Original article
Evaluation of the effects of neem leaf (Azadirachta indica) extract
on the morphology, development, biochemical composition and
digestive enzyme activity in Drosophila melanogaster
R. Devisree , J.B. Hyzil
Postgraduate and Research Department of Zoology, Government College for Women,
Thiruvananthapuram, Kerala, India
Corresponding author: J.B.Hyzil, Email: hyzilsyam@gmail.com
Journal of Experimental Biology and Zoological Studies. 2(1): p 66-75, Jan-Jun 2026.
Received: 20/11/2025; Revised: 10/12/2025; Accepted: 11/12/2025; Published: 01/01/2026
__________________________________________________________________________
Abstract
The present study investigates the impact of neem (Azadirachta indica) leaf extract on
Drosophila melanogaster. Flies were reared on 50 mL of food medium supplemented with
varying concentrations of neem extract (1%, 5%, and 10%). Observations were made on
changes in body size and external morphology, development and metamorphosis, and
biochemical responses. Significant alterations were recorded in the levels of total proteins and
amino acids, as well as in the activity of key digestive enzymes such as amylase, protease, and
trehalase. Larval development was adversely affected, and severe morphological deformities
were observed when the highest concentration of the extract was used. Neem extracts caused
varying levels of stress responses depending on their concentrations. The study thus
demonstrates that neem extract exerts dose-dependent effects on Drosophila melanogaster,
potentially affecting digestion, metabolism, growth, larval development, and metamorphosis,
and may serve as a natural and eco-friendly biopesticide alternative.
Keywords: Amylase, Azadiracta indica, biopesticide, Drosophila melanogaster, protease,
trehalase,
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Introduction
The increasing global demand for food security has led to intensive agricultural practices that
heavily rely on synthetic pesticides for crop protection. These chemical insecticides have
improved agricultural productivity; however, their widespread and indiscriminate use causes
numerous environmental and health concerns. Synthetic pesticides persist in the environment,
contaminate soil and water resources, disrupt ecological balance by affecting non-target
organisms, and pose serious health risks to humans through bioaccumulation in the food chain.
Furthermore, the development of pesticide resistance in target pest populations has necessitated
the application of higher concentrations and more frequent treatments, further exacerbating
these problems. In response to these concerns, there has been a shift towards developing
sustainable and eco-friendly alternatives to conventional pest control methods. Natural plant-
based insecticides have emerged as promising candidates due to their biodegradable nature,
lower environmental persistence, and reduced toxicity to non-target organisms. Neem
(Azadirachta indica) has gained considerable attention as a potential source of natural
insecticides.[1] Azadirachta indica, commonly called ‘Indian Lilac’ belongs to the family
Meliaceae.[2] Neem exhibits antiallergenic, antidermatic, antifeedant, antifungal, anti-
inflammatory, antipyorrhoeic, antiscabic, cardiac, diuretic, insecticidal, larvicidal, nematicidal,
oviposition-deterrent, and spermicidal activities among others. These biological properties are
attributed to the presence of bioactive compounds such as azadirachtin, nimbin, salannin, and
other limonoids.[3] These compounds interfere with various physiological processes in insects,
including hormone regulation, feeding behaviour, reproduction, and development, making
neem extracts highly effective as natural pest control agents. A neem-based fertilizer has been
effective against the pest, southern armyworm. Neem cake is often used as a fertilizer.
Azadirachta indica has been found to be toxic to non-target organisms as well.[4]
Drosophila melanogaster, commonly known as the fruit fly can be used as an ideal model
organism for investigating the biochemical effects of potential insecticides.[5] This species has
been extensively used in biological research due to its short life cycle, well-characterized
genetics, ease of culturing, low cost, short generation time, and physiological similarities to
other insects.[6] The wealth of genetic and molecular tools available for Drosophila
melanogaster makes it particularly suitable for understanding the mechanisms of action of
bioactive compounds and their effects on insect physiology and behaviour.[7]
Understanding the biochemical mechanisms underlying the insecticidal activity of neem leaf
extract is important for optimizing its application as a natural pesticide. It may contribute to
the development of more effective formulations, appropriate dosage recommendations, and
targeted application methods. The development of natural insecticides represents a critical step
towards sustainable agricultural practices that minimize environmental impact while
maintaining effective pest control.The present study aims to investigate the biochemical effects
of neem leaf extract on development and metamorphosis and various physiological and
biochemical parameters in Drosophila melanogaster, with a view to providing insights into the
cautious and environmentally safe use of neem-based formulations as pesticides. The findings
from this investigation are expected to contribute to the growing body of knowledge on the
application of biopesticides in pest management.
