Effects of Phytochemicals in the Post Embryonic Development of the Lesser Mealworm - Alphitobius Diaperinus (Panzer)(Insecta: Coleoptera: Tenebrionidae)

ISSN 2349-7823
International Journal of Recent Research in Life Sciences (IJRRLS)
Vol. 3, Issue 1, pp: (25-33), Month: January - March 2016, Available at: www.paperpublications.org
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Paper Publications
Effects of Phytochemicals in the Post
Embryonic Development of the Lesser
Mealworm - Alphitobius Diaperinus (Panzer)
(Insecta: Coleoptera: Tenebrionidae)
Aziza Yasmin1
, N. C. Ghorai2
, A. Islam3
1,2
Department of Zoology, W. B. State University, Barasat, N. 24-Paraganas, W.B., India
3
Department of Zoology, K. C. College, Hetampur, Birbhum, W.B., India.
Abstract: Coleopteran insects constitute the major insects causing havoc damage to the stored food grains both for
human consumption and sowing. Lesser mealworm (Alphitobius diaperinus) is considered as a minor pest causing
damage to different stored grains especially in poorly maintained grain processing warehouses. Additionally it is
an important pest of poultry houses where it lives in poultry droppings and litter. Its larvae and adults are also
suspected to be health risk to humans as they are the vector of pathogens of many viruses, bacteria and protozoa
causing serious diseases in birds. Synthetic insecticides are widely used for the control of stored grain coleopteran
pests but development of pesticide resistance, accumulation of toxic residues in food and consequent health
hazards creating more and more problems than resolving them due to indiscriminate use of broad-spectrum
insecticides has been creating serious health hazards. These problems have led the scientists to introduce new
insecticides that are equally lethal to the insects but very less harmful to the consumers. With a view to this
objective the insecticidal efficacy of neem, eucalyptus oil, sesame oil and mustard oil have been tested to report
their various physiological effects on A. diaperinus during post embryonic developmental stages. Adults are
broadly-oval, moderately convex, black - 5.8 to 6.3 mm long in length. Eggs are about 1.5 mm in length, creamy
white with slightly rounded ends. Their incubation period is 6-7 days. Larvae show 6 instars attaining 7 - 11 mm
long at last instar. Pupae are creamy white to tan coloured, 6-8 mm long, with 6 days duration; the pharate adults
appear brown that gradually become black. The larvae show decline in survivality; the size and weight gradually
increase in successive instars. Treatment with different neem, eucalyptus oil, sesame oil and mustard oil depicted
substantial decline in survival rate and sizes of larval instars starting from 4th instars. Increased duration of
respective instars actually starts from third instars and increased duration of respective instars is maximum
following nimbecidine treatment and minimum in mustard oil treatment.
Keywords: Lesser mealworm, Alphitobius, Phytoproducts, Neem, Eucalyptus oil, Sesame oil, Mustard oil.
1. INTRODUCTION
A variety of stored grain insect pests render the stored food completely unfit for consumption as well as for sowing that
affect the quality and quantity of the concerned seeds. According to FAO report 10% of the stored foods are lost in this
way [1]. Such high levels of losses are attributed to the unscientific post-harvest management practices and poorly
designed storage programme. With the advent of chemical insecticides, the most commonly used method of controlling
stored grain pests is the application of synthetic contact insecticides and fumigants [2]. The indiscriminate use of broad-
spectrum insecticides has created more problems than resolving them. The development of pesticide resistance,
accumulation of toxic residues in food, serious health hazards for human applicators and undesirable environmental
pollution are acute critical problems that have drawn attention of the scientists to adopt alternative, natural pesticides that
are safer and environmentally acceptable [3], [4]. Constant scientific survey eventually have recommended use of eco-

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friendly alternatives, with a special focus on the plant-derived insecticidal compounds [5], [6]. Botanical insecticides have
long been treated as alternative to synthetic chemical insecticides for pest management, because these are less harmful to
the environment due to their biodegradation [3]. The use of these traditional materials has recently stimulated research to
establish the scientific basis for their continued use regarding their efficacy, active constituents and effective application
technology [7]. But in spite of the huge potentialities of phytoproducts as insecticides, only a handful of such compounds
are currently used in post harvest management of seeds [7].
