and maintained in the laboratory. The

earthworm was cultured at an average ambient temperature of 28.9 ± 0.36 ºC, using the crop

residues were amended with cattle dung as the food source. Earthworms and cocoons,

produced during the experiment, was separated from the soil by hand sorting, after which

worms were washed in normal water to remove adhering material from their body, and

subsequently weighed, live weight basis. Then all measured earthworms were individually

kept in a separate glass vial. Separated cocoon was counted and introduced in separate

bedding containing the same material in which their parents were reared.

2.9.2. Contact toxicity using filter paper method

The contact filter paper test was performed with a slight modification of OECD (1984). A

piece of filter paper was placed in a 9 cm Petri plate dish and treated with an extract made

from NGNF and pesticides. The treated filter paper was remoistened with 2 ml distilled

water, and one earthworm was placed on it. The petriplates were incubated in the dark at 20 ±

1 ºC for 48 h and mortality was recorded. A preliminary test was carried out to determine the

mortality concentration range for each chemical. Three different concentrations of NGNF,

pesticides and a control were included. Five replicates per concentration were performed.

Treated earthworms were inserted to plates held at 20 ± 1 ºC under 80-85% relative humidity

in the dark, for 48 h.

2.9.3. Artificial soil toxicity analysis

According to OECD (1984), artificial soil comprised of 10% ground sphagnum peat, 20%

kaolinite clay, 70% fine sand was used for artificial soil tests. A small amount of calcium

11
carbonate was added to adjust the pH to 6.0 ± 0.5. The water content was adjusted to 35% of

the dry weight in toxicity test. The desired amount of chemical insecticide (cypermethrin) or

NGNF was dissolved and mixed into a small quantity of fine sand for every test

concentration. The sand was mixed for 1 h to evaporate the solvents and then mixed

thoroughly with the pre-moistened artificial soil in a house hold mixer. The final moisture

content of artificial soil was adjusted to the appropriate level by the addition of distilled

water. A total of 0.65 kg soil was placed in a 500 ml glass jar, with 10 adult earth worms

added to each jar. Polypropylene lids, were used to cover the jars loosely to allow the

exchange of air, and stored at 20 ± 1 0C with 80-85% RH under constant light. Mortality was

measured at 2, 7 and 14day post treatment of 0.1% chemical insecticides or NGNF. Two

concentrations of NGNF (0.5 and 1.0%), were used to determine a mortality concentration,

tests replicated five times. For each concentration, ten adult earthworms were used.

Earthworms were held for 24 h under controlled conditions as described above in the

untreated soil, before use.

2.10. Chemical characterization of the NGE

The chemical compositions of NGE were analyzed using GC-MS (GCD-HP1800A

system, Hewlett-Packard, USA) equipped with a split/splitless capillary injection port. For

GC-MS detection, an electron ionization system (quadruples analyzer; mass range, 10-425

amu) with ionization energy of 70 eV was used. Each of these steps was carried out under

high vacuum (10-4 to 10-8 Torr). Helium gas was used as a carrier at a constant flow rate of 1

mL/min. Injector and mass transfer line temperatures were set at 250°C and 280°C,

respectively. The NGE constituents were identified after comparison with those available in

the computer library (NIST) attached to the GC-MS instrument and reported.

2.11. Statistical analysis

12
 The data related to antifeedant, larvicidal and pupicidal activities were analyzed using

one-way ANOVA. Significant differences between treatments were determined using

Tukey’s multiple range tests (P ≤ 0.05) (Benelli, 2017). Analyses were performed with the

original data since even after transformation with various approaches (the arcsin, logarithmic,

and square root methods), the distribution of the data did not show significant deviations

from normality. Since data in days was normally distributed, log transformation was not

done. Analyses were performed with the original data. LC50 and LC90 values were calculated

using probit analysis (Finney, 1971).