Materials and Methods
Preparation of neem leaf extract
Fresh Azadirachta indica leaves were collected and washed with running tap water and rinsed
with distilled water to remove dust and debris. The neem leaves were crushed using a mortar
and pestle, adding distilled water (4 mL of water /g). The extract of the crushed leaves was
filtered through Whatman filter paper and the filtrate obtained was used for further
experiments.[8]
Preparation of Drosophila food medium
A base culture medium was prepared by adding corn flour (5g) and agar (1g) in 100 mL distilled
water. The mixture was boiled and subsequently cooled. Before solidification, yeast and
sucrose were added. Test food media containing 1%, 5%, and 10% neem leaf extracts were
prepared by adding the respective extracts to the base medium, in the proportions required,
maintaining the final volume at 50 mL. Each of these were then mixed thoroughly, and allowed
to solidify. The base medium without the neem leaf extract was used as the negative control.
Collection of Drosophila melanogaster
Drosophila melanogaster specimens were collected using a beaker with curd as an attractant.
The beaker was covered with cling film perforated with small pores, allowing the flies to enter
while preventing their escape.
Rearing of Drosophila
Adult Drosophila (20–50 individuals per vial) were introduced into culture vials containing the
test food medium treated with a specific concentration of neem extract. The flies were
maintained in these vials for 2–3 days to allow females to lay eggs. Thereafter, the adults were
removed, and the vials were plugged with cotton. The eggs were allowed to hatch, and the
newly emerged larvae were maintained on the same treated food medium until the end of the
experiment.
Biochemical estimations
Estimation of protein and amino acid
Protein and amino acid estimation was done using Lowry method.[9] Drosophila adults that
were previously exposed to different concentrations of neem leaf extract were used for the
estimation of protein and amino acid. For this, 50 mg of Drosophila adults were cold
anaesthetised and homogenized in 1mL ice cold water. 250 µL of the homogenate was taken
in an Eppendorf tube. 250 µL of 10 % trichloroacetic acid was added to it and shaken well and
centrifuged at 3000 rpm for 10 min. The pellet obtained was used for protein estimation and
the supernatant for amino acid estimation.
For protein estimation, the pellet was dissolved in 0.1N NaOH and made up to 1mL. One mL
of distilled water was taken as the blank. To the sample, 3.5 mL of alkaline copper sulphate
solution was added and mixed well and kept it for 10 min. To this, 0.5 mL of Follin’s reagent
was added. It was mixed well by vigorous shaking and kept at room temperature for 30 min.
2.5% Bovine serum albumin was used as the standard. Optical density (OD) was recorded at
660 nm.
Supernatant was used for amino acid estimation. To 0.5 mL of supernatant, 0.5 mL of NaOH
was added. To this, 3.5 mL of alkaline copper sulphate solution was added and mixed well and
kept it for 10 min followed after adding 0.5 mL of Follin’s reagent. It was mixed well by
vigorous shaking and kept it at room temperature for 30 min. One mL of distilled water
similarly treated with reagents was taken as the blank. A 2.5 % solution of tyrosine was used
as the standard. Optical density was measured at 540 nm. The concentration of protein / amino
acid in the sample was calculated by the formula:
(Concentration of standard) (OD of test)
OD of standard
Digestive enzyme assay
Preparation of enzyme extract
Cold anaesthetised 50 mg Drosophila adults, after washing thoroughly with insect saline were
transferred to 1 mL of ice-cold distilled water in an Eppendorf tube, homogenized and
centrifuged at 5000 rpm for 10 min at 4⁰C. The supernatant obtained was used for digestive
enzyme assay.
Amylase
The method used by Sreekumar and Prabhu (1988)[10] was followed for determining amylase
activity. To 0.2 mL of enzyme extract, 0.2 mL of Tris- HCl buffer (pH 8.2) and 0.4 mL of 1%
starch solution were added and incubated at 37° C for 30 min. The reaction was stopped by
adding 1.2 mL of 3, 5-dinitrosalicylic acid followed by heating at 50°C for 5 min. The
absorbance of the solution was read at 550 nm and µg of maltose equivalents liberated was
calculated using 0.01% of maltose solution as standard.
