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Free amino acids in thoraces of flown honey bees, Apis mellifera L. (Hymenoptera: Apidae)
Comp. Biochem. Physiol., 1972, VoL 43B, pp. 163 to 169. Pergamon Press. Printed in Great Britain
FREE AMINO ACIDS IN THORACES OF FLOWN HONEY BEES, APIS MELLIFERA L. (HYMENOPTERA: APIDAE)* ROY J. BARKER and YOLANDA L E H N E R Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, Tucson, Arizona 85719t
(Received 14 ~anuary 1972) Abstract--1. The free amino acids were compared in the thoraces of flown and unflown honey bees, Apis mellifera L. Both workers and drones were tested. 2. The amino acids showed little change with flight detectable by thinlayer chromatography on silica gel. By spectrophotometric analysis, a slight depletion in proline content with flight was observed. 3. Evidently, proline is a minor source of flight energy in bees fed sucrose. INTRODUCTION THE ~m~TIONSmP between dance tempo of returned forager bees and either the distance or the duration of a flight is not a direct relationship (von Frisch, 1967). Bees compensate for load, wind and other stresses. Consequently, von Frisch calculated and experimentally demonstrated that honey bees, Apis rnellifera L., can estimate distance by the energy they expend. The relationship between energy expended and dance tempo is nonlinear and polyfactorial. This association suggests to us that there is more than one source of flight energy. Glucose is utilized during flight by honey bees (Sotavalta, 1954). However, a role for proline as an additional energy reserve may be widespread in insects (Gilmour, 1965). Proline is prominently metabolized in the flight of blow flies (Sacktor & Childress, 1967), tsetse flies (Bursell, 1963, 1966) and locusts (Kirsten et al., 1963; Brosemer & Veerabhadrappa, 1965; Mayer & Candy, 1969). Free proline is also conspicuously abundant in pollen (Auclair & Jamieson, 1948), bee larval food (Pratt & House, 1949; Rembold, 1965; Barker et al., 1971) and bee hemolymph (Pratt, 1950; Fujii et al., 1962; Florkin & Jeuniaux, 1965; Lue & Dixon, 1967; Gilliam & McCaughey, 1971). The present experiment explores whether proline or other specific amino acids are depleted during flight in honey bees. MATERIALS AND METHODS
A direct means of determining materials that supply metabolic fuel for flight is to measure those materials which diminish during prolonged flight. Two sets of experiments * Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the U.S.D.A. t Co-operating with the University of Arizona Agricultural Experiment Station. 163
164
RoY J. BARKERAND YOLANDA LEHNER
were performed to measure amino acid depletion. In the first set, mature foraging workers were caged 24 hr in the laboratory and fed a sucrose solution and water a d lib. Individual bees were then attached by their thoraces to the head of an insect pin with melted paraffin. Sixteen bees were made to fly by twirling the pins between our fingers until the bees would no longer respond. T h e resulting flight intervals ranged from 6 to 128 min. Fifteen other bees were killed and analyzed immediately after being attached to a pin. Since the caged bees did not fly while caged, these bees were considered unflown. In the second series of experiments, drones were used instead of worker bees. Four categories of energy expenditure were selected: Rested = caged upon return from a flight and held in the hive overnight, Leaving = collected as they left the hive on a flight, not necessarily the first flight of the day, Returning = captured at the hive entrance upon return from a flight, Exhausted = returning drones screened from the hive and collected from the ground below the hive entrance when they would no longer fly. As the bees were flown, hemolymph