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Indian Journal of Experimental Biology
Vol. 50, March 2012, pp. 232-238
Mortality and testicular derangements in red flour beetles, Tribolium castaneum
(Herbst) exposed to hen’s egg white proteins
Ranjit K Parshad* & Megha Kansal
Department of Zoology, College of Basic Sciences & Humanities, Punjab Agricultural University, Ludhiana 141 004, India.
Received 1 August 2011; revised 27 December 2011
Red flour beetle (T. castaneum) is a major pest of stored grains and is known for its adaptability to all classes of
insecticides. The present study was carried out to determine the insecticidal potential of egg white proteins to manage beetle
population. Protein samples obtained through salt fractionation were lyophilized and were used separately and
simultaneously in different concentrations by adding them to wheat flour and milk powder. The results indicated that the
mortality rate of the adult beetles was dependent on the type of treatment, concentration of protein samples and duration of
feeding. In multiple-choice feeding trials beetles showed their movement towards the control section as the concentration of
treatment increases. Marked abnormalities were observed in appearance and dimensions of the testes which indicated that
the egg white proteins caused considerable effect on the process of spermatogenesis and sperm functions. SEM study
revealed the formation of deep wrinkles and folds on the testicular surface of the testes of beetles fed on treated diets, points
towards the depletion of internal cellular material. The results suggest that egg white protein affects the survival and cause
subsequent derangements in the testis of red flour beetle.
Keywords: Egg proteins, Mortality, Red flour beetle, Testis
The red flour beetle, Tribolium castaneum (Herbst)
(Coleoptera: Tenebrionidae), a major pest of the
processing and storage of grain-based products, it has
a long history of exposure to pesticides and has
proved to be readily adaptable to all classes of
insecticides and fumigants, having developed
resistance via oxidative and hydrolytic metabolism,
target insensitivity and other mechanisms1-3
.
Therefore, the importance of using some alternative
methods in pest control has been increasing, which
should also be ecofriendly due to environmental
problems caused by chemical products. The egg white
proteins possess unique multifunctional properties and
hence being extensively used by medical, cosmetic,
pharmaceuticals and biotechnology industries4.
Numerous biological activities including antibacterial,
antiviral, immunomodulatory and anti-cancer have
now been associated with egg components indicating
the importance of egg and egg components in human
health, disease prevention and treatment4. Avidin a
glycoprotein present in egg white protects the chicken
embryo from disease causing organisms and has also
been shown to provide resistance to wide spectrum of
insect pests in crops like maize, tobacco and potato
after the insertion of gene coding for avidin
production5-8
. However, no information is available
on the insecticidal potential of this protein against
pests in stored products. Therefore, this study has
been undertaken to determine the effect of hen’s egg
white proteins against T. castaneum.
Materials and Methods
Insect stock culture—Beetles obtained from
infested samples were maintained in the laboratory by
placing them in glass jars containing sterilized wheat
flour/milk powder mixture (75:25). The colonies were
reared under an experimental conditions (30° ± 1°C
and 75% RH).
Preparation of egg white protein samples—The
shell of the eggs was pricked carefully and whole egg
whites (WEW) were taken out in a flask. Pooled egg
whites were lyophilized till dried. From collected egg
whites two protein fractions (PPT-I and PPT-II) were
isolated through salt precipitation method9 and then
these fractions were used after freeze drying.
Feeding trials
No-choice feeding trials using adult beetles—
Feeding trials were conducted in three treatment
——————
*Correspondent author :
Telephone: 9872185327
E-mail: [email protected]
PARSHAD & KANSAL : EFFECTS OF HEN’S EGG WHITE PROTEINS ON RED FLOUR BEETLE
233
groups (WEW, PPT-I, PPT-II) with three subgroups
within each group using rearing medium containing 2,
5, 10% of each of the protein sample. Simultaneously
a group without egg white proteins was run which
served as control. One hundred and ten beetles were
released in each bottle and the mouth of each bottle
was covered with muslin cloth. The bottles were kept
in the incubator at 30° ± 1°C and 75% RH. Mortality
of adult beetles was observed on 15th, 23
rd, 33
th, 41
th,
48th, 55
th day of the experiment. The trial was
performed four times.
