Efficiency of Azadirachtin, A Chitin Synthesis Inhibitor on Growth, Development and Reproductive Potential of Tribolium confusum after Adult Treatment
Najat Aly Khatter
The present study was established to evaluate the potential of insect growth regulator on the growth and development of Tribolium confusum Azadirachtin, a neem derivative which considered as insect growth regulator due to its chitin inhibitor activity was tested topically at four doses (0.5, 1, 1.5 and 2 μg insect-1) on adults of Tribolium confusum (Coleoptera: Tenebrionidae) and first evaluated on fecundity, hatchability and viability of eggs, longevity and morphometric of oocytes. Results show that the compound reduces the longevity and the fecundity. Generally, treatment with azadidrachtin affected growth and development of oocytes and egg-viability as shown by calculating the number of oocytes per ovaries and the size of basal follicle. The width of the follicle has not been affected significantly by these treatments. In the second group of experiments, this IGRs dont induce significant reduction in percentage of hatchability, the hatching rate of eggs laid by F1 females was not affected by the treatment (F = 2.86, DF = 99, p = 0.5) but it affect very significantly the viability rate of eggs laid by first female generation F1 (F = 7.5, DF = 99, p>0.0001). However, the azadirachtin lengthened very significantly the total duration of life cycle. Duration of the embryonic and post embryonic development of eggs laid by treated female increase when the concentration varied from 0 to 2 μg μL-1. This data confirm the efficiency of growth regulators derived from neem plant, on the development of stored product insect pests.
Received: November 01, 2010;
Accepted: March 30, 2011;
Published: May 30, 2011
In recent years the use of synthetic organic insecticides in stored product
pest control programs around the world has resulted in damage to the environment,
pest resurgence, pest resistance to insecticides and lethal effects on non target
organisms (Abudulai et al., 2001). Insect growth
regulators, botanical insecticides and microbial pesticides are highly effective,
safe and ecologically acceptable (Weinzierl and Henn, 1991;
Nathan et al., 2005a,b;
Nathan and Kalaivani, 2005; Sadeghian
and Mortazaienezhad, 2007; Radhakrishnan et al.,
2007; Suman et al., 2010).
The common trend in the past to decades toward reducing reliance on synthetic
insecticides for control of insect pests in agriculture, stored products, forestry
and human health has renewed worldwide interest in the neem derivative, azadirachtin,
as an environmentally desirable alternative (Paoletti and
Pimentel, 2000; Nderitu et al., 2008). Because
of secondary effects of conventional insecticides, the Insect Growth Regulators
(IGRs) are receiving more practical allention to provide for safer tools and
a cleaner environment.
Among these compounds the neem product, azadirachtin cause abnormal circular
deposition and abortive molting in insects by interfering with chitin biosynthesis
(Casida and Quistad, 1998). It was found to be effective
on several insect species (Khebeb et al., 1997;
Ben Jannet et al., 2001; Flamini
et al., 2003; Chaieb et al., 2007).
These compound have been found to interfere with chitin biosynthesis (Fields
et al., 2001; Soltani et al., 1983,
1996a). Other IGRs were reported to inhibit ecdysteroid
synthesis (Chebira et al., 2000; Soltani-Mazoun
et al., 2000; Al-Fifi, 2006) or mimic the
action of 20-hydroxy-ecdysone (Soltani et al., 2002).
Saponin extract from alfalfa roots, azadirachtin from the neem seed oil, synthetic
ecdysteroid agonist RH-2485 and juvenoid hydroperene disturb the development
and reproduction of Tropinota squalid (Coleoptera: Scarabeidae) (Hussein
et al., 2005; Facknath, 2006). Tribolium
confusum is a major insect pest of stored grain in all over the world and
whose larval development occurred in flour. In additions laboratory and field
tests have evaluated the toxicity of several IGRs against some stored product
pests (Abo El-Ghar, 1992; Soltani-Mazouni,
1994; Soltani-Mazoun and Soltani, 1995; Soltani
et al., 1996b; Oberlander et al., 1997;
Peppuy et al., 1998). The aim of the present
study was to investigate the effect of azadirachtin on growth and development,
adult reproductive potential and egg hatchability of T. confusum. This
IGR is tested for first time for control insects that develop in stored flour.
MATERIALS AND METHODS
Rearing of insects: Stock cultures of T. confusum were maintained in glass jars (20 cm in height, 10 cm diameter) containing wheat flour. Insects were kept at 30±1°C and 70±5% R.H. under almost continuous darkness. The laboratory experiments were carried out in July 2010 and extended to January 2011.