became more difficult to obtain, and its total volume in bees is not satisfactorily definable. Once this problem was recognized, whole thoraces were used for analysis instead of a customary hemolymph aliquot. T h e thorax of bees is predominantly muscle. Each bee thorax, with appendages and gut removed, was homogenized in 2 ml of water and centrifuged at 1700g for 5 min. T h e supernatant solution was then chromatographed on a 5-ram dia. column containing 0"35 g of moist Dowex50W-X4 ® (100-200 mesh; ionic form H +) by the method of Harris et al. (1961). Six bee thoraces did not overload this column. Free amino acids eluted from the column were dried in a vacuum over sulfuric acid and redissolved in 0"10 ml of distilled water. A 5-/~1 aliquot of each sample was spotted on a 20 x 20-cm chromatography plate coated with 0"3 m m of Silica Gel-G. The plates were developed in two dimensions according to Pataki (1969) in chloroform-methanol-17% ammonia (2 : 2 : 1) followed by phenol-water (3 : 1) in the second direction. Plates were dried overnight at room temperature and then sprayed with 0-3 % ninhydrin in butanol with 3% acetic acid. T h e plates were warmed on a hot plate immediately after spraying, and the major amino acid spots were visualized and marked. T h e plates were then heated at 110°C for 10 min. Ninhydrin spots change within an hour, so the plates were photographed in color as soon as they cooled to about 70°C. T h e thin-layer chromatography (TLC) plates gave us an indication of which particular free amino acids were changing in relation to flight. Proline was measured selectively and quantitatively; 20-/zl aliquots of the samples used for T L C were analyzed in duplicate by the spectrophotometric method of Bergman & Loxley (1970), with volumes scaled down to 0"1 and the pigment extracted in 2 ml of benzene. RESULTS W h e n w e flew b e e s to a p p a r e n t e x h a u s t i o n on t h e p i v o t e d w i r e a p p a r a t u s o f S o t a v a l t a (1954), t h e b e e s r e s u m e d flight w h e n t h e y w e r e r e m o v e d f r o m t h e w i r e . C o n t i n u e d flight for m o r e t h a n 2 h r has b e e n o b t a i n e d b y o u r s i m p l e r t e c h n i q u e . W h e n p h o t o g r a p h s o f t h e c h r o m a t o g r a p h e d a m i n o acids f r o m e i g h t flown a n d e i g h t u n f l o w n b e e s w e r e c o m p a r e d , n o n e o f t h e c h r o m a t o g r a m s f r o m flown b e e s h a d definite v i s u a l i n d i c a t i o n s of p r o l i n e d e p l e t i o n ( F i g . 1). T h e c o n c e n t r a t i o n o f a s p a r t i c a c i d a n d o f g l u t a m i c acid s e e m e d to d e c r e a s e w i t h flight, as w o u l d b e e x p e c t e d f r o m a role for t h e s e a c i d s in t h e t r i c a r b o x y l i c a c i d cycle. T h e c o n c e n t r a t i o n o f fl-alanine a p p a r e n t l y i n c r e a s e d . T h e s e differences
UNFLOWN
----
Proline
---
Hydroxyprotine
~II~----
Glutomic
a c i d -- -- - j
Aspar~c
acid . . . .
,
~--alanine
;
~'~
FLOWN
:O
FIG. 1. C o m p a r i s o n of thin-layer c h r o m a t o g r a m s of the free a m i n o acids f r o m t h e thoraces of a typical unflown worker h o n e y bee a n d a bee flown 27 min. C h r o m a t o g r a p h y plates were developed first f r o m r i g h t to left, t h e n f r o m b o t t o m to top. F o r recording, t h e y were i l l u m i n a t e d w i t h a H o n e y w e l l P r o x - O - L i t e 7 ® electronic flash at 45 c m a n d p h o t o g r a p h e d at f6 with E k t a c h r o m e ® X. Black a n d white negatives for these p h o t o g r a p h s were m a d e on K o d a k ® E l e c t r o n microscope film a n d p r i n t e d o n No. 4 Ilfoprint® p a p e r at f 2 2 for 25 sec.
O phe pro
{,~) hypro his thr
0
cys
asp
FI(:.
