Studies on the incorporation of Carbon-14 in tissues of Galleria mellonella

Technical notes 9. AMONENKO V. M., KRUGLYKIt A. A., PAVLOV V. S. and TIKHINSKII G. F. Izv. Akad. Nauk SSSR. Metal. 1, 190 (1966). 10. KRUGLYKHA. A., PAVLOVV. S. and TIKHINSKII G. F. Zh. Neorgan. Khim. 10, 285 (1965).

International Journal of Applied Radiation and Isotopes, 1975, Vol. 26 pp. 567-569. Pergamon Press. Printed in Northern Ireland

567

The autoradiographs were prepared using stripping film (Kodak A R 10) according to technique of ROORRSIs). The grains were counted using an oil immersion objective and an eye piece graticule. The microscope was always focused on the level from which the maximum grain count was obtained. The grains under 5 preselected squares were counted from each focus.

Results Studies on the Incorporation of Carbon-14 i n T i s s u e s o f Galleria meUoneUa (Received 26 July 1974; and in revisedform 25 November 1974)

Introduction IN THE study of the effect of ~4C on insects, specially on oogenesis and egg development one of the problems encountered is to identify a suitable carrier of 14C that can be used as an internal source of betaradiation. From that point of view the study of general distribution of 14C in different tissues of insects needs special attention. Objective of this study is to introduce 14C through different carriers separately in Galleria mellonella in one stage of its development and to observe and compare the pattern of distribution of a4C in the key tissues of the insect.

The radioactivity observed in different tissues 15 days after feeding is shown in Fig. 1. Radioactivity in the cuticle and in the wing was very low, compared with that of other tissues. Overall, low radioactivity was observed in the glycine-14C group. The radioactivity in the fatbody and in the unlaid eggs of insects fed glycerol-14C was greater than for insects fed glucose-a4C or glycine-14C. In the laid eggs of the glycerol-14C group the activity was only slightly lower than for the unlaid eggs. However, radioactivity in the lald eggs of the glucose-14C and glycine-x4C groups was 47 and 54 per cent higher than in unlaid eggs; these differences were statistically significant (P values for glucose and glycine groups were <0.02 and <0.01 respectively). Radioactivity decreased in the laid eggs on successive days of oviposition (Fig. 2). The percentages of decrease in radioactivity from the first-day eggs to

Materials and Methods

12ooo~

Three different radioactive substances uniformly labelled with x4C vim, glucose-14C, glycine-14C and glycerol-14C were made into separate solutions of similar activity using distilled water. Each of the ten mature larvae of G. mellonella used in each experiment was anaesthetized with CO 2 and forcefed with a dose equivalent to 2 #Ci of radioactivity using an Agla syringe fitted with a No. 30 needle. After emergence of adult the insect was either killed or allowed to lay eggs. Key tissues were removed, washed, weighed and dissolved in NCS solution (Nuclear Chicago Corporation) for liquid scintillation counting. The method was essentially that of HANSEN and Bust~(x). The laid eggs had to be disintegrated with an ultrasonic drill after NCS treatment while wings and cuticle were digested with conc. H N O 3 according to the method of O'BRxENc2). The radioactivity of prepared samples was measured in a Packard Tri-Carb liquid scintillation counter, Results are expressed as counts/mg of wet tissue. The distribution of radioisotope in ovarioles and eggs treated with glucose-X4C was determined by autoradiography using a dose of 47 #Ci radioactivity of glucose-a4C per female. The sections used for autoradiography were prepared by wax embedding.

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Fatbody

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FIG. 1. Distribution of 3 radioactive compounds in adult tissues of G. mellonella 15 days after inoculation of mature larvae.