3. Results

3.1. FTIR analysis

The FTIR spectrum of TiCl4, NGE, and NGNF are shown in Fig. 1A, B, and C

respectively. The functional group of the TiCl4 showed characteristic bands at 3430 and 1643

cm-1 which correspond to the surface water and a hydroxyl group (Fig. 1A). The band

intensities of the spectrum for the NGE which were found to be 3424 (O–H stretch, H–

bonded; alcohols, phenols), 2923 (C–H stretch; alkanes),1727 (C=O stretch;α,β –unsaturated

esters),1618 (N–H bend; 1° amines),1382 (C–H bend; alkanes), 1060 (C–N stretch; aliphatic

amines), 823 (C–Cl stretch; alkyl halides), 796 (C–Cl stretch; alkyl halides) and 598 cm−1

(C–Br stretch, alkyl halides) (Fig. 1B). Similarly, Fig. 1C revealed the characteristic

functional group of biosynthesized NGNF at 3423 (O–H stretch, H–bonded; alcohols,

phenols),2929 (C–H stretch; alkanes), 2126 (–C≡C– stretch; alkynes), 1612(N–H bend; 1°

amines), 1522 (N–O asymmetric stretch; nitro compounds),1442 (C–H bend alkanes),1289

(C–H wag (–CH2 X) alkyl halides), 1036 (C–N stretch aliphatic amines) and 546 cm−1 (C–Br

stretch alkyl halides).

13
3.2. X-ray diffraction analysis

The crystalline nature of the NGNF analyzed using XRD. The mean particle diameter of

the nanoparticles was calculated from the XRD pattern using the Scherrer equation. XRD

pattern for the control sample (TiCl4) (Fig. 2A) and synthesized NGNF (Fig. 2B) were

recorded, and the data were compared. The XRD pattern of the synthesized NGNF revealed

intense peaks at 31.92º, 36.14º, 41.15º, 54.39º, 56.55º and 68.00º corresponding to (1 0 0), (1

0 1), (1 1 1), (2 1 1), (2 2 0) and (3 0 1) Bragg’s reflection, respectively. Which is found to be

that of the rutile form as when compared with the JCPDS data (File No. 99-101-0954). The

main peak of 2 θ = 36.14 matches the (101) crystallographic plane of rutile form of NGNF,

indicating that the nanoparticles structure produced is only rutile form and not of anatase or

brookite form. The crystallite sizes were calculated using Scherrer’s formula applied to the

major intense peaks and found to be in the range of 31.27.

3.3. SEM analysis

The surface morphology of NGNF was investigated using SEM. The NGNF were

distributed uniformly on the surface with formation of aggregated nanoparticles. It shows that

the nanoparticles were densely dispersed with a narrow range of dispersion. Particles were of

nanosize with a smooth surface. Fig. 3A, B, and C show that the size of the NGNF was very

consistent at different magnifications. EDX is showing chemical composition and purity of

the NGNF (Fig. 3D). The particle size of the nanoparticles ranges in size from 20 to 40.83 nm

and the individual size of 31.27 nm. The results indicate that these surface planes having

different packing structures were energetically similar to each other. The formation of NGNF

synthesized from Neem gum with two insets showing the spherical and uneven nanoparticles.

The surface of NGNF was analyzed, and the aggregated structures possess considerable

surface roughness.

14
3.4. Larvicidal activity

The present investigation revealed that the NGNF exhibited highest larval mortality

against second, third and fourth instar larvae of H. armigera and S. litura at 100 ppm with

percentage mortality of 100, 96.40 and 92.46 %; 100, 90.57 and 86.80%, followed by neem

gum extract (NGE) showed 76.56, 74.48 and 68.77%; 76.29, 72.65 and 68.43%; TiCl4 found

to be low activity 4.06, 4.33 and 2.00%; 3.60, 4.46 and 1.00 % , against H. armigera and S.

litura respectively at 100ppm (Fig.4.). The NGNF and NGE showed distinct toxicity effect

on the larvae of H. armigera and S. litura. The larvae which had consumed less amount of

treated diet showed a higher amount of larval mortality. The LC50 values were 10.20, 38.36,

12.49 and LC90 values were 32.68, 120.59, 36.68, and 92.88 ppm, respectively at 100 ppm

against H. armigera and S. litura (Table 1).