Over the past three decades, neem, Azadirachta indica has come under close scientific scrutiny as a source of natural
pesticide [8]. Neem products have been shown to be effective protectants of grains against infestation by grain weevils,
grain borers armyworm and several species of storage moths [9]. The characteristic odour of neem mate rials permitting
the closed storage environment presumably repelled insects and bitter compounds in neem materials mixed with the stored
grain discouraged insect feeding. Probably, the oil present in neem seed or kernel also inhibits egg deposition on grains.
Singh (1993) [10] has reviewed the role of neem products on the behaviour, growth, development, survival and
reproduction of stored grain insects. The advantage of neem treatment is that it does not impair the germination of stored
seed [11].
In addition to neem, attentions have also been drawn for the pesticidal properties of eucalyptus oil [12]. [13], [14], [15];
oil of Sesamum indicum [16], [17]; Cucurmin [18], Mustard [19]; Garlic is considered as the heavenly spice having
innumerable medical use. It has proved to be a potent antimicrobial and insecticidal agent [20], [21], [22], [23]. Essential
oil from chenopodium, sesame and garlic were also tested against different insect pests [24].
Of all the stored grain insects, Coleopteran insects constitute a major percentage of the stored grain major and minor
pests. The lesser mealworm, Alphitobius diaperinus (Panzer), is a cosmopolitan stored product minor pest of wheat,
barley, millet, cottonseed, oatmeal, soybeans, cowpeas, oilseed products, and tobacco especially in poorly maintained
grain processing warehouses. It is well adapted for warm, humid conditions. Additionally it is an important inhabitant of
poultry or brooder houses where it lives in poultry droppings and litter and is considered a significant pest in the poultry
industry [25]. The beetle also consumes birds' feed and irritates them by biting them [26].
Alphitobius is also known to be vector of many pathogens. It spreads many bird diseases [27]. It also transmits different
animal viruses [28]. It also transmits bacteria like Campylobacter jejuni, Salmonella typhi, E. coli, and
Staphylococcus [29]. This beetle can become a household pest if old litter is used as compost.
In spite of different attempts to explore different possible measures to control Alphitobius, [30], [31], [32], [33], no
attempt has been made to show deleterious effects of phytoproducts on this pest. With a view to this unexplored field,
present investigation was undertaken to report the effects of nimbecidine, sesame oil, mustard oil and eucalyptus oil on
the post embryonic developmental stages and fecundity of A. diaperinus.
2. MATERIALS AND METHODS
Experimental insect: The insect (both larvae and adults) were collected from a neighbouring warehouse of the Food
Corporation of India. They were specifically collected from damp, almost dark sites from the underneath of the polythene
sheets of the wheat stacks. In the laboratory, they were maintained in dark containers at 28 +/- 2o
C and 70 – 75% RH. on
wheat. To check fungal infection, 2% Nepazin was mixed with the wheat. Temperature and humidity were routinely
examined by thermometer and hygrometer. By routine examination from the lower stratum of the culture pots pupae were
separated and hatched adults were kept in separate pots to assess the normal developmental profiles. Their incubation
period is 4 - 7 days and it takes 40 – 50 days to complete the life cycle. After mating, a female beetle can lay over 2,000
eggs (with an average 200 - 400). Females produce eggs for most of their life. Adults can live up to 3-12 months;
Preparation of phytoproducts:
Preparation of neem extracts: Leaves, fruits, and bark were collected from neem trees (Azadirachta indica) and were
used to prepare water extract of neem. Respective parts of neem were weighed and emulsified in 1000 ml of distilled
water with a blender and the suspension was sieved through a muslin cloth. A powder was prepared by drying respective
parts at 35–40°C in an oven and these were grinded to form powder.

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Preparation of sesame oil extracts: Sesame (Sesamum indicum L.) is an oleaginous seed of the family Pedaliaceae.
Sesame seeds are allowed to be grinded in a traditional Ghani and the extracted oil was refined to be used for treatment.