1--(cont,)
Amino acid standards
~I
FREE AMINO ACIDS IN THORACES OF FLOWN HONEY BEES
165
were detected on the TLC plates by three independent observers but have not been verified with quantitative analysis. The specific proline measurements by the method of Bergman & Loxley (1970) did indicate proline depletion during flight of both workers and drones. The proline concentrations in flown and unflown worker bees are listed in Table 1. Analysis of variance indicated a reduction of proline in flown bees as compared with unflown bees. However, the magnitude of the difference is not TABLE
l-THE
PROLINE CONTENT OF THORACES OF WORKER HONEY BEES
f'g Proline/mg wet wt. Unflown 1·66 2·44 1·36 2·32 1·77 1·86
1·45 1'64 1·54 1-18 1·34 1·80 1·76 1·60 1·71
Flown 22 July 1970 1-60 1-19 1·30 1·41 12 November 1970 1·19 1·04 1·75 1·20 18 November 1970 1·74 1'46 1·33 1·04 30 November 1970 1·25 2·05 1-19
Time flown (min) 128 24 83 51 10 9 9 82 11 46 6 32
28 13 45
Means 1·39
very satisfying in view of the variability present in unflown bees. The difference between flown and unflown worker bees is significant at the 8·5 per cent level (F = 6·44). Regression analysis of relationship between time flown by individual bees and content of proline gave a coefficient of - 0·40 (P < 0·05). The likelihood of a difference in flown vs. unflown workers combined with the significant negative correlation of flight time vs. content of proline supports a statement that the proline content of the thorax of worker bees does decrease during flight. The linear regression of proline content in /Lg/mg wet weight (Y) on time in minutes (X) is Y = 1·642-0·0059X. The intercept (1·642) is a little below the mean (1· 71) for unflown worker bees. Logarithmic transformation gave a lower correlation coefficient which indicated a poorer fit.
166
Roy
J.
BARKER AND YOLANDA LEHNER
The results of experiments in which treatments of drones were compared are shown in Table 2. There were four treatments with three cells missing and either five or six drones in each sample. TABLE 2-THE PROLINE CONTENT OF THORACES OF DRONE HONEY BEES
(f.Lg
PROLINE/mg
wet wt.)
Condition of drones Rested
2·01 2·08 1-18 1·98 1·94 1·59
Leaving
Returning
8 March 1971 1·38 1·33 1·54 1·17 1·44 1·08 18 March 1971 2·66 2·12 1·66 1·27 1·39 0·86 1·10 1·83 1·48 1·14 1'49 7 April 1971 2·50 3·37 2·89 2·73 2·63 2·45 3·07 2·97 1·97 1·67 2·16
Exhausted
0·86 1·53 1·20 1·20 1·53 1·44 1·12 0·57 0·65 0·97 1·89 1'04 2·19 2·72 2·40 2·80 2'06 2·30
Because of variability between days, a two-way classification with unequal numbers and no interaction (Kempthorne, 1952) was used for analysis of the data. This analysis of variance gave an F-value for treatment difference of 4·1 (P < 0·05). The adjusted means for {tg proline/mg thorax are as follows (x± S.E.): Rested = 2·207 ± 0'189, Leaving = 1·799 ± 0'134, Returning = 1·654 ± 0'098, Exhausted = 1·464 ± 0·097.
Paired comparisons gave the following results: Pair Leaving-returning Leaving-exhausted Leaving-rested Returning-exhausted Returning-rested Exhausted-rested
Difference
t test
= 0·1450 ± 0·1691 0·857 2·015 = 0·3351 ± 1·663 = 0·4081 ± 0·2974 -1,372 = 0·1902 ± 0·1389 1·369 -2,189 = -0·5530 ± 0·2526 -2,940 = - 0·7432 ± 0·2528 * to' 06; 46 d.f. = 2·013
Significance N.s.
*
N.s. N.s.