3.5. Antifeedant activity

The results presented in Table 2, the antifeedant activity revealed that the all tested

components except TiCl4 showed more than 50% feeding deterrent activity. The Higher

antifeedant activity of 100% and 100% was found with NGNF at 100 ppm concentrations

respectively. The minimum antifeedant activity of 74.82% and 82.21% was observed at 100

ppm concentrations with NGE. Were positive control Azadirachtin exhibited 68.26 and 76.80

% antifeedant activity against larvae of H. armigera and S. litura, respectively. In the present

study, NGNF revealed a strong antifeedant activity against H. armigera and S. litura at 100

ppm concentration and the activity was statistically significant over control.

3.6. Pupicidal activity

15
 The higher pupicidal activity of 100% and 100% was observed in NGNF at 100 ppm

concentrations, followed by 68.40% and 72.09% at 100 ppm in NGE (Table 3). 100% pupal

mortality was recorded with higher concentrations of NGNF, similar to azadirachtin the

positive control showed 78.85 and 86.20, at 100 ppm concentration. The lowest pupal

mortality of 2.06 and 3.42 % was observed in TiCl4 at 100 ppm concentration, respectively H.

armigera and S. litura larvae. There was no pupicidal effect in control. Different degrees of

abnormalities such as larval-pupal intermediate and pupal-adult intermediate were observed.

It was also observed that most of the treated larvae were not able to cross further

developmental stages. Pupicidal activities were statistically significant with increasing

concentrations of the tested components. In general, prolonged larval-pupal durations were

directly proportionate to the increase in pupicidal activities. Different degrees of

abnormalities such as larval, pupal and adult were noticed in both the insects.

3.7. Metal detoxifying enzymes activities

The glucosidase enzyme activity was a decrease in the H. armigera and S. litura larval gut

in both the treatments, in all the doses of NGNF, NGE and TiCl4 except at 100 ppm

treatment, where there was an increase in the activity as compared to control larvae (Fig. 5A).

With respect to S. litura, there was a decrease in the glucosidase activity except at 100 ppm

concentration of NGNF fed through the leaf diet, whereas an increase in the glucosidase

activity in the larval gut was recorded on NGNF. Carboxyl esterase enzyme activity in H.

armigera larval gut decreased and was statistically significant on treatment with NGNF and

NGE when compared to control except at 100ppm of TiCl4 (Fig. 5B). Contrary to H.

armigera, there was an increase in the CaE activity in S. litura larval gut with statistical

significance (p < 0.001). Glutathione-S-transferase activity increased at the 100 ppm NGNF

treatments in H. armigera larval gut and similar results were obtained with S. litura at 100

16
ppm concentrations (Fig. 5C). On nano formulation treatment, an increase in the GST activity

was observed at 100ppm concentrations in H. armigera larval gut. In the case of S. litura

there was an increase in the GST activity in various concentrations.

3.8. Earthworm toxicity

3.8.1. Filter paper test

The present observations filter paper bioassays on earthworms demonstrated a wide

variety of responses to NGNF, NGE, and chemical insecticides. Concentrations of 6.25 to

100ppm NGNF, NGE, and the insecticide: cypermethrin (at 100ppm) were analyzed.

Cypermethrin had the greatest toxicity compared to NGNF and NGE at 100ppm. In contrast,

the NGNF and NGE were not toxic to earthworms for 72hr treatment (Fig. 6).

3.8.2. Artificial soil test

In a 72hr exposure period in artificial soil test mortality of earthworms was not observed

for control groups. In contrast, increased mortality occurred in earthworms from artificial soil

tests treated with chemical pesticide (cypermethrin). Cypermethrin resulted in greater

mortality rates versus NGNF, NGE, and control. Whereas, earthworms in trials treated with

NGNF had lower mortality rates and improved biomass of adult earthworms even when

exposed for 72d (Fig. 6). Earthworms treated with 100ppm chemical pesticide cypermethrin

had a decreasing mean percentage of weight (biomass) across the 24, 48 and 72hr trials. The

greatest decline of weight was observed in the cypermethrin at 24, 48 and 72hr exposures at

100ppm concentrations, respectively.