Multiple-choice trials—Sterilized petriplates were
taken and the base of each petriplate was divided into four equal parts. Four diets were tested at a time for their preference. Rearing medium without and with 2, 5, 10% WEW, PPT-I, PPT-II was sprinkled into the petriplates and 50 adult beetles were released in the centre of each petriplate. The number of beetles in each patch was
observed at intervals of 24 h for 5 consecutive days and this experiment was repeated three times.
Beetle dissection and measurement of testicular
dimensions—The plan of the experiment for different treatment groups and subgroups was the same as
mentioned earlier under no-choice feeding trials. Fifty beetles were released in each bottle. After 30 days of treatment equal number of beetles were dissected from each group in a drop of Ringer’s physiological solution on a wax-fixed petridish under dissecting microscope. A pair of surgical pins was used to open
the abdominal cavity and then the pair of testes was taken out
10. The surrounding tracheoles and
fat bodies were removed. Size of the testes was measured with the help of ocular micrometer pre-calibrated with stage micrometer. Testes were also processed for scanning electron microscopy
and histological studies.
Scanning electron microscopy (SEM)—The testes of specimens after 30 days of feeding of treated and untreated rearing medium were removed and fixed in 2.5% glutaraldehyde for 2 h and post fixation was
done in osmium tetraoxide for an hour followed by three buffer washings of 15 min each. The tissues were then dehydrated in a graded ethanol series (50-100%) for 15-20 min in each grade. After drying, the samples were assembled on aluminium stubs, coated with gold and were examined for SEM
11.
Histological studies—After 30 days of treatment 15 beetles were taken from each bottle, decapitated with surgical blade and fixed the abdomens in alcoholic bouins fixative for 24 h. The material was
washed with distilled water and then dehydrated using graded ethanol series (50 - 100%). Paraffin wax blocks were prepared and serial sections of 7 µm thickness were obtained and stained with haematoxylin and eosin.
Preparation of sperm smears—After 30 days beetles
from each treatment groups and from control
were dissected and the testes were teased on a slide in a
drop of saline solution, observed under microscope
to check the presence of sperms. The smears was
allowed to dry and fixed in the methanol for 5 min. The
slides were then allowed to dry and smears were stained
with 4% Giemsa solution for 15-20 min and again slides
were dried and examined under microscope.
Statistical analysis—Data have been presented as
mean±SE and differences between the control and treated
groups were determined using analysis of variance
(ANOVA) after angular transformation of % data.
Results and Discussion
Mortality of adult beetles—Significant mortality
was recorded in beetles fed on diet containing
different treatments (WEW, PPT-I, PPT-II) as
compared to that in beetles fed on diet containing
rearing medium without protein samples (Table 1).
After 33 days of treatment more than 90% mortality
was observed in beetles fed on diet containing 10%
PPT-I and 10% PPT-II while 100% mortality of
beetles were observed between 41-48 days of
treatment in diet containing 10% PPT-I, 5% PPT-II
and 10% PPT-II and 48 to 55 days, in diet containing
5% PPT-I. Analysis of data revealed that the
significant effect (P<0.05) on the mortality rate of the
adult red flour beetles was dependent on the type of
treatment, concentration of protein samples and
duration of feeding.
Feeding preference—Observations on movement
of red flour beetle after 24-hours of intervals for
five consecutive days in different diets were made
and the results were depicted Fig. 1. The movement of
the beetles across different diets varying in protein
concentration in multiple-choice trials reflects that
the food containing whole egg white (2, 5%) was
preferred over other diets containing PPT-I and
PPT-II. However, when the concentration of whole
egg white was increased to 10%, the beetles showed
their movement towards the control section i.e. having
food without the egg proteins.
Morphological study and measurement of
testicular dimensions—Male reproductive system of
INDIAN J EXP BIOL, MARCH 2012
234
T.castaneum consists of a pair of oval testes. From
each testis a short, vas efferens arises which was
connected with the vas deferens. A portion of vas
deferens was enlarged as the seminal vesicle. The two
vasa deferentia were connected to a common median
ejaculatory duct. The shape, size and appearance of
testes varied considerably in control and treatment
groups. The shape of testes were nearly oval in
majority of the beetles dissected from the control
group (Fig. 2 a) whereas testes appeared oval to
elongated in structure, in different groups of beetles
kept on diets containing variable concentrations of
different protein samples (Fig.2 b-d). Length of the
testes decreased significantly (P < 0.05) in all the
treatment groups as compared to control except the
length of testes of beetles fed on 5% WEW and 10%
PPT-I, where the decrease in size was observed
to be non-significant (Table 2). No marked change
Fig 1—Multiple-choice feeding trials showing the movement of
adults of red flour beetle at interval of 24 h for 5 consecutive days
on medium without or with variable concentrations of different egg
white proteins. [a, 2% protein; b, 5 % protein; c, 10 % protein].