Protease
Protease activity was determined using the method of Birk et al. (1962).[11] To 0.2 mL of
enzyme extract, 0.2 mL glycine NaOH buffer (pH 9) and 0.4 mL 1% casein solution were
added. Enzyme activity was terminated after 30 min of incubation at 37°C by adding 1.5 mL
of 5% trichloroacetic acid, and centrifuged at 13000 rpm for 15 min. The supernatant was read
at 250 nm using 0.005% of tyrosine as standard.
Trehalase
The method of Friedman (1996)[12] was used for the estimation of trehalase activity based on
the rate of glucose generation from trehalose. To 0.2 mL of enzyme extract, 0.2 mL of 60 mM
citrate buffer and 0.2 mL 10 mM trehalose were added and incubated for 15 min at 32oC. The
reaction was stopped by the addition of 1 mL of Ba(OH)2 and 1 mL of 0.5 M ZnSO4. The
volume was made up to 5 mL by adding distilled water. The reaction mixture was centrifuged
at 6000 rpm for 5 min. The supernatant was used for the estimation of glucose. To 1 mL of
supernatant, 4 mL of anthrone reagent was added, mixed well and kept in a water bath for 15
min. For the blank, 1 mL of distilled water was used instead of the enzyme extract and 0.1%
glucose was used as standard. OD was measured at 620 nm.
Statistical analysis of data
Student’s t-test was employed to evaluate the statistical significance of the data obtained from
the experiments.
Observations and Results
Life cycle of Drosophila
The life cycle of Drosophila melanogaster includes four stages, viz., egg, larvae comprising
three instars, pupa and adult (Figure 1). The duration of various instars with their duration, and
morphometric parameters are presented in Table 1.
Figure 1: The developmental stages of Drosophila melanogaster. Above, from left to right: egg, first
instar larva, and second instar larva; below, from left to right: third instar larva, pupa and adult.
Table 1: Duration of developmental stages and related morphometric parameters in Drosophila
melanogaster
Developmental stages*
Duration
Length (mm)
Width (mm)
Egg
24 h
0.51±0.02
0.21 ±0.03
First instar
24 h
1.02±0.03
0.24 ±0.03
Second instar
24 h
2.40±0.10
0.52 ±0.02
Third instar
48 h
4.50±0.20
1.02 ±0.03
Pupa
4 -5 days
3.27±0.26
1.21 ±0.04
Adult
40-50 days
2.96±0.50
1.00 ±0.20
*The values are presented as mean + standard deviation of 8 observations.
Figure 2: Malformed larvae and adults of Drosophila melanogaster, treated with neem leaf
extract
Effect of neem leaf extract treatment on Drosophila
In Drosophila larvae reared on food media containing neem leaf extract, development and
growth were found to be impaired depending on the concentration of the extract. The effects
were less pronounced when the concentration of the extract was low at 1%. During the early
stages, the larvae appeared pale and slender, indicating a clear alimentary canal devoid of food.
As development progressed, the body became darker, and the cuticle showed a wrinkled
appearance. These effects were more pronounced at higher concentrations of the extract,
adversely affecting pupation and adult emergence (Figure 2). The adults exhibited
developmental deformities such as crumpled bodies or malformed wings, as shown in Figure
2. The duration of the life cycle revealed an increase in treated larvae, compared to the control.
Effect of neem leaf extract treatment on protein and amino acid levels in Drosophila
Neem leaf extract caused a significant reduction in both protein and amino acid levels. The
decrease was evident at all tested concentrations, with a pronounced decline in protein content
observed at the highest concentration (10%) of the extract. (Table 2 and Figure 3).
Table 2: Effect of varying concentrations of neem leaf extract on protein and amino acid
levels in Drosophila melanogaster
Concentration of extract
(%)
Protein level
(mg/ mL)
Amino acid level
(mg/mL)
Control
8.07± 0.88
11.20 ± 0.64
1
4.63 ±0.31*
5.65 ± 1.39*
5
5.89 ±0.52*
8.44 ± 0.69*
10
3.81 ±0.34*
7.05 ± 0.30*
*Significant at 0.05 level with respect to control and the values are presented as mean + standard deviation of 6
observations.