3.9. Chemical characterization through GC-MS

17
 The results identified ten chemical compounds in the NGE, molecular formula, molecular

weight (g/mol), retention time (RT) and chemical structures presented in (Table 4), with their

peak area. The peak area correlated to concentration and was greater for Oleic acid (31.45

%). Compounds also identified included: 3, 7, 11, 15- tetramethyl-2 hexadecen-1-ol (8.60 %),

3, 7-dimethyl-1, 7-octadien-3-amine (6.40 %), Dimethyl 4-aminobenzene-1,2-dioate (28.26

%), Acetic acid, 1-[2-(2,2,6-trimethyl-bicyclo[4.1.0]hept-1-yl)-ethyl]-vinyl ester (5.10 %), n-

hexadecanoic acid (6.40 %), Linoleic acid (9.53 %), 1,4 Dioxaspiro[4.5]decane (4.60 %) and

Ricinoleic acid methyl ester (16.84%).

4. Discussion

Chemical pesticides can cause increased toxicity to valuable organisms in a treated

ecosystem. This can affect parasitoids, predators, soil organisms, including earthworms, and

soil-borne microbes. The health and vitality of soils are strongly linked to the presence of the

soil fauna (Kinney et al., 2012). Chemical pollution by pesticides primarily due to their

improper use is a recurring problem in the management of various pests or weeds (Chen et

al., 2014). Whereas resistance to various insecticides is a worldwide problem (Jit et al., 2011;

Yadav et al., 2015), this is especially the case for H. armigera and S. litura, which most

insecticides have failed to adequately control (Zaka et al., 2014). While there are reports on

the sustenance of plant populations to nanoparticles, but studies on the effect of titanium

dioxide nanoparticles (TiO2NPs) treatments on herbivore feeding remain scarce. The authors

have not come across any publication reporting effect of TiO 2NPs treatments on the

development and growth of lepidopteran insect pests of castor plants when fed upon treated

diets. Hence the present study is supposed to be the first to investigate the effect of NGNF

through feeding in H. armigera and S. litura.

18
 The insecticidal properties of nanoparticles are due to their morphological features that

cause physiological changes (Nel et al., 2006) and their use is new in the field of insect pest

management (Bhattacharyya et al., 2010). NPs prepared with aqueous leaf extract of

Manilkara zapota have been tested for insecticidal properties against Musca domestica

(Kamaraj et al., 2012). The present study showed reduction in growth and development of H.

armigera and S. litura larvae when fed exclusively on NGNF treated castor leaves. On

comparing the growth of larvae fed with NGE and TiCl4 treated and untreated castor leaves, it

was observed that NGNF treatment slowed down insect growth and prolonged the larval

period by 4 d. The delayed growth in lepidoptera larvae when fed on artificial diets enriched

with other metals, such as zinc, cadmium, copper, and lead is on record (Kramarz and Kafel,

2003; Stone et al., 2001).

NGNF used in the experiments were dispersed in the Millipore water to ensure the natural

conditions, as water forms the major mode for nanoformulations entry into the environment.

Hence no manipulations were made to make NGNF dissolve in the solution with other

additives (e.g. use of solvents). The FT-IR spectrum of synthesized NGNF showed the

presence of different functional groups viz., alkane, methylene, alkene, amine, and carboxylic

acid may have been contributed in the process of nanoformulations synthesis. Functional

groups associated with these were the cause for the bioreduction of TiO(OH) 2 to TiO2 NPs

(Rajakumar et al., 2015). The XRD pattern of the synthesized NGNF revealed intense peaks

at (1 0 0), (1 0 1), (1 1 1), (2 1 1), (2 2 0) and (3 0 1) Bragg’s reflection, respectively. The

crystallite sizes were calculated using Scherrer’s formula applied to the major intense peaks

and found to be in the range of 31.27. The broadening of the peaks clearly shows the small

size of the nanoparticles (Moroni et al., 2005). SEM measurements basically depend on the

hydrodynamic diameter which is not only based on the core of the nanoparticles but also on

surface coating (i.e. NGE capping agent) and the concentration of ions in the medium. From

19
the quality reports obtained using SEM, some data represent high polydispersity nature of the

NGNF which may be due to its dispersion in Millipore water and their tendency to

agglomerate. To prevent agglomeration of the NGNF, sonication was done for 30 min before

application onto the castor leaf disc used for larval feeding.