*
*
FREE AMINO ACIDS IN THORACES OF FLOWN HONEY BEES
167
The results indicate that proline is used during flight. Our judgement on whether bees were going on a flight or returning from one was poor. The data from rested or exhausted drones were convincing evidence of a decline in proline with increased exertion. DISCUSSION
It is reasonable to assume that the proline decrease during flight represents utilization of proline to supply energy. If the linear regression for proline content with flight time of worker bees is used as an approximation of proline consumption, flying worker bees consume 0·354 ILg of proline/mg of thorax per hr. The mean weight of thoraces of worker bees flown in this experiment was 27 mg. Each bee thorax then used about 10 ILg of proline/hr. Sotavalta (1954) found that honey bees consumed 7·9 mg of sugar/hr. These estimates suggest that about 0·1 per cent of the flight energy of worker honey bees is supplied by proline. Even though proline predominates in the free amino acids of bees and in their foods, the utilization pathway of this amino acid by bees is still speculative. Proline is synthesized from glucose (Lue & Dixon, 1967) and is therefore nonessential. Through a reversible transformation to glutamate and a-ketoglutarate, proline can enter the tricarboxylic acid cycle. It can also be formed from arginine. The addition of proline to bee rations failed to make an artificial diet the nutritive equivalent of pollen (Barker, 1971). Honey bees have low titers of proline dehydrogenase (Crabtree & Newsholme, 1970). This should limit proline oxidation for the provision of energy in muscle. Now we can add that in bees proline does supply energy but as a minor source. These findings support the contention of Sacktor (1965) and Weiss-Fogh (1967) that the availability of carbohydrate influences the portion of the metabolic fuel that is supplied by amino acids. Bees evidently have sufficient carbohydrate available from nectar and honey to fill most of their flight energy requirements. In the customary separation techniques, ,a-alanine contaminates a-amino acids. Although ,a-alanine responds to similar reagents, its metabolic role differs; it terminates peptide chains and does not form proteins. ,a-alanine can arise from metabolism of pyrimidines by several possible pathways. If ,a-alanine is considered as a fragment of the pantothenic acid moiety of coenzyme A or as a degradent of uracil, then its increase with flight is not surprising. When von Frisch (1967) correlated distance flown vs. tempo of dancing, he obtained a nonlinear relationship. He discussed the correction factors which had been used to obtain a linear relationship. Heran (von Frisch, 1967) introduced two constants, but neither had a physiological basis. Von Frisch accepted one factor which he termed a constant of forgetfulness. But he also wrote, "When bees flying from the hive to the feeding station meet with a headwind, then its hindrance is balanced by a tailwind on the return." In terms of energy expended, this balance needs a correction. More time is spent in the headwind than in the tailwind. Perhaps the tempo of dancing is related to sugar utilization, and the two constants of Reran represent windage correction and proline metabolism to energy. At
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Roy J. BARKER AND YOLANDA LEHNER
least we have new insight for a physiological basis for a nonlinear polyfactorial relationship between dance tempo and flight range. Acknowledgements-John J. Corrigan of Indiana State University was a wellspring of information during our preliminary trials. John R. Wharrie produced the photographs from color slides; John Edwards suggested the use of drones; Judson U. McGuire, Jr., analyzed the data; and George D. Butler, Jr., helped calculate correlations. All four are members of the Agricultural Research Service of the U.S.D.A., and their support is deeply appreciated. REFERENCES AUCLAIR J. L. & JAMIESON C. H. (1948) A qualitative analysis of amino acids in pollen collected by