Toxic material (azadirachtin) and treatments: Azadirachtin was kindly supplied by Dow Elanco (Iniamapolis, USA). Serial dilutions were prepared in acetone and several doses (0, 0,5, 1, 1.5 and 2 μg insect-1) were applied topically on newly emerged adults (24-old). Acetone (1 μg individual-1) was used as control. The bioassay was conducted with four replicates each of 50 adult insects (25 males and 25 females) placed in glass box (20 cm diameter, 5 cm height) containing food (100 g of dry wheat flour).
Measurements of ovaries: Females were sampled from control and treated series at different moments during the adult life (0, 1, 2 and 3 days) and ovaries dissected. The number of oocystes per ovaries and the size of basal oocytes were determined.
Determination of longevity, fecundity and hatchability rate: Newly emerged adults of T. confusum were collected from rearing containers control and treated series were reared under standard conditions. The longevity of both males and females, the total number of eggs laid per female (Fecundity and the percentage of egg hatching were determined.
Growth and development: Laid eggs were collected from controls and treated series and placed under standard conditions. The time taken for adult emergence was determined. The number and the body weight of first generation were determined in all series, the survival rate during the post-embryonic development was calculated (number of emerged adults on number of eggs laidx100).
Statistical analysis: Data are presented as the Mean±SD. The age and the number of insects tested per series are given with the results. To detect azadirachtin among treatments, data were subjected to analysis of variance and one way ANOVA test.
Effects on morphometric parameters of ovaries
Number of ovocytes per paired ovaries: Results in Table
1 shown that the number of oocytes by pair of ovaries in treated females
was decreased significantly in females (1-3 days), when these females treated
with 2 μg of azadirachtin it record (58 to 52.3) when compared with control
groups which record a mean varied from (55.9 to 58.3) according to the age of
The oocytes basal size: The length of basal oocytes decreased just significantly according to the age of females descended from treated parents (2 μg). The width has not been affected significantly by this treatment compared with control group which show no difference in width of the basal oocytes size according to the age of the female (Table 2).
Effects on reproductive capacity: Results represented in Table
3 shows the effect of the tested material, azadirachtin on the fecundity
of resulted females which indicate a highly significant reduction (p<0.0001)
of number of eggs laid by each female treated with the higher dose (2 μg
μL-1), compared with the control. The number of eggs laid by
the first generation females was higher than the one of the females treated
especially with the higher doses.
||Effect of azadirachtin on the number of oocytes per paired
ovaries in F1(first generation) female adults of Tribolium confusum
developed from previously treated adults with dose of 2 μg Insect-1
|Values represent Mean±SE (n = 50 females per age),
ns: No significant difference, *Significant, ***Highly significant at (p<0.05)
||Activity of azadirachtin on the width and the length (mm)
of basal oocytes of F1 adult females of Tribolium confusum resulted
from treated adults (2 μg Insect-1)
|Values represent Mean±SE (n = 50 females per age).
ns = No significant difference, *Significant, ***Highly significant at (p<0.05)
The treatment undergone by the parents did not affect the egg laying of the
F1 females. The hatching rate of eggs in the control did not differ significantly
the one of the eggs laid by the females treated with dose of 0.5 μg μL-1
(F = 20.19, DF = 99, p>0.0001). The hatching of eggs laid by the females
treated with the doses higher than 0.5 μg μL-1 has been
reduced more than the one of the eggs laid by the F1 females (Fig.
1). The hatching rate of eggs laid by F1 females was not affected by the
treatment (F = 2.86, DF = 99, p = 0.5).
Also data illustrated in Fig. 2 the effect of the tested growth regulator, the neem product (azadirachtin) on the viability or post-embryonic survival rate of eggs laid by the treated females which was significantly reduced (F = 27.04, DF = 99, p>0.0001) different between the control and the treatments.
Effects on growth and development: Results in Table 4 showed the duration of the life cycle, (eggs to adults), did not differ significantly between the control and the treatment with dose of 0.5 μg. However, the azadirachtin lengthened very significantly the total duration of the embryonic and post-embryonic development, when the dose was higher than 0.5 μg μL-1.