Sub-lethal concentrations of NGNF against H. armigera and S. litura extended the larval

and pupal development. Methanol extracts and sterols from Myrtillocactus geometrizans

produced abnormalities in the larvae and pupae of Spodoptera frugiperda and Tenebrio

molitor (Cespedes et al., 2005). Tanzubil and McCaffery (1990) demonstrated that aqueous

neem seed extracts and azadirachtin inhibit the growth and development of larva and pupae

of Spodoptera exempta. Koul et al. (2013) reported that acute toxicity of plant essential oils

against larvae of H. armigera, S. litura, and Chilo partellus. The present study provides

further evidence that NGNF can also reduce the growth rate of larva and pupae of H.

armigera and S. litura.

Metals cause toxic effects by entering into biochemical reactions in which they are not

normally involved (Hopkin, 1989) and also larvae feeding on high-metal diets may require

more energy for metal-detoxification (Kramarz and Kafel, 2003). The estimation of NGNF

concentration in larvae of H. armigera and S. litura revealed that accumulation of NGNF in

larval body mass was in lower amounts, whereas the concentration of NGNF in feces was

high. The NGNF concentration in larval exuviae was higher as compared to that of gut

contents which represent that the molting does not form an efficient pathway for metal

detoxification (Hopkin, 1989; Kozlov et al., 2000). In the present studies, an NGNF treatment

through diet had affected the pupal mass of the insects. The effect on adult mass is

comparable with the data obtained in other reports (Gintenreiter et al., 1993; Ortel et al.,

1993; Kramarz and Kafel, 2003).

20
 Some of the metals may be accumulated or hardly assimilated by lepidopteran larvae

(Gintenreiter et al., 1993) that are generally controlled by regulation mechanisms (Ortel et al.,

1993). Regulation of metal contents in larvae is usually achieved through lysis of gut cells

(where metals are accumulated) into the lumen of the digestive system for excretion in the

feces and through excretion of metals via the malpighian tubules (Hopkin, 1989; Dallinger,

1993). In our studies, it is found that not only the larval growth but also the activity of

detoxification enzymes was altered in lepidopteran species fed with NGNF incorporated

diets. Increased detoxification enzyme activities in insect midgut and fat body tissues are

often associated with enhanced detoxification of insecticides (Valles et al., 1999).

In environmental ecotoxicology assessments, earthworms have been utilized as an

essential end point organism for the last two decades (Ando et al., 2002; An and Lee, 2008;

Wu et al., 2011). Eudrilus eugeniae is a large worm that grows extremely rapidly and is

reasonably prolific (Edwards and Bater, 1992). The results of in-vitro evaluation of NGNF on

E. eugeniae revealed that earthworms can survive well, and gain weight in the presence of

NGNF treatments. The positive effects are in contrast to the results from cypermethrin

treatments which led to increased mortality and decreased body weight. Pesticides can show

both direct toxicity against earthworms and produce latent effects on their growth and fertility

(Paoletti, 1999; Wang et al., 2012). Thus, these results demonstrate an example of NGE and

NGNF not producing significant harm to earthworms.