bees. Science 108, 357-358. BARKER R. J. (1972) Whether the superiority of pollen in diet of honey bees is attributable to its high content of free proline. Ann. ent. Soc. Am. 65, 270 BARKER R. J., ICE K. K., SPRATT G. & MCCAUGlmY w. F. (1972) Unusually high proline in royal jelly of honey bees. Ann. ent. Soc. Am. (Submitted for publication.) BERGMAN I. & LoXLEY R. (1970) New spectrophotometric method for the determination of proline in tissue hydrolysates. Analyt. Chem. 42, 702-706. BROSEMER R. W. & VEERABHADRAPPA P. S. (1965) Pathway of proline oxidation in insect flight muscle. Biochim. biophys. Acta 110, 102-112. BURSELL E. (1963) Aspects of the metabolism of amino acids in the tsetse fly, Glossina (Diptera). J. Insect Physiol. 9, 439-452. BURSELL E. (1966) Aspects of the flight metabolism of tsetse flies (Glossina). Compo Biochem. Physiol. 19, 809-818. CRABTREE B. & NEWSHOLME E. A. (1970) The activities of proline dehydrogenase, glutamic dehydrogenase, aspartate-oxoglutarate aminotransferase and alanine-oxoglutarate aminotransferase in some insect flight muscles. Biochem.J. 117, 1019-1021. FLORKIN M. & JEUNIAUX C. (1965) Hemolymph: composition. In The Physiology of Insecta (Edited by ROCKSTEIN M.), Vol. 3, pp. 110-152. Academic Press, New York. VON FRISCH K. (1967) The Dance Language and Orientation of Bees, pp. 114-129. Harvard University Press, Cambridge. FuJll N., KAWANO I., KUWAHARA H. & IWAMURA I. (1962) Studies on the free amino acids in honey bees at different stages of development-I. Observations on the free acids present in adults and nymphs of drones. Bull. Miyazaki Univ. Fac. Agr. 7, 1-4. GILLIAM M. & MCCAUGHEY W. F. (1972) Total amino acids in developing worker honey bees (Apis mellifera L.). Experientia. (In press.) GILMOUR D. (1965) The Metabolism of Insects, pp. 16-17. Oliver & Boyd, Edinburgh. HARRIS C. K., TlGANE E. & HANES C. S. (1961) Quantitative chromatographic methods-7. Isolation of amino acids from serum and other fluids. Can. J. Biochem. Physiol. 39, 439-451. KEMPTHORNE O. (1952) Design and Analysis of Experiments, pp. 79-87. Wiley, New York. KIRSTEN E., KIRSTEN R. & ORESE P. (1963) Das Verhalten von freien Aminosiiuren, energiereichen Phosphorsaure-Verbindungen und einigen Glykolyse- und Tricarbonsiiure-cyclus-Substaten in Muskeln von Locusta migratoria bei der Arbeit. Biochem. Z. 337, 167-178. LUE P. F. & DIXON S. E. (1967) Studies in the mode of action of royal jelly in honeybee development-VII. The free amino acids in the haemolymph of developing larvae. Can.J. Zool. 45, 205-214. MAYER R. J. & CANDY D. J. (1969) Changes in energy reserves during flight of the desert locust, Schistocerca gregaria. Compo Biochem. Physiol. 31, 409-418.
FREE AMINO ACIDS IN THORACES OF FLOWN HONEY BEES
169
PATAK I G. (1969) Techniques of Thin-layer Chromatography in Amino Acid and Peptide Chemistry, 2nd English edn (revised), p. 90. Humphrey Science, Ann Arbor, Michigan. PRATT J. J., JR. (1950) A qualitative analysis of the free amino acids in insect blood. Ann. ent. Soc. Am. 43, 573-580. PRATT J. J., JR. & HOUSE H. L. (1949) A qualitative analysis of the amino acids in royal jelly. Science 110, 9-10. REMBOLD H. (1965) Biologically active substances in royal jelly. Vitams Horm. 23, 359-382. SACKTOR B. (1965) Metabolism of amino acids in muscle. In The Physiology of Insecta (Edited by ROCKSTEIN M.), Vol. 2, pp. 525-528. Academic Press, New York. SACKTOR B. & CHILDRESS C. C. (1967) Metabolism of proline in insect flight muscle and its significance in stimulating the oxidation of pyruvate. Archs Biochem. Biophys. 120, 583-588. SOTAVALTA O. (1954) On the fuel consumption of the honeybee (Apis mellifica) in flight experiments. Ann. Zool. Soc. Zool.-botan. Fenn. Vanamo 16, 1-27. WEIS-FoGH T. (1967) Metabolism and weight economy in migrating animals, particularly birds and insects. In Insects and Physiology (Edited by BEAMENT J. W. L. & TREHERNE J. E.), pp. 143-159. Academic Press, New York.
Key Word Index-Apis mellifera L.; proline; flight; muscle; energy; ammo acid; ,a-alanine; drones; honey bee.