568

Technical notes

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Fro. 2. Radioactivity in laid eggs from 1 4 C treated females in relation to days of oviposition. the fifth-day eggs were 37-1, 41.2 and 41.2 in glucose, glycine, glycerol groups respectively. The decrease in each case was highly significant (P values for glucose, glycine and glycerol groups were <0.01, <0-02, <0.002 respectively). Variation in incorporation in glycerol-14C group was much higher (Glycerol 5049 ~ 2547: Glucose 5687 5_ 348: Glycine 2990 ± 502). T h e radioactivity in three different parts of an overiole is shown in Fig. 3. The differences in radioactivity between the " p r o x i m a l " eggs the "germ a r i u m " eggs were only 24 and 12 per cent in the glycineJaC and glycerol-XaC groups respectively. These differences were, however, not statistically significant. No data for the glucoseJaC group were taken. Autoradiography showed that the isotope was uniformly distributed in the central yolk but with higher concentration at the periphery and in the outer and inner layers of the chorion (Fig. 4). The mean number of grains per 6.8 x 10 a square micron was 31.7 ± 0.996 (standard error, sample of 40) at the centre of the yolk compared with 39-5 - 0.996 (standard error, sample of 40) at the periphery of the yolk; and the difference was statistically significant (P < 0-02). In the ovary the follicular layer appeared to have a slightly greater concentration of isotope than the nurse cells or the oocyte. Discussion

Results showing high content of glycerol in fatbody support the view of GILuS~aTM that storage of it is maintained in fatbodies of insects. The difference between the radioactivity in unlaid

eggs and laid eggs in the groups of glucose-laC and glycine-14C indicated that a considerable amount of radioactivity was incorporated during the period between ovulation and oviposition. It may be mentioned that in Lepidoptera a sticky secretion is added to the surface of the eggs during oviposition.
4[ o.

Proximal eggs Middle eggs

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]

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FIo. 3. Distribution of radioactivity in 3 different parts of ovariole. A schematic diagram of an ovariole with parts taken for radioactivity measurement shown also.

(a)

b

ch

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+e

% l:v<;. I+. l)istvil+tntioll

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:

+I+I

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"~++~]k. ;"+/j~

Technical notes radioactivity in glucose:4C group was slightly less (37-1 per cent) than that in glycine-14C group (41.2 per cent) during the similar period. SONENSHINE suggested that the decrease could be due to different rates of uptake of metabolites by the eggs in the oviduct compared with those in the ovary. It is more probable that this decrease in the activity in eggs of Galleria, may be due to a difference in the amount of labelled nutrient available for developing eggs as oogenesis and oviposition proceeded. Another explanation for the difference in radioactivity in eggs laid at different times is that it was due to different amounts of secretions added to the surface of the eggs prior to oviposition, as already mentioned. Autoradiographic study shows that glucose-a4C was distributed homogenously both in the mainbody of egg and in the chorion region. However, concentration of the isotope was higher in the latter.

Acknowledgements--I wish to thank Dr. F. D. MORGAN and Dr. GILLIAN RO~ERTSON of Waite Agriculture

569

Research Institute, South Australia for their constant interest in this research project. MOSLEH U . M A L L I K

Radiobiology Division Atomic Energy Centre P.O. Box 164, Ramna Dacca

Bangladesh References I. HANSEN D. L. and BOsH E. T. Analyt. Biochem. 18, 320 (1967). 2. O'BRIEN R. D. Analyt. Biochem. 7, 251 (1964). 3. ROOERS A. W. Techniques of Autoradiography. Elsevier, Amsterdam (1969). 4. GILBERT L. I. Adv. Insect. Physiol. 4, 117 (1967). 5. WIGOLESWORTIt V. B. Principles of Insect Physiology, p. 464. Methuen, London (1961). 6. SONENSHINE D. E. and CLARK G. M. J. med. Entomol. 5, 229 (1968).

International Journal of Applied Radiation and Isotopes, 1975, Vol. 26, p. 569. PergamoIl Press. Printed in Northern Ireland

BOOK R E V I E W D e t e r m i n a t i o n of S e q u e n c e s in RNA by G. G. B r o w n l e e . Laboratory Techniques in Biochemistry and Molecular Biology. General Editors T. S. WORK and E. WORK (North-Holland/ American Elsevier Publishing Companies, Amsterdam, London, New York) 1972. Tins IS a boratory Biology quately

very valuable monograph of the series LaTechniques in Biochemistry and Molecular (edited by T. S. and E. Work). It adecompiles the methodology* involved in

* Including the use of radioactive tracers--Editor.

determining the nucleotide sequences of ribonucleic acids. Many of the techniques reported here have been developed by the author in collaboration mith Dr. Sanger at Cambridge, England. T h e book is written clearly and gives sufficient details for a novice in the field. It would have been of interest if Dr. Brownlee had reviewed some of the methodology developed in the sequencing of deoxyribonucleic acids, where considerable progress is being made.

INDER VERMA