Chemical characterization of NGE through GC-MS studies revealed ten pure compounds

which have been previously identified in other plants and reported to have insecticidal

properties, all of which are also present in the leaves of neem. Srinivasan et al. (2016) have

identified the Eudesm-7(11)-en-4-ol compound as the major component from Piper betle

which showed activity against S. litura. Similar results by Pavela (2005) reported that

essential oils of Nepeta cataria and Thuja occidentalis were highly toxic towards the third

21
instar of S. littoralis. The identified compounds like Ethyl hexadecanoate is recommended to

be antioxidant, hypocholesterolemic, nematicide, pesticide and sequesters spleen function and

shows anti- inflammatory effect (Saeed et al., 2012). Oleic acid ethyl ester is suggested to be

plasticizer and lubricant in pharmaceutical industries. Stearic acid ethyl ester can be used as

anti-viral and anti- inflammatory drugs and linoleic acid ethyl ester help to prevent

cardiovascular diseases (Sudha et al., 2013). Methyl rincinoleate and ricinoleic acid exerts

analgesic and anti-inflammatory effects (Vieira et al., 2000). Koul et al. (2013) reported that

acute toxicity of essential oils against larvae of H. armigera, S. litura and Chilo partellus,

also support these conclusions.

5. Conclusions and recommendations

Based on the findings, neem gum nano formulation (NGNF) could provide an important

new control product to reduce populations against H. armigera, S. litura. The physiological

changes occurred in the larvae are due to the accumulation of NGNF and localization in the

larval body. Hence, we found that the titanium suspensions induced stress alters the

detoxifying enzymatic levels in the larval midgut tissues of Furthermore, reduced

environmental toxicity provides a pest management approach that reduces harm to beneficial

organisms, like earthworms. Chemical analyses using GC-MS revealed ten compounds in

NGE, and these potentially provide novel pest management chemistries which should be

further examined.

Acknowledgements

This paper was supported by the KU-Research Professor Program of Konkuk

University, Seoul, South Korea. We are grateful to Sophisticated Test & Instrumentation

Centre (STIC) Cochin University of Science and Technology, Cochin 682 022, Kerala, India

for providing the facilities to carry out the characterization studies, like XRD, FTIR and

22
SEM-EDX. CK acknowledges the Department of Science & Technology, Science &

Engineering Research Board (SERB), New Delhi, India, for the grant of National Post-

Doctoral Fellowship (PDF/2016/000496).

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31
 (A)

(B)

(C)

Fig. 1. FTIR spectrum of: (A) TiCl4, (B) Neem Gum Extract (NGE) and (C) Neem gum nano

formulation (NGNF).

32
Fig. 2. X-ray diffraction patterns of: (A) TiCl4 and (B) Neem gum nano formulation (NGNF).

33
A B

C
D

Fig. 3. (A, B and C) SEM images of the different magnifications of synthesized Neem gum nano

formulation (NGNF), (D) EDX showing chemical composition.

34
Fig.4. Percentage mortality of second, third and fourth instar larvae of H. armigera and S.

litura post treatment with Neem gum nano formulation (NGNF) and Neem gum extract

(NGE) Means (±(SE) standard error) there is no significant difference (P ≤0.05).

35
A

36
 C

Fig.5. Enzyme activities of (A) b-glucosidase (Glu), (B) carboxylesterase (CaE) and (C)

glutathione-S-transferase (GST) H. armigera and S. litura larvae on treatment with Neem

gum nano formulation (NGNF), Neem gum extract (NGE) and control TiCl4. Same letters

above each column indicate no significant differences (P>0.05), Different letters indicates

significant differences among columns among treatments. T-bars represent standard errors.

37
Fig.6. Percentage mortality of earthworm in filter paper test (FPT) and artificial soil test

(AST) after treatment with Neem gum nano formulation (NGNF), Neem gum extract (NGE)

and chemical pesticide (cypermethrin). Means ±SE (standard error) indicate significant

difference (P ≤ 0.05).

38
 Table 1

Larvicidal activity of effective concentrations (LC50 and LC90 ppm) of Neem Gum Nano

Formulation (NGNF) and Neem Gum extract against H. armigera and S. litura.