Table 1―Percentage mortality of red flour beetles after15, 23, 33, 41, 48, 55 days in rearing medium without or with variable
concentrations of different egg white proteins in no-choice feeding trials using adult beetles.
[Values are mean ± SE]
Treatment Protein
(%)
Beetle mortality (%)
15 days 23 days 33 days 41 days 48 days 55 days
Gr 1 0 12.72±1.05 19.99±5.25 28.17±2.09 43.63±6.29 45.45±6.29 49.99±7.87
2 22.26±6.03* 24.54±2.36* 55.90±8.13* 62.26±11.80* 62.26±11.80* 62.72±12.07*
5 15.45±1.05* 23.18±2.88* 28.45±3.93 33.63±1.05* 37.26±3.14* 45.45±5.77*
Gr 2
10 29.54±10.23* 39.09±5.24* 45.45±7.87* 52.26±10.75* 57.27±12.07* 61.36±10.23*
2 23.63±0.52* 39.08±2.62* 50.90±9.44* 55.45±6.82* 62.72±2.62* 71.36±1.83*
5 11.36±2.36 27.27±1.05* 48.62±6.56* 87.72 ± 0.26* 97.72±0.26* 100±0*
Gr 3
10 9.99±3.14* 60.45±4.46* 94.08±2.07* 100±0* - -
2 9.54±1.31* 39.09±2.62* 55.45±2.10* 60.90±2.62* 70.90±2.62* 79.54±0.13*
5 28.17±3.14* 46.36±2* 77.72±0.26* 97.27±1.05* 100±0* -
Gr 4
10 21.36±3.41* 54.40±5.24* 94.08±0.26* 99.54±0.26* 100±0* -
* Significant differences (P<0.05) between control and treatment groups on different days
Gr 1, control; Gr 2, WEW; Gr 3, PPT-I; Gr 4, PPT-II
Table 2 ― Measurement of testicular dimensions of red flour
beetles reared on medium without or with variable
concentrations of different egg white protein.
[Values are mean ± SE ]
Treatment Protein
(%)
Size of testes (µm)
Length Breadth
Gr 1 0 525±14.43 256.25±6.25
2 275±10.20* 250±15.72
5 475±53.03 400±42.08*
Gr 2
10 362.5±38.86* 275±28.86
2 362.5±23.93* 228.125±33.49
5 356.25±58.07* 275±62.08
Gr 3
10 456.25±161.55 325±50
2 368.75±44.92* 243.75±57.41
5 256.25±15.72* 206.25±23.66
Gr 4
10 431.25±21.34* 381.25±8.52*
* Significant differences (P<0.05) between control and treatment
groups
Gr 1, control; Gr 2, WEW; Gr 3, PPT-I; Gr 4, PPT-II
PARSHAD & KANSAL : EFFECTS OF HEN’S EGG WHITE PROTEINS ON RED FLOUR BEETLE
235
was recorded in the breadth of the testes but with
few exceptions. The breadth of testes increased
significantly (P < 0.05) in beetles fed on 5% WEW
and 10% PPT-II. These changes in the shape,
appearance and size of the testes indicated that these
egg white protein samples caused considerable effects
indirectly or directly in the growth and differentiation
of testis. Whole egg white protein sample appeared to
promote cellular aggregations as a result of which two
zones within a testis i.e. denser and translucent
became conspicuous (Fig. 2 b). While PPI-I leads to
deformation in the testicular shape (Fig.2 c). On the
other hand, PPT-II caused asymmetry, increase in size
and testes appeared as sacs filled with fluid (Fig. 2 d).