FREE AMINO ACIDS IN THORACES OF FLOWN HONEY BEES, APIS MELLIFERA L. (HYMENOPTERA: APIDAE)* ROY J. BARKER and YOLANDA L E H N E R Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, Tucson, Arizona 85719t
(Received 14 ~anuary 1972) Abstract--1. The free amino acids were compared in the thoraces of flown and unflown honey bees, Apis mellifera L. Both workers and drones were tested. 2. The amino acids showed little change with flight detectable by thinlayer chromatography on silica gel. By spectrophotometric analysis, a slight depletion in proline content with flight was observed. 3. Evidently, proline is a minor source of flight energy in bees fed sucrose. INTRODUCTION THE ~m~TIONSmP between dance tempo of returned forager bees and either the distance or the duration of a flight is not a direct relationship (von Frisch, 1967). Bees compensate for load, wind and other stresses. Consequently, von Frisch calculated and experimentally demonstrated that honey bees, Apis rnellifera L., can estimate distance by the energy they expend. The relationship between energy expended and dance tempo is nonlinear and polyfactorial. This association suggests to us that there is more than one source of flight energy. Glucose is utilized during flight by honey bees (Sotavalta, 1954). However, a role for proline as an additional energy reserve may be widespread in insects (Gilmour, 1965). Proline is prominently metabolized in the flight of blow flies (Sacktor & Childress, 1967), tsetse flies (Bursell, 1963, 1966) and locusts (Kirsten et al., 1963; Brosemer & Veerabhadrappa, 1965; Mayer & Candy, 1969). Free proline is also conspicuously abundant in pollen (Auclair & Jamieson, 1948), bee larval food (Pratt & House, 1949; Rembold, 1965; Barker et al., 1971) and bee hemolymph (Pratt, 1950; Fujii et al., 1962; Florkin & Jeuniaux, 1965; Lue & Dixon, 1967; Gilliam & McCaughey, 1971). The present experiment explores whether proline or other specific amino acids are depleted during flight in honey bees. MATERIALS AND METHODS
A direct means of determining materials that supply metabolic fuel for flight is to measure those materials which diminish during prolonged flight. Two sets of experiments * Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the U.S.D.A. t Co-operating with the University of Arizona Agricultural Experiment Station. 163
164
RoY J. BARKERAND YOLANDA LEHNER
were performed to measure amino acid depletion. In the first set, mature foraging workers were caged 24 hr in the laboratory and fed a sucrose solution and water a d lib. Individual bees were then attached by their thoraces to the head of an insect pin with melted paraffin. Sixteen bees were made to fly by twirling the pins between our fingers until the bees would no longer respond. T h e resulting flight intervals ranged from 6 to 128 min. Fifteen other bees were killed and analyzed immediately after being attached to a pin. Since the caged bees did not fly while caged, these bees were considered unflown. In the second series of experiments, drones were used instead of worker bees. Four categories of energy expenditure were selected: Rested = caged upon return from a flight and held in the hive overnight, Leaving = collected as they left the hive on a flight, not necessarily the first flight of the day, Returning = captured at the hive entrance upon return from a flight, Exhausted = returning drones screened from the hive and collected from the ground below the hive entrance when they would no longer fly. As the bees were flown, hemolymph became more difficult to obtain, and its total volume in bees is not satisfactorily definable. Once this problem was