Species & 95 % 95 %
χ2 (df=4)
Test Compounds LC50±SE UCL-LCL LC90±SE UCL-LCL

H. armigera

NGNF 10.20±1.29 41.56-32.28 32.68±2.39 58.16-34.27 7.42

Neem Gum 38.36±2.85 64.17-46.18 120.59±3.65 112.50-78.85 12.68

S. litura

NGNF 12.49±3.90 24.88-16.85 36.68±4.62 64.19-38.92 8.49

Neem Gum 42.80±4.74 63.98-47.20 92.88±3.09 110.80-86.27 10.83

Control = no mortality ; LC50 lethal concentration that kills 50% of the exposed larvae ; LC90

lethal concentration that kills 90% of the exposed larvae ; UCL upper confidence limit

LCL lower confidence limit ; χ2 = chi-square ; d.f. = degree of freedom

39
Table 2

Antifeedant activity of Neem Gum Nano Formulation (NGNF) and Neem Gum extract

against H. armigera and S. litura

Concentration (ppm) Antifeedant activity (%)a

NGNF H. armigera S. litura

6.25 22.63±0.14a 26.32±1.20A

12.5 48.14±3.83b 40.31±0.84B

25 64.08±4.29c 62.24±2.34C

50 94.36±3.60d 88.46±4.66D

100 100.00±0.00e 100.00±0.00e

Neem Gum extract

6.25 6.46±0.29f 12.68±2.44F

12.5 14.82±2.64g 24.43±3.40G

25 26.32±3.44h 48.38±2.88H

50 42.63±2.26i 54.24±1.63I

100 74.82±2.48j 82.21±2.68J

Control

D2H2O 0.00±0.00k 0.00±0.00k

TiCl4 2.40±1.06l 4.38±1.93L

Azadirachtin (Positive control-100ppm) 68.26 ± 3.53m 76.80 ± 4.23M

a
Mean value of five replicates

Different letters within each group showed significant differences; same letters with in the

group showed no significant differences at P ≤ 0.05 (ANOVA, Tukey’s HSD test)

40
Table 3

Pupicidal activity of Neem Gum Nano Formulation (NGNF) and Neem Gum extract against

H. armigera and S. litura.

Concentration (ppm) Pupicidal activity (%)a

NGNF H. armigera S. litura

6.25 16.76±5.78a 22.62±3.03g

12.5 25.60±5.21b 33.79±4.65A

25 64.04±3.52c 75.47±4.08B

50 88.46±4.78d 82.24±2.68C

100 100.00±0.00e 100.00±0.00e

Neem Gum extract

6.25 18.42±2.09f 20.62±1.48f

12.5 22.99±3.48g 37.68±5.37E

25 43.14±2.26h 40.09±4.06F

50 54.39±4.11i 66.84±4.50j

100 68.40±2.58j 72.09±3.04G

Control

D2H2O 0.00±0.00k 0.00±0.00k

TiCl4 2.06±0.62l 3.42±1.08l

Azadirachtin (Positive control-100ppm) 78.85 ±4.03m 86.20 ±3.04d

a
Mean value of five replicates P ≤ 0.05.
Different letters within each group showed significant differences; same letters with in each

group showed no significant differences at P ≤ 0.05 (ANOVA, Tukey’s HSD test)

41
Table 4

Chemical compositions of Neem gum extract (NGE)

S.No. RT Compound Name Mol. For./ Mol. Wit. /Structure Peak area total %

1 9.0 3, 7, 11,15- C20H40O (296) 8.60

tetramethyl-2
hexadecen-1-ol

2 12.6 3,7-dimethyl-1,7- C10H19N(153) 6.40

octadien-3-amine

3 13.5 Dimethyl 4- C10H11NO4 (209) 28.26

aminobenzene-1,2-
dioate

4 13.5 Acetic acid, 1-[2- C16H26O2 (250) 5.10

(2,2,6-trimethyl-
bicyclo[4.1.0]hept-1-
yl)-ethyl]-vinyl ester

5 14.1 n-hexadecanoic acid C16H32O2 (256) 6.40

6 22.5 Oleic acid C20H38O2 (310) 31.45

7 30.7 Linoleic acid C19H34O2 (294) 9.53

8 34.2 1,4 C8H14O2 (142) 4.60

Dioxaspiro[4.5]decane

9 38.4 Ricinoleic acid methyl C19H36O3 (312) 16.84

ester

10 40.6 Stearic acid, ethyl C20H40O2 (312) 8.4

ester

42