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: rkparshad@yahoo.co.in

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 ].

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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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