recognized, whole thoraces were used for analysis instead of a customary hemolymph aliquot. T h e thorax of bees is predominantly muscle. Each bee thorax, with appendages and gut removed, was homogenized in 2 ml of water and centrifuged at 1700g for 5 min. T h e supernatant solution was then chromatographed on a 5-ram dia. column containing 0"35 g of moist Dowex50W-X4 ® (100-200 mesh; ionic form H +) by the method of Harris et al. (1961). Six bee thoraces did not overload this column. Free amino acids eluted from the column were dried in a vacuum over sulfuric acid and redissolved in 0"10 ml of distilled water. A 5-/~1 aliquot of each sample was spotted on a 20 x 20-cm chromatography plate coated with 0"3 m m of Silica Gel-G. The plates were developed in two dimensions according to Pataki (1969) in chloroform-methanol-17% ammonia (2 : 2 : 1) followed by phenol-water (3 : 1) in the second direction. Plates were dried overnight at room temperature and then sprayed with 0-3 % ninhydrin in butanol with 3% acetic acid. T h e plates were warmed on a hot plate immediately after spraying, and the major amino acid spots were visualized and marked. T h e plates were then heated at 110°C for 10 min. Ninhydrin spots change within an hour, so the plates were photographed in color as soon as they cooled to about 70°C. T h e thin-layer chromatography (TLC) plates gave us an indication of which particular free amino acids were changing in relation to flight. Proline was measured selectively and quantitatively; 20-/zl aliquots of the samples used for T L C were analyzed in duplicate by the spectrophotometric method of Bergman & Loxley (1970), with volumes scaled down to 0"1 and the pigment extracted in 2 ml of benzene. RESULTS W h e n w e flew b e e s to a p p a r e n t e x h a u s t i o n on t h e p i v o t e d w i r e a p p a r a t u s o f S o t a v a l t a (1954), t h e b e e s r e s u m e d flight w h e n t h e y w e r e r e m o v e d f r o m t h e w i r e . C o n t i n u e d flight for m o r e t h a n 2 h r has b e e n o b t a i n e d b y o u r s i m p l e r t e c h n i q u e . W h e n p h o t o g r a p h s o f t h e c h r o m a t o g r a p h e d a m i n o acids f r o m e i g h t flown a n d e i g h t u n f l o w n b e e s w e r e c o m p a r e d , n o n e o f t h e c h r o m a t o g r a m s f r o m flown b e e s h a d definite v i s u a l i n d i c a t i o n s of p r o l i n e d e p l e t i o n ( F i g . 1). T h e c o n c e n t r a t i o n o f a s p a r t i c a c i d a n d o f g l u t a m i c acid s e e m e d to d e c r e a s e w i t h flight, as w o u l d b e e x p e c t e d f r o m a role for t h e s e a c i d s in t h e t r i c a r b o x y l i c a c i d cycle. T h e c o n c e n t r a t i o n o f fl-alanine a p p a r e n t l y i n c r e a s e d . T h e s e differences
UNFLOWN
----
Proline
---
Hydroxyprotine
~II~----
Glutomic
a c i d -- -- - j
Aspar~c
acid . . . .
,
~--alanine
;
~'~
FLOWN
:O
FIG. 1. C o m p a r i s o n of thin-layer c h r o m a t o g r a m s of the free a m i n o acids f r o m t h e thoraces of a typical unflown worker h o n e y bee a n d a bee flown 27 min. C h r o m a t o g r a p h y plates were developed first f r o m r i g h t to left, t h e n f r o m b o t t o m to top. F o r recording, t h e y were i l l u m i n a t e d w i t h a H o n e y w e l l P r o x - O - L i t e 7 ® electronic flash at 45 c m a n d p h o t o g r a p h e d at f6 with E k t a c h r o m e ® X. Black a n d white negatives for these p h o t o g r a p h s were m a d e on K o d a k ® E l e c t r o n microscope film a n d p r i n t e d o n No. 4 Ilfoprint® p a p e r at f 2 2 for 25 sec.
O phe pro
{,~) hypro his thr
0
cys
asp
FI(:.