Preparation of eucalyptus oil: Fresh eucalyptus leaves were taken in a pot and were allowed to boil to extract oil which
were collected and used to treat the insect.
Preparation of mustard oil: Mustard seeds (Brassica compestris) were allowed to be grinded in a traditional Ghani and
the extracted oil was refined to be used for treatment.
All the developmental stages were divided into control and four treated groups and their morphological and durational
changes were recorded separately. Mortalities were recorded at every 24 hours until all larvae or pupae had either died or
developed to the next stage.
3. RESULTS
Alphitobius is a holometabolous insect showing adult, eggs, larvae and pupae in its life cycle. Characteristic features of
these stages are as follows:
Adults: Lesser mealworm adults are broadly-oval, moderately convex, black or brownish-black and usually shiny in
appearance. Colour can be variable depending on age or 'strain' of lesser mealworm. Length is approximately 5.8 to 6.3
mm. Antennae are densely clothed with short yellowish hairs, with the terminal segment lighter in colour. The head is
deeply emarginate in front, has a distinct clypeal groove, and the surface is coarsely punctured. Eyes are also emarginated.
The prothorax is twice as broad as long, slightly narrowed from base to apex, with sides feebly curved and narrowly
margined. There is a shallow impression on each side of the pronotum in the middle near the base. The pronotal disk is
finely and sparsely punctured, with punctures much coarser laterally.
The elytra have moderately impressed striae with finely punctured, feebly convex intervals. Elytral punctures are sparse
and nearly as large as those of the striae. The ventral surface of the lesser mealworm is dark reddish-brown, with the
prosternal process horizontal between coxae and having a prominent apex.
Eggs: Eggs are about 1.5 mm in length, creamy white to tan coloured and slender with slightly rounded ends.
Larvae: Larvae hatch in 6-7 days and complete development to the adult stage in 40 to 100 days, depending on
temperature and food quality. Several studies have been conducted on A. diaperinus development on varied diets, at
varying ambient temperatures and microhabitats, and among other poultry house inhabiting species. Larvae consume
spilled feed, manure, and in some cases dead or sick birds and cracked eggs.
Larvae have a segmented body with three pairs of legs and tapering abdominal tip posteriorly. They are approximately 7 -
11 mm in length at last instar. Freshly-emerged larvae are a milky colour but gradually a shade of brown colour are
observed; before moulting they again assume dark colour. There are 6 larval instars. The duration of each instar is 3, 5, 5,
6, 8 and 12 days respectively.
Pupae: Pupae are approximately 6 to 8 mm in length, creamy white to tan coloured, with legs tucked alongside the body.
Pupal period is 6 days. At the end of pupal period, the whole body appears brownish and newly hatched pharate adults
appear brown that gradually turns into black.
Declining survival rate: Following the rule of natural selection gradually the number of surviving individuals in
successive instars decline by 34%. The size and weight however gradually increase in successive instars; it shows 200
times increase in size and substantial increase in weight (Table 1).
Effects of different phytoproducts: Treatment with different phytoproducts depicted substantial decline in survival rate
showing 44%, 44%, 44% and 42% decline in number in respect of treatment with nimbecidine, eucalyptus oil, sesame oil
and mustard oil. Increased duration of respective instars actually starts from third instars and duration increase is
maximum following nimbecidine treatment and minimum in mustard oil treatment. Declining size of respective instars in
different treatments appeared significant from 4th instar onwards (Table 2).
Effect of different doses of Nimbecidine against different developmental stages: 50 gm semi crushed wheat grains
were treated with 0.5ml, 0.4 ml, 0.2 ml, 0.1 ml of nimbecidine and 20 adults were subjected to toxic effects of

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nimbecidine for several weeks to report the detrimental effects of this phytoproduct on the post embryonic developmental
stages of this beetle (Table – 3, 4).