Figure 3: Effect of different concentrations of neem leaf extract on protein and amino acid
concentration in Drosophila melanogaster
Table 3: Effect of varying concentrations of neem leaf extract on protease, amylase and
trehalase levels in Drosophila melanogaster
Concentration of
extract (%)
Amylase activity
in units b
Trehalase activity
in units c
Control
11.54 ± 1.15
43.67 ± 1.53
1
9.50 ± 1.095*
39.89 ± 1.39*
5
27.67 ± 2.52*
39.11 ± 1.02*
10
14.89 ± 0.86*
34.74 ± 2.29*
aµg of tyrosine liberated / min /mL of extract; bµg of maltose equivalents liberated /min /mL of extract; cµg
of glucose liberated / min /mL of extract.
*Indicates values that are significant with respect to control at 0.05 level and the values are presented as mean
+ standard deviation of 6 observations.
0
10
20
30
40
50
60
70
80
90
100
Control 1 5 10
Concentration mg/mL
Concentration of neem extract (%)
Protein level mg/mL
Amino acid level mg/mL
Figure 4: Effect of different concentration of neem leaf extract on digestive enzyme activity of
Drosophila melanogaster
Effect of neem leaf extract treatment on digestive enzyme levels in Drosophila
Protease activity increased with increasing concentrations of neem leaf extract from 1% to
10%. Amylase activity showed fluctuations at different concentrations of the extract while
Trehalase showed a dose dependant decrease in activity when compared with controls (Table
3 and Figure 4).
Discussion
Drosophila melanogaster being a holometabolous insect, the life cycle includes a larval stage
comprising three instars, followed by pupal and adult stages. Neem contains a large number of
biologically active compounds, and more than 140 compounds have been isolated from
different parts of the neem. Neem leaf constituents are found to have immunomodulatory, anti-
inflammatory, hyperglycemic and antibiotic properties.[13]
It was observed in this study that the presence of neem leaf extract in food media impaired the
growth and development of Drosophila larvae; this effect was dependent on the concentration
of the extract. Effects were minimal at a low concentration of 1%. Initially, larvae appeared
pale and slender, suggesting an empty alimentary canal and reduced feeding. At the later
stages, the body darkened, and the cuticle became wrinkled. These adverse effects intensified
with higher extract concentrations, severely inhibiting pupation and adult emergence. The
initiation of metamorphosis depends on several external and internal cues. In Oryctes
rhinoceros, it has been observed that the final instar larvae must either attain a critical age[14]
or reach a threshold body weight [15] to successfully pupate. Neem-treated larvae often fail to
undergo metamorphosis, as they are unable to achieve these critical conditions. This results in
a prolonged larval instar duration. The present study shows that surviving adults of Drosophila
treated with neem extract, exhibit severe developmental deformities, such as crumpled bodies
or malformed wings as may be seen in Figure 2. Such deformities are also observed in other
insects. [16,17] The prolonged life cycle observed in larvae treated with neem extract, compared
to the control, observed in the present study, can be attributed to the interference of certain
neem compounds with the endocrine system. This interference may be mediated by certain
compounds present in the neem extract that may mimic or block hormones such as ecdysone
and juvenile hormone, thereby disrupting critical processes like moulting, metamorphosis, and
overall growth. In addition to its direct toxicity, azadirachtin influences several physiological
processes in insects, including growth regulation, protein synthesis, reproduction, and
hormonal balance, by affecting both ecdysteroid and juvenile hormone titres.[18,19]
In the present study, a decrease in whole-body protein content was observed in neem-treated
larvae. This reduction may be attributed to the neem compound azadirachtin, which can
interfere with ribosomal function, enhance proteolysis, and trigger stress and energy demand,
causing insect to break down proteins by proteolysis.[20] Neem compounds are also known to
induce oxidative stress, resulting in protein denaturation and degradation. An earlier study
reports that treatment with neem extract causes a significant reduction in total protein,
carbohydrate, and lipid levels in the lepidopteran larvae of Pericallia ricini.[21]
0
10
20
30
40
50
60
70
80
90
100
Control 1 5 10
Enzyme activity
Concentration of extract (%)
Protese activity units
Amylase activity units
Trehalasa activity units
Insects maintain a rich pool of amino acids that act as anti-stress agents, protecting them from
various environmental stressors. Under stress conditions, these amino acids are utilized for the
synthesis of specific stress-related proteins, such as heat shock proteins, antimicrobial
peptides[22] or other novel peptides, which may lead to an elevation in total protein content.