Scanning electron microscopy of testicular
surface—Testicular surface was observed to be lined
by two layers; peritoneal sheath and below follicular
epithelium (Fig. 3 a). Higher resolution of the
testicular surface revealed the presence of interwoven
meshwork of band-like structure which probably
provides stiffness to the organ (Figs 3 a and b). Testes
of beetles reared on diet containing treatment got
shriveled and wrinkles (Figs 3 c and d). Formation of
deep wrinkles and folds in the testes of treated beetles
Fig. 2—(a): Oval and dense testes of beetle fed on rearing medium containing wheat flour and milk powder (75:25) without egg white
proteins. (b): Elongated testes with denser and translucent zones of beetle fed on rearing medium containing 2% WEW. (c): Elongated,
asymmetrical and dense testes of beetle fed on rearing medium containing 10% PPT-I. (d): Dense, asymmetrical and hypertrophied testes
of beetle fed on rearing medium containing 10% PPT-II [ bar = 100µm ].
IND
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236
Fig. 3—(a): Testicular surface was lined by two layers; peritoneal sheath and follicular epithelium. (b): Higher resolution of the testicular surface at different site revealing the
presence of interwoven meshwork of band-like structures. (c): Shriveled and wrinkled testicular surface of testes of beetle fed on rearing medium containing treatment.
(d): Higher resolution of the testicular surface revealing large number of wrinkles, of beetle fed on rearing medium containing treatment.
PARSHAD & KANSAL : EFFECTS OF HEN’S EGG WHITE PROTEINS ON RED FLOUR BEETLE
237
points towards the regression and depletion of
internal cellular material. It is suggested that the
spermatogenic cells or the displacement of later lead
to the formation of cellular clumps and zonation in
testicular appearance (dense and translucent).
Spermatogenesis and spermatozoa—Similar to
other insect species, the testes were formed by
round follicles and they were six in number in
T. castaneum, 175 µm in diameter, connected to short
efferent tubes. The follicles were coated and
interconnected by a peritoneal sheath. Beneath this
membrane, a follicular epithelium covered each
follicle11
. The testicular tubule contain spermatogenic
cells at different stages and exhibit cystic pattern of
spermatogenesis i.e. many cysts in various
developmental stages and each cyst contained cells at
the same developmental stage.
From the longitudinal section of the pair of testes,
location of different germ cells and the zones of
progressive development of the sperm formation from
the spermatogonia, spermatocytes and spermatozoa
can be traced starting from the apex to the base of
the testis (Fig. 4 a). Spermiogenesis involves process
such as nuclear elongation, chromatin condensation
and flagella development. Differentiation of the
spermatids occurs within cyst and bundles of
spermatozoa can be seen within a testis in part of the
stained section (Fig. 4 b). Feeding on egg white
proteins affected the number of spermatogenic cells
and number of spermatozoa per bundle (Fig. 4 c).
Treatment with different egg white proteins also
induced structural abnormalities particularly those
fed on variable concentrations of WEW and
PPT-II. Large number of spermatozoa with coiled
Fig. 4—(a): Distal cysts (arrows) in the testis of beetle. (b): Sperm bundles in the testis of beetle. (c): Sperm bundles in the testis of beetle
fed on diet containing treatment [ bar = 50 µm ]. (d): Coiling of flagella of spermatozoa from testes of beetle fed on rearing medium
containing WEW or PPT-II [ bar = 100 µm ].
INDIAN J EXP BIOL, MARCH 2012
238
flagella can be seen in all the three concentrations
of WEW and PPT-II (Fig. 4 d).
The results of this study have shown that all the protein fractions of egg white cause mortality and disrupt testicular functions in red flour beetle. Fraction PPT-II appeared to be more effective in inducing mortality and degenerative changes in the testis than the whole egg white proteins and PPT-I. The insecticidal properties of avidin, a egg white protein are well established through genetic engineering particularly in transgenic maize
6-8. This
protein binds to biotin as a result of which growth and development of the insect remain inhibited and cause death. Higher rate of mortality of beetles observed in PPT-II fraction may be due to avidin, which is expected to be present in this fraction, however, further studies need to be carried out to ascertain the biopesticidal properties of egg white proteins. In addition, whole egg white and PPT-I fractions as indicated in this study also affected the survival and reproductive process in males.
Acknowledgement
Thanks are to the Director of Electron Microscopy
and Nanoscience Laboratory, Punjab Agricultural
University, for scanning microscopy facilities.
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