4. DISCUSSION
In spite of the tremendous advent of the chemical insecticides, due to their hazardous side effects, different phytoproducts
have long been treated as alternative harmless insecticides that has minimum side effects and from the view point of
imparting resistance to insects and toxic residual effects in grains and for their biodegradable property, use of
phytoproducts has been gaining importance day by day. The insecticidal activity of different essential oils and plant
extracts against different stored-product pests has been evaluated [34], [35], [36], [37]. [38]. In spite of the wide-spread
recognition that many phytoproducts possess insecticidal properties, only a handful of phytopesticides are directly in use
because the commercialization of new botanical insecticides can be hindered by a number of issues [39]. These products
presently constitute 1% of the world insecticide market [40]. Essential oils from different plant species possess ovicidal,
larvicidal, and repellent properties against various insects and are regarded as environmentally compatible pesticides [41].
Neem (Azadirachta indica) has long treated as the miracle plant for its spectacular role in traditional medicine for the
cure of multiple diseases including cancer for centuries [42], [43], [44]. Apart from its medicinal roles, it has long been
used in controlling the infestation of stored grains. Different vegetative parts of neem, like flower [45], wood chipping
[46], neem oil from seed and kernel [47] etc. have been tested against different insects for their control.
Nimbecidine is a totally natural neem-oil based product with Azadirachtin as the most effective insect growth regulator
molecule in addition to Meliantriol, Salanin, Nimbin. It acts as an insect repellant, antifeedant, growth regulator and
mating disruptor and has been proved to be really effective against a wide range of pests [48], [49], [07], [50], [51], [52],
[53]. It also corroborates the findings of Jilani et al (1988) {54] showing significant reduction in the number of progeny
emergence, delayed development, failure of pupation and abnormal pupae and adults of T. castaneum treated with
Calneem oil.
Data of present investigation showed a dose dependent effect (Table -1) which might not due to the Azadirachtin content
since other active ingredients of nimbecidine may be responsible for such dose-dependent effect [48]. Present data
showing reduced progeny production in A. diaperinus corroborates the observation of Lale and Mustapha (2000) [55]
where they reported significant reduction in oviposition and adult emergence in Callosobruchus maculatus treated with
neem seed oil. Mortality of A. diaperinus in nimbecidine treated wheat corroborates the findings of Dunkel et al (1990)
[56] who noticed significant mortality of S. oryzae in neem-based insecticide Margosan-O treated wheat.
As the pupae are non feeding stage, their mortality could not be explained by any of the known modes of action of neem
i.e. antifeedant effects and growth regulator effects. Treating stored grain with nimbecidine can disrupt insect feeding by
making the treated materials unattractive or unpalatable and as a consequence, insect growth, survival and reproduction
are adversely affected [57]. Azadirachtin, inhibits the release of prothoracicotropic hormones and allatotropins, thereby
affecting metamorphosis in insects [58].
Neem has been treated as an anti-feedant, an oviposition deterrent (anti-egg laying), a growth inhibitor and a
chemosterilizer. [59]. Azadirachtin, a tetranortriterpenoid compound, closely mimics the hormonal action of Edison,
which is necessary for reproduction in insects. Substantial decline in the level of ovarian protein corroborates this data.
Disruption of feeding process may be attributed to the decline in the concentration total body protein in other organs.
Since essential oils of different plants are a complex mixture of components (unlike chemical pesticides which are based
on a single product), they work together within a plant and it is unlikely that pests will become resistant to them. Since
eucalyptus oil is particularly strong when in vapourized form, they could also be used commercially as a fumigant
(gaseous pesticide) for stored products and impregnated into packaging to prevent insect infestation. The study explains
that eucalyptus essential oil could have a large role in the control of pests and provide an alternative to chemical
pesticides.
The vapour of mustard oil can penetrate the grain and kill, the internal larvae and the pupa of S. zeamais within 24 h, like
other grain fumigants such as phosphine and methyl bromide. Of all the developmental stages, pupae proved to be most
resistant to the fumes of oil because they have the least respiratory rate [60] because the developmental stage of low

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respiration rate absorbs less quantity of the fumigant in a given time period. In this study also, the bioactive compound of
mustard oil seem to be penetrated the larvae or pupa through spiracles.