Amino acids are essential for protein synthesis, and most insects obtain their amino acid
requirements from dietary proteins. In the present study, the total free amino acid levels in adult
Drosophila melanogaster cultured on food media containing different concentrations of neem
extract were estimated. The results showed that treatment with neem extract significantly
decreased the total free amino acid supporting the observations of the aforementioned report.
Feeding inhibition, disruption of protein metabolism, or mild oxidative stress may also explain
the decrease in amino acid concentration.
Among the most important benefits of neem application are the insecticidal and feeding
deterrent characteristics of the products.[19,23] In insects, feeding activity strongly influences the
secretion of digestive enzymes. The act of feeding itself, or the presence of food materials in
the midgut—particularly proteins, which serve as major secretagogues—can stimulate enzyme
release. While feeding is regulated by neural mechanisms, enzyme secretion by secretagogues
is mediated through neuroendocrine pathways, aided by midgut hormones.[24] Azadirachtin, in
neem extracts, is known to disrupt insect physiology and digestion. In Drosophila larvae, neem
treatment may induce a state of starvation by reducing feeding activity, thereby adversely
affecting enzyme secretion.[25]
Amylase is an enzyme that degrades starch, first into oligosaccharides and then into maltose
and glucose, by hydrolyzing α-1,4-glycosidic bonds.[26] Khosravi and Sendi (2013) reported
that treatment of fifth-instar Glyphodes pyloalis larvae with azadirachtin resulted in a reduction
in α-amylase activity, with the decrease becoming more pronounced at higher concentrations
of the plant extract.[27] Similarly, sublethal concentrations of pyrethroids were found to
decrease α-amylase activity in the larval gut of the beetle Tribolium castaneum.[28] Treatment
with Artemisia annua extract also caused a reduction in α-amylase activity in Eurygaster
integriceps, and this reduction increased with higher concentrations of the extract.[29] . In the
elm leaf beetle treated with Artemisia annua extract, α-amylase activity decreased after 24
hours but sharply increased after 48 hours, indicating a possible loss of potency of the extract
over time.[30] In the present study, treatment of Drosophila melanogaster with a low
concentration (1%) of neem leaf extract caused a reduction in amylase activity. The reduction
in α-amylase activity caused by plant extracts could be attributed to plant defence compounds
that act on insect gut enzymes or to the cytotoxic effects of the extract on amylase-secreting
cells of the midgut.[31] At lower concentrations, neem’s active constituents (azadirachtin) might
slightly inhibit enzyme synthesis or activity. However, when the concentration of neem extract
was increased to 5%, amylase activity showed an increase, followed by a decrease at the highest
concentration tested. These results suggest that both the concentration of neem extract and the
duration of exposure may influence amylase secretion through multiple physiological
mechanisms.
The present study revealed a dose-dependent decrease in trehalase activity at different
concentrations of neem extract. Trehalase plays a vital role in maintaining haemolymph sugar
levels, which are primarily composed of trehalose, a disaccharide. An increase in trehalose
concentration raises the osmotic pressure of the haemolymph, which can inhibit gut emptying
and indirectly suppress feeding.[32] The decrease in trehalase activity, which remained
relatively constant across all concentrations of the extract, observed in the present study, may
represent a response to physiological stress, for maintaining homeostasis.
Azadirachtin inhibits peristalsis, reduces enzyme production as food passes through the gut,
interferes with midgut cell renewal, and suppresses feeding.[33] It is reported that botanical
insecticides can interfere with the production of certain proteases, impairing the digestion of
ingested proteins.[34] Azadirachtin A inhibits the growth and development of Bactrocera
dorsalis larvae by releasing cathepsin in the midgut.[35] Similarly, azadirachtin causes a
reduction of the protease activity in Glyphodes pyloalis.[27,36] It has also been reported that
digestive protease of Cnaphalocrocis medinalis was suppressed by extracts of Vitex negundo
and Azadirachta indica.[37] Similar to their effects on α-amylase activity, plant defence
compounds can also influence the secretion of proteinases.[38,39] Heliothis virescens larvae
reared on an artificial diet containing azadirachtin exhibited increased digestibility,[37]
presumably due to a reduced rate of food passage through the gut.[40] In the present study,
Drosophila melanogaster larvae showed a significant decrease in protease activity when
treated with a low concentration of neem extract. However, protease activity increased as the
concentration of neem extract was raised. This increase could represent a physiological
response to stress induced by azadirachtin or other bioactive compounds present in the extract.