The weight of adult males derived from treated adults didnt differ significantly
between the control and the treatments (0.5 and 1 μg). This weight decreased
just significantly (F = 3.33, DF = 99, p = 0.0218) if the dose of the parents
treatment was higher than 1 μg (Fig. 3). The azadirachtin
did not affect significantly the weight of the F1 adult females (F = 1.23, DF
= 99, p = 0.3011) (Fig. 3). The adult individual (males and
females) lived between 8.2 to 8.5 days in the control.
|| Effect of azadirachtin on the viability rate (Fecundity)
of Tribolium confusum females
|Values represent Mean±SE (n = 50 females per age),
ns = No significant difference, *Significant, ***Highly significant at (p<0.05)
|| Hatchability rate of eggs laid by treated females and F1
of Tribolium confusum
|| Effect of azadirachtin on the duration of the developmental
cycle (egg to adult) in Tribolium confusum
|Values represent Mean±SE (n =100 eggs for each dose),
***Highly significant at (p<0.05)
|| Post-embryonic survival rate in Tribolium confusum
|| Weight of adult progeny of Tribolium confusum refernined
to the dose of parents treatments
The azadirachtin reduced significantly (F = 19.63, DF = 29, p = 0.002) the
adult longevity when the flour beetle was treated with the highest dose. On
the other hand, this growth regulator did not affect the longevity of the first
generation adults (Fig. 4).
Using of IGRs into management strategies to control T. confusum in flour
warehouses presents different issues than the use of IGRs in stored grain commodities.
The familiarity and visibility of this pest to consumers, its ability to penetrate
food packing and the essentially zero tolerance for insects in finished food
products are challenges that be faced when using IGRs for any other insecticide.
Because IGRs can be considered reduced-risk insecticides, they represent excellent
options for control in the warehouses environment.
|| Effects of azadjrachtin treatments (μg Insects-1)
on adult longevity of Tribolium confusum
The tests in which T. confusum larvae were exposed on concrete treated
with azadirachtin show the potential of using IGRs to control this species in
food warehouses, retail stores and urban storage situations. Arbogast
et al. (2002) showed reduction in population of several stored-product
pests including T. confusum, when hydroprene was applied as a spot-treatment
in a botanical warehouses. In laboratory tests, exposure of T. confusum
adults prolonged the development time of the eggs and caused mortality in the
dose-dependent manner similar to the results described for larval exposure (Mohandass
et al., 2006).
The results of our study confirm the efficiency of the growth regulators, derived
of the neem plant, azadirachtin on the development of insect pest of stored
products whose known as a steroid like tetra-notriteroenoid, isolated in 1968
from neem seeds (Butterworth and Morgan, 1971) along
with about 40 biner principles components belonging to diterpenoid, triterpenoids
and flavinoid groups from different parts of neem tree. The structural geometry
of azadirachtin was revealed by Nakanishi (1975). These
neem compounds have shown broad range of bioactivity against insects such as
reproductive fitness oviposition, hatchability, antifeedent, repellent, metamorphosis
disruption and death (Su and Mulla, 1998; Kumar
and Parmar, 1998; Mulla and Su, 1999; Mital
and Subbarao, 2003; Garcia et al., 2006;
Abdullah and Subramanian, 2008), these findings were
in agreement of present results. Feng and Ishman (1995)
showed the development of resistance in Peach potato aphid Myzans persicae
to pure azadirachtin over 4 generation but the same did not happen with neem
extract. The multiple mode of action or complexity of mechanism may be responsible
for avoiding the development of resistance in insects against mixture of neem
compounds. Hami et al. (2005) study the comparative
toxicity of three ecdysone against insecticide against the Mediterranean flour
moth, Ephestia kuehniella. They concluded that using bisacylhdrazine
derivatives are non steroidal ecdysteroid agonists that mimic the action of
moulting hormone. Also, our findings agree with that obtained by Arthur
et al. (2009) who concluded that pyriproxyfen gave effective residual
control of primary stored-product insect species by inhibition emergence of
exposed larvae and Sengottayan et al. (2009)
concluded that the neem based insecticide (Parker oil and Neema) containing
low lethal concentration, can be used effectively to inhibit the growth and
survival of Nilaparavata lugens.
Kumari (2010) found that IGRs cause a reduction in chitin,
also defects in the mobility (leg movement) was observed, these observations
also detected during our laboratory experiments. In contrast, Athanassiou
et al. (2005) found that neemazal was not very effective against T.
confusum where adult mortality was very low. It is clear from many investigations
that azadirachtin-based insecticides can be used with success for protection
of stored products, Kavallieratos et al. (2007)
found that the use of these substances is not comparable to the use of traditional
In summary, there are many opportunities for the increased use of IGRs in management programs to control stored product insects. In storage sites where the primary pest is Tribolium confusum and control is targeted towards the larval and adult stages, IGRs used alone may be sufficient to give complete control.
In this study, we have demonstrated that azadirachtin has a latent effect on T. confusum progenies as manifested by the reduced growth and development of immature and mature stages and lesser production of adults. The present results imply that the use of natural products like azadirachtin as pesticide would help controlling the storage pests in an environment-friendly way.
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