During the pupal period oxygen consumption initially declines and then rises again, due to changes in the enzymes
systems regulating energy release. It is well accepted that the developmental phases of insects with higher metabolic rate
are more susceptible to gas exchange incited by an insecticide resulting in the higher energy expense for homeostasis
maintenance. (Emekci et al., (2002) [61]. reported that in cockroaches (Blattella germanica) CO2 production rate
increased when exposed to sub lethal concentrations of AITC (the major component of the mustard oil), which led to
conclude that AITC might also act on the energy metabolism and may be acting as inhibitor of oxidative phosphorylation,
and thus interrupting ATP formation. All the phytoproducts used in this investigation act as anti-feedant, oviposition
deterrent (anti-egg laying), a growth inhibitor and chemosterilizer and these phytoproducts indirectly act as
phytoinsecticide.
ACKNOWLEDGEMENT
This investigation has been carried out by the financial grant received from the University Grant Commission funded by
Moulana Azad National Fellowship for Minority Students as Aziza Yasmin is regarded as Ministry of Minority Affairs
scholar. In addition, we are indebted to Sandipan Chatterjee for his active cooperation in carrying out the technical aspects
of the investigation.
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APPENDIX - A
Table – 1. Gradual decline in the survival rate (number), duration of different post embryonic stages (days), size (mm), colour
and weight (mg) of different stages:
Parameters Egg 1st Instar 2nd
Instar
3rd
Instar
4th
Instar
5th
Instar
6th Instar Pupa Adult
Survival rate 50+/- 9 48+/- 8 47+/- 9 46+/- 7 44+/- 9 42+/-8 40+/-8 35 +/-10 33+/-12
Duration 6-7 3-4 5-6 5-6 6-7 8-9 12-14 6-7
Size (mm) 1.5 2.2+/-0.1 3.2+/-0.2 4.3+/-0.4 5.4+/-0.5 7.7+/-0.2 8.8 +/-0.3 5.8+/-0.6 6.1+/-0.5
Body Color Milky
white
Creamy
white
Light
brown
Brown Deep
brown
Dark
Brown
Dark
Brown
Off
white
Black
Weight (mg) 0.0004 0.0005 0.0010 0.0018 0.0036 0.007 0.009 0.010 0.015

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Paper Publications
Table – 2. Gradual decline in the survival rate (number), duration (days), size (mm) and weight (mg) of different post
embryonic stages in response to nimbecidine, eucalyptus oil, sesame oil and mustard oil treatment against untreated insects:
Parameters Egg 1st
Instar
2nd
Instar
3rd
Instar
4th
Instar
5th
Instar
6th
Instar
Pupa Adult
Survival rate
Nimbecidine
Eucalyptus oil
Sesame oil
Mustard oil
50+/- 9
50 +/- 6
50 +/- 4
50 +/- 6
50 +/- 4
48+/- 8
42+/- 5
45+/- 6
44+/- 4
45+/- 4
47+/- 9
45+/- 4
44+/- 8
44+/- 6
43+/- 4
46+/- 7
41+/- 6
44+/- 6
45+/- 3
44+/- 4
44+/- 9
40+/- 5
43+/- 6
42+/- 5
43+/- 4
42+/- 8
36+/- 4
35+/- 3
35+/- 4
38+/- 4
40+/- 8
35+/- 4
35+/- 3
35+/- 4
36+/- 2
35 +/-10
29+/- 4
31+/- 3
30+/- 5
31+/- 4
33+/-12
28+/- 4
28+/- 3
28+/- 4
29+/- 3
Duration
Nimbecidine
Eucalyptus oil
Sesame oil
Mustard oil