Digestive physiology plays a critical role in insect adaptation to stress factors. Although insects
exhibit remarkable plasticity in their digestive systems, a comprehensive understanding of how
digestive physiology mediates adaptation to various stressors remains limited.[41]
Phytophagous insects are known to regulate their digestive enzyme activity in response to plant
defence proteins such as chitinases, lectins, and enzyme inhibitors.[42-44] Such responses may
not always follow the usual pattern of enzyme regulation. For example, the midgut trypsin
activity of Ephestia kuehniella larvae decreased following treatment with Inga vera trypsin
inhibitor, whereas chymotrypsin-like activity increased.[45] Tenebrio molitor compensates for
trypsin inhibition by enhancing cathepsin activity, thereby maintaining digestive efficiency.[46].
Similarly, Hyphantria cunea larvae exposed to α-amylase inhibitors showed decreased midgut
α-amylase activity but increased trypsin activity.[41] Some insects also employ homeostatic
strategies to modulate digestive enzyme activity when exposed to plant secondary metabolites.
Exposure to azadirachtin significantly inhibited the growth of Bactrocera dorsalis larvae but
was accompanied by a notable increase in cathepsin activity.[35] The elevated cathepsin activity
in Bactrocera dorsalis may represent a compensatory digestive response, enabling larvae to
meet the heightened energy demands associated with metamorphosis under azadirachtin-
induced stress. The results of the present study indicate that digestive enzyme secretion in
Drosophila larvae exhibits variable responses to neem extract depending on both the duration
of exposure and the concentration of the extract. It is possible that at low concentrations, the
insects did not initiate a compensatory stress response. However, at higher concentrations
(around 5% or above), enzyme activity may increase, as moderate or elevated stress levels
could induce adaptive metabolic adjustments, including the upregulation of digestive enzymes
to counteract the physiological challenge. The effects of neem application are not always
consistent, as they depend on several factors such as insect species, timing and method of
application, and the concentration used. [47] Excessive concentrations can be toxic, highlighting
the importance of dose optimization in potential pest control applications. For this reason, it is
necessary to have more information on the gut enzymatic activity of insects to devise a rational
strategy for insect pest control utilizing plant extracts.[27]
Conclusion
This study characterizes the bioactive effects of neem leaf extract on Drosophila melanogaster
larvae. When incorporated into the culture medium, the extract acts as a potent antifeedant. It
appears to induce physiological stress, leading to adverse alterations in key biochemical
parameters, including haemolymph protein and amino acid concentrations, as well as digestive
enzyme secretion. These disruptions result in arrested larval development and metamorphosis,
confirming the extract’s strong potential as a natural insect growth regulator.
Acknowledgement
The authors gratefully acknowledge the help rendered by the affiliating institute for the
successful completion of work.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
1. Nicoletti M, Mariani S, Maccioni O, Coccioletti T, Murugan K. Neem cake: chemical composition and larvicidal activity
on Asian tiger mosquito. Parasitol Res 2012; 111(1): 205-13.
2. Wiart C. Medicinal plants of Asia and the Pacific. 1st ed. Boco Raton (Florida):CRC Press; 2006.
3. Isman MB. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world.
Annu Rev Entomol 2006; 51(1): 45-66.
4. Siriwattanarungsee S, Sukontason KL, Kuntalue B, Sukontason K. Ultrastructural alteration of larvae and puparia of
blow fly Chrysomyamegacephala (F.) (Diptera: Calliphoridae) and house fly Musca domestica L. (Diptera: Muscidae)
exposed to neem extract. Trop Biomed 2008; 25(2): 107-116.
5. Wilson T G. Drosophila melanogaster (Diptera: Drosophilidae): a model insect for insecticide resistance studies. J Econ
Entomol 1988; 81(1), 22-27.
6. Baenas N, Wagner AE. Drosophila melanogaster as an alternative model organism in nutrigenomics. Genes Nutr 2019;
14(1):14.
7. Perry T, Batterham P. Harnessing model organisms to study insecticide resistance. Curr Opin Insect Sci 2018; 27:61-7.
8. Vaishna VV, Padmaja Devi MS, Prasobh GR. A Review on extraction of neem leaves and study the antimicrobial
activity. Int J Pharm Res Appl 2022;7(2): 466-70.
