Mortality and testicular derangements in red flour beetles, Tribolium castaneum (Herbst) exposed to hen s egg white proteins

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1 Indian Journal of Experimental Biology Vol. 50, March 2012, pp 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 , 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 mechanisms 1-3. 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 industries 4. 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 treatment 4. 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 *Correspondent author : Telephone: rkparshad@yahoo.co.in insect pests in crops like maize, tobacco and potato after the insertion of gene coding for avidin production 5-8. 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 method 9 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

2 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 15 th, 23 rd, 33 th, 41 th, 48 th, 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 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 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 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

3 234 INDIAN J EXP BIOL, MARCH 2012 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 ± ± ± ± ± ±7.87 Gr 2 Gr 3 Gr ±6.03* 24.54±2.36* 55.90±8.13* 62.26±11.80* 62.26±11.80* 62.72±12.07* ±1.05* 23.18±2.88* 28.45± ±1.05* 37.26±3.14* 45.45±5.77* ±10.23* 39.09±5.24* 45.45±7.87* 52.26±10.75* 57.27±12.07* 61.36±10.23* ±0.52* 39.08±2.62* 50.90±9.44* 55.45±6.82* 62.72±2.62* 71.36±1.83* ± ±1.05* 48.62±6.56* ± 0.26* 97.72±0.26* 100±0* ±3.14* 60.45±4.46* 94.08±2.07* 100±0* ±1.31* 39.09±2.62* 55.45±2.10* 60.90±2.62* 70.90±2.62* 79.54±0.13* ±3.14* 46.36±2* 77.72±0.26* 97.27±1.05* 100±0* ±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 (%) Length Size of testes (µm) Breadth 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]. 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 Gr ± ±6.25 Gr 2 Gr 3 Gr ±10.20* 250± ± ±42.08* ±38.86* 275± ±23.93* ± ±58.07* 275± ± ± ±44.92* ± ±15.72* ± ±21.34* ±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 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

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.

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

4 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 ].

(b): Higher resolution of the testicular surface at different site revealing the presence of interwoven meshwork of band-like

5 236 INDIAN J EXP BIOL, MARCH 2012 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.

Spermatogenesis and spermatozoa Similar to other insect species, the testes were formed by round follicles and they were six in number in T.

6 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 follicle 11. 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 ].

7 238 INDIAN J EXP BIOL, MARCH 2012 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. References 1 Andreev D, Rocheleau T, Phillips T W, Beeman R W & Constant F R H, A PCR diagnoistic for cyclodiene insecticide resistance in the red flour beetle, Tribolium castaneum, Pest Sci, 41 (1994) Beeman R W & Stuart J J, A gene for lindene and cyclodiene resistance in the red flour beetle (Coleoptera: Tenebrionidae), J Econ Entomol, 83 (1990) Beeman R W & Nanis S M, Malathion resistance alleles and their fitness in the red flour beetle (Coleoptera: Tenebrionidae), J Econ Entomol, 79 (1986) Ibrahim H R, Sugimoto Y & Aoki T, Ovotransferrin antimicrobial peptide (OTAP-92) kills the bacteria through membrane damage mechanism, Biochim Biophys Acta, 1523 (2000) Kizek R, Masarik M, Kramer K J, Potesil D, Bailey M, Howard J A, Klejdus B, Mikelova R, Adam V, Trnkova L & Jelen F, An analysis of avidin, biotin and their interaction at attomole levels by volummetric and chromatographic techniques, Anal bioanal Chem, 381 (2005) Cooper S G, Douches D S & Grafius E J, Insecticidal activity of avidin combined with genetically engineered and traditional host plant resistance against Colorado potato beetle (Coleoptera: Chrysomelidea) larvae, J Econ Entomol, 99 (2006) Masarik M, Kizek R, Kramer K J, Billova S, Brazdova M, Vacek J, Bailey M, Jelen F & Howard J A, Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize, Anal Chem, 75 (2003) Kramer K J, Morgan T D, Throne J E, Dowell F E, Bailey M & Howard J A, Transgenic avidin maize is resistant to storage insect pests, Nat Biotechnol, 18(2000) Bernardi G & Cook W H, An electrophoretic and ultracentrifugal study on the proteins of the high density fraction of egg yolk, Biochem Biophys Acta, 44 (1960) Anwar M, Chambers D L, Ohinata K & Kobayashi K, Radiation-sterilization of the Mediterrean fruit fly (Diptera: Tephritidae); comparison of spermatogenesis in flies treated as pupae or adults, Ann Entomol Soc Am, 64 (1971) Almeida M C & Landim C C, Spermiogenesis in Palembus dermestoides (Coleoptera: Tenebrionidae) with emphasis on the formation of mitrochondrial derivatives, Braz J Morphol Sci, 17 (2000) 75.

IN normal male fowls, four developmental stages of spermatogenetic activity

IN normal male fowls, four developmental stages of spermatogenetic activity Development of the Testis Tubule in the Fowl By GAMAL A. R. KAMAR (From the Animal Production Department, Faculty of Agriculture, Cairo University, Giza, Egypt) With three plates (figs. 1-3) SUMMARY Three