6-7
6-7
6-7
6-7
6-7
3-4
3-4
3-4
3-4
3-4
5-6
5-6
5-6
5-6
5-6
5-06
5-11
5-09
5-10
5-08
6-07
6-13
6-11
6-12
6-11
8-09
8-15
8-14
8-13
8-12
12-14
12-19
12-18
12-17
12-17
6-07
6-11
6-10
6-10
6-09
Size (mm)
Nimbecidine
Eucalyptus oil
Sesame oil
Mustard oil
1.5
1.5
1.5
1.5
1.5
2.2+/-0.1
2.2+/-0.2
2.2+/-0.1
2.2+/-0.1
2.2+/-0.2
3.2+/-0.2
3.2+/-0.2
3.2+/-0.2
3.2+/-0.2
3.2+/-0.2
4.3+/-0.4
4.0+/-0.2
4.1+/-0.2
4.1+/-0.3
4.2+/-0.4
5.4+/-0.5
4.8+/-0.2
4,9+/-0.1
5.1+/-0.2
5.0+/-0.3
7.7+/-0.2
6,9+/-0.3
7.1+/-0.1
7.0+/-0.2
7.2+/-0.3
8.4+/-0.3
7.6+/-0.1
7.9+/-0.3
7.8+/-0.2
8.1+/-0.3
5.8+/-0.6
5.1+/-0.3
5.2+/-0.4
5.3+/-0.4
5.4+/-0.2
6.1+/-0.5
5.8+/-0.2
6.0+/-0.1
5.9+/-0.3
6.0+/-0.2
Body Color Milky
white
Creamy
white
Light
brown
Brown Deep
brown
Dark
Brown
Dark
Brown
Off white Black
Weight (mg)
Nimbecidine
Eucalyptus oil
Sesame oil
Mustard oil
0.0004
0.0004
0.0004
0.0004
0.0004
0.0005
0.0005
0.0005
0.0005
0.0005
0.0010
0.0010
0.0010
0.0010
0.0010
0.0018
0.0015
0.0016
0.0018
0.0017
0.0036
0.0032
0.0035
0.0033
0.0033
0.0071
0.0066
0.0068
0.0069
0.0070
0.0092
0.0081
0.0086
0.0086
0.0088
0.014
0.012
0.013
0.014
0.013
0.015
0.011
0.013
0.014
0.012
Table 3. Mean number of Alphitobius diaperinus larvae, pupae and adults that emerged from wheat treated with different
dosages of Nimbecidine at different times after oviposition period. Data are mean ± SE (n=5 taking 20 insects).
Doses
% (v/v)
1st instar
larva
2nd instar
larva
3rd instar
larva
4th instar
larva
5th instar
larva
6th instar
larva
Pupa Adult
Control 53±5.2 48.6±5.81 45±4.04 44±3.51 42.6±3.8 39±4.72 23±2.64 32±4.16
0.2ml 31±3.2 26±3.05 24.6±0.3 23±0.57 22±1.15 20±1.15 15±2.88 11±2.08
0.1ml 34.6±2.4 31±2.08 30.3±1.8 27.6±1.2 24.3±2.3 23±2.64 17.6±1.4 16±1.15
0.05ml 38.3±1.2 37±1.9 34.3±1.4 33±1.52 29±2.08 26±3.05 18.6±1.8 16.3±2.1
0.025ml 39±0.57 36.6±0.88 35.3±0.3 32.3±1.2 30±.6.7 28.3±2.2 26.3±2.0 24.6±2.6
Number of progeny emergence (mean ± S.E.). Means in the same column followed by the same lower case letter do not
differ significantly, means in the same rows followed by the same uppercase letter do not differ significantly at P < 0.05
(Holm–Sidak test).
Table 4. Toxicity of different dosages of Nimbecidine in wheat grains to Alphitobius diaperinus adults at a temperature of
25°C and 65–70% RH. Data are means ± SE (n=5 of 20 insects each.Exposure period (d)% mortality (mean ± SE)
* Means in the same column followed by the same lowercase letter do not differ significantly, means in the same rows
followed by the same uppercase letter do not differ significantly at P < 0.05 (Holm–Sidak test).
Dosage % (v/v) ml 2 days 6 days 10 days
Control 0±0.00 0±0.00 0±0.00
0.5 6.66±3.52 11.66±0.88 18±1.154
0.4 6±1.15 8.66±0.66 12±1.15
0.2 3.33±0.88 6±0.57 9±0.57
0.1 1±0.57 1.66±0.88 3.33±0.88

Effects of Phytochemicals in the Post Embryonic Development of the Lesser Mealworm - Alphitobius Diaperinus (Panzer)(Insecta: Coleoptera: Tenebrionidae)

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