9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem
1951; 193(1): 265-75.
10. Sreekumar S, Prabhu VKK. Probable endocrine role of midgut tissue in stimulation of digestive enzyme secretion in
Oryctes rhinoceros (Coleoptera: Scarabaeidae). Proc Indian Acad Sci (Amin Sci) 1988; 97(1):73-8.
11. Brik Y, Harpaz I, Bondi A. Studies on proteolytic activity of the beetles Tenebrio and Tribolium. J Insect Physiol 1962;
8: 417-427.
12. Friedman M. Chemistry, biochemistry, and dietary role of potato polyphenols. A review. J Agric Food Chem 1997;
45(5):1523-40.
13. Subapriya R, Nagini S. Medicinal properties of neem leaves: a review. Curr Med Med Anticancer Agents 2005: 5(2):
149-56.
14. Mini A, Prabhu VKK. Significance of critical developmental stage of starvation-induced endocrine mediated precocious
metamorphosis in Oryctes rhinoceros (Coleoptera; Scarabaeidae). Proc Indian Acad Sci (Amin Sci) 1986; 95:379-85.
15. Shobhita-Rani S, Jaya S, Deepa- Chandran, Sreekumar S, Effect of antibiotics in larval growth and metamorphosis in
Oryctes rhinoceros (Coleoptera; Scarabaeidae). J Plantation Crop 2002:30(3):63-5.
16. Veena O. Regulation of feeding in the coconut pest Oryctes rhinoceros (Coleoptera; Scarabaeidae. Ph.D Thesis, 2013;
University of Kerala, Thiruvananthapuram.
17. Aerts RJ, Mordue AJ. Feeding deterrence and toxicity of neem triterpinoids. J Chem Ecol 1977; 23: 2117-32.
18. Garcia JF, Grisoto E, Vendramim JD, Botelho PSM. Bioactivity of neem, Azadirachta indica, against spittlebug
Mahanarva fimbriolata (Hemiptera: Cercopidae on sugarcane. J Econ Entomol 2006; 99:2010-14.
19. Morgan ED. Azadirachtin, a scientific gold mine. Bioorg Med Chem 2009; 17:4096-105
20. Huang Z, Shi P, Dai J, Xu F. Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide
azadirachtin. Pestic Biochem Physiol 2004; 80(2):85-93.
21. Gnanamani R, Dhanasekaran S. Efficacy of azadirachta indica leaf extract on the biochemical estimation of a
lepidopteran pest Pericalliaricini (Lepidoptera: Arctiidae). World Appl Sci J 2017; 35(2):177-81.
22. Lowenberger C, Bulet P, Charlet M, Hetru C, Hodgeman B, Christensen BM, et al. Insect immunity: isolation of three
novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti. Insect Biochem Mol Biol 1995;
25(7):867-73.
23. Mordue AJ, Blackwell A. Review: Azadirachtin-an update. J Insect Physiol 1993; 39:903-24.
24. Prabhu VKK and Sreekumar S. Endocrine regulation of feeding and digestion in insects. In Agrawal OP, editor.
Perspectives in entomological research. Jodhpur: Scientific Publishers, 1994.pp117-35.
25. Sreekumar S. Digestive enzyme secretion consequent to starvation and refeeding in the final instar larvae of Oryctes
rhinoceros (Coleoptera; Scarabaeidae). Proc Indian Natn Sci Acad B 1991; 61(6) :441-6
26. Terra WR, Ferriera C. Biochemistry of Digestion: Insect Molecular Biology and Biochemistry. In: Gilbert LI, Iatrou
K, Gill SS, Editors. Comprehensive Molecular Insect science Vol 4. Oxford: Elsavier; 2005: pp 171-224.
27. Khosravi R, Sendi JJ. Effect of neem pesticide (Achook) on midgut enzymatic activities and selected biochemical
compounds in the hemolymph of lesser mulberry pyralid, Glyphodes pyloalis. J Plant Prot Res 2013;53(3):238-47.
28. Saleem MA, Shakoori AR. Point effects of Dimilin and Ambush on enzyme activies of Tribolium castaneum larvae.
Pestic Biochem Physiol 1987; 29:127–37.
29. Zibaee A, Bandani AR, Malagoli D. Purification and characterization of phenoloxidase from the hemocytes of
Eurygaster integriceps (Hemiptera: Scutelleridae). Comp Biochem Physiol B Biochem Mol Biol 2011; 158: 117-23.
30. Shekari M, Sendi M J, Etebari K, Zibaee A, Shadparvar A. Effects of Artemisia annua L. (Asteracea) on nutritional
physiology and enzyme activities of elm leaf beetle, Xanthogaleruca luteola Mull (Coleoptera: Chrysomellidae). Pestic
Biochem Physiol 2008;91: 66-74.
31. Jbilou R, Amri, H, Bouayad N, Ghailani N, Ennabili A, Sayah F. Insecticidal effects of extracts of seven plant species
on larval development, α-amylase activity and offspring production of Triboliumcastaneum (Herbst) (Insecta:
Coleoptera: Tenebrionidae). Bioresour Technol 2008; 99(5): 959-64.
32. Bernays EA, Simpson SJ. Control of food intake. Advances in Insect Physiology 1982;16: 59-118.
33. Anuradha A, Annadurai RS. Biochemical and molecular evidence of azadirachtin binding to insect actins. Curr Sci
2008; 95(11): 1588–93.
34. Johnson DE, Brookhart GL, Kramer KJ, Barnett BD, McGaughey WH. Resistance to Bacillus thuringiensis by the
Indian meal moth Plodia interpunctella: Comparison of midgut proteinase from susceptible and resistant larvae. J Inver
Pathol 1990; 55(2): 235–44.
35. Zhao T, Lai D, Zhou Y, Xu H, Zhang Z, Kuang S, Shao X. Azadirachtin A inhibits the growth and development
of Bactrocera dorsalis larvae by releasing cathepsin in the midgut. Ecotoxicol Environ Saf 2019;183: 109512. .
36. Nasiruddin M, Mordue- Luntz AJ. The effect of azadirachtin on the midgut histology of the locusts, Schistocerca
gregaria and Locusta migratoria. Tissue Cell 1993; 25: 875-84.
37. Senthil-Nathan S, Chunga PG, Muruganb K. Combined effect of biopesticides on the digestive enzymatic profiles of
Cnaphalocrocis medinalis (Guenee) (the rice leaffolder) (Insecta: Lepidoptera: Pyralidae). Ecotoxic Environ Saf 2006;
64(3): 382–9.
38. Ryan CA. Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev
Phytopath 1990; 28: 425–49.
39. Franco OL, Riggen DJ, Melo FR, Bloch C, Silva C, Grossi de sa MF. Activity of wheat α-amylase inhibitors towards
Bruchid α-amylases and structural explanation of observed specificities. Eur J Biochem 2002; 267(8): 2166–73.
40. Barnby M A, Klocke JA. Effects of azadirachtin on the nutrition and development of the tobacco budworm, Heliothis
virescens (Fabr.) (Noctuidae). J Insect Physiol 1987; 33 :69-75.
41. Zhang AY, Li T, Yuan LS, Tan MT, Jiang D, Yan SC. Digestive characteristics of Hyphantria cunea larvae on different
host plants. Insects. 2023; 14:463.
42. Vaghela B, Vashi R, Rajput K, Joshi R. Plant chitinases and their role in plant defense: a comprehensive review. Enzyme
Microb Technol 2022; 159: 110055.
43. Chao H, Li YT, Liu YX, Hao GF, Yang XQ. Molecular interaction network of plant-herbivorous insects. Adv Agrochem
2024; 3(1):74–82.
44. Peumans WJ, van Damme EJ. The role of lectins in plant defence. Histochem J 1995; 27(4):253–71.
45. Bezerra CDS, Oliveira CT, Macedo MLR. Inga vera trypsin inhibitor interferes in the proteolytic activity and nutritional
physiology of Ephestia kuehniella larvae. Entomol Exp Appl 2017;165:109–19.
46. Ajaha A, Bouayad N, Aarab A, Rharrabe K. Effect of 20-hydroxyecdysone, a phytoecdysteroid, on development,
digestive, and detoxification enzyme activities of Tribolium castaneum (Coleoptera: Tenebrionidae). J Insect Sci 2019;
19(5):18.
47. Hail K.S, John LC., Nawaf M F. Effects of Neem-Based Insecticides on Consumption and Utilization of Food in Larvae
of Spodoptera eridania (Lepidoptera: Noctuidae). J Insect Sci 2015; 15(1) 152.
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