1--(cont,)
Amino acid standards
~I
FREE AMINO ACIDS IN THORACES OF FLOWN HONEY BEES
165
were detected on the TLC plates by three independent observers but have not been verified with quantitative analysis. The specific proline measurements by the method of Bergman & Loxley (1970) did indicate proline depletion during flight of both workers and drones. The proline concentrations in flown and unflown worker bees are listed in Table 1. Analysis of variance indicated a reduction of proline in flown bees as compared with unflown bees. However, the magnitude of the difference is not TABLE
l-THE
PROLINE CONTENT OF THORACES OF WORKER HONEY BEES
f'g Proline/mg wet wt. Unflown 1·66 2·44 1·36 2·32 1·77 1·86
1·45 1'64 1·54 1-18 1·34 1·80 1·76 1·60 1·71
Flown 22 July 1970 1-60 1-19 1·30 1·41 12 November 1970 1·19 1·04 1·75 1·20 18 November 1970 1·74 1'46 1·33 1·04 30 November 1970 1·25 2·05 1-19
Time flown (min) 128 24 83 51 10 9 9 82 11 46 6 32
28 13 45
Means 1·39
very satisfying in view of the variability present in unflown bees. The difference between flown and unflown worker bees is significant at the 8·5 per cent level (F = 6·44). Regression analysis of relationship between time flown by individual bees and content of proline gave a coefficient of - 0·40 (P < 0·05). The likelihood of a difference in flown vs. unflown workers combined with the significant negative correlation of flight time vs. content of proline supports a statement that the proline content of the thorax of worker bees does decrease during flight. The linear regression of proline content in /Lg/mg wet weight (Y) on time in minutes (X) is Y = 1·642-0·0059X. The intercept (1·642) is a little below the mean (1· 71) for unflown worker bees. Logarithmic transformation gave a lower correlation coefficient which indicated a poorer fit.
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J.
BARKER AND YOLANDA LEHNER
The results of experiments in which treatments of drones were compared are shown in Table 2. There were four treatments with three cells missing and either five or six drones in each sample. TABLE 2-THE PROLINE CONTENT OF THORACES OF DRONE HONEY BEES
(f.Lg
PROLINE/mg
wet wt.)
Condition of drones Rested
2·01 2·08 1-18 1·98 1·94 1·59
Leaving
Returning
8 March 1971 1·38 1·33 1·54 1·17 1·44 1·08 18 March 1971 2·66 2·12 1·66 1·27 1·39 0·86 1·10 1·83 1·48 1·14 1'49 7 April 1971 2·50 3·37 2·89 2·73 2·63 2·45 3·07 2·97 1·97 1·67 2·16
Exhausted
0·86 1·53 1·20 1·20 1·53 1·44 1·12 0·57 0·65 0·97 1·89 1'04 2·19 2·72 2·40 2·80 2'06 2·30
Because of variability between days, a two-way classification with unequal numbers and no interaction (Kempthorne, 1952) was used for analysis of the data. This analysis of variance gave an F-value for treatment difference of 4·1 (P < 0·05). The adjusted means for {tg proline/mg thorax are as follows (x± S.E.): Rested = 2·207 ± 0'189, Leaving = 1·799 ± 0'134, Returning = 1·654 ± 0'098, Exhausted = 1·464 ± 0·097.
Paired comparisons gave the following results: Pair Leaving-returning Leaving-exhausted Leaving-rested Returning-exhausted Returning-rested Exhausted-rested
Difference
t test
= 0·1450 ± 0·1691 0·857 2·015 = 0·3351 ± 1·663 = 0·4081 ± 0·2974 -1,372 = 0·1902 ± 0·1389 1·369 -2,189 = -0·5530 ± 0·2526 -2,940 = - 0·7432 ± 0·2528 * to' 06; 46 d.f. = 2·013
Significance N.s.
*
N.s. N.s.
*
*
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167
The results indicate that proline is used during flight. Our judgement on whether bees were going on a flight or returning from one was poor. The data from rested or exhausted drones were convincing evidence of a decline in proline with increased exertion. DISCUSSION
It is reasonable to assume that the proline decrease during flight represents utilization of proline to supply energy. If the linear regression for proline content with flight time of worker bees is used as an approximation of proline consumption, flying worker bees consume 0·354 ILg of proline/mg of thorax per hr. The mean weight of thoraces of worker bees flown in this experiment was 27 mg. Each bee thorax then used about 10 ILg of proline/hr. Sotavalta (1954) found that honey bees consumed 7·9 mg of sugar/hr. These estimates suggest that about 0·1 per cent of the flight energy of worker honey bees is supplied by proline. Even though proline predominates in the free amino acids of bees and in their foods, the utilization pathway of this amino acid by bees is still speculative. Proline is synthesized from glucose (Lue & Dixon, 1967) and is therefore nonessential. Through a reversible transformation to glutamate and a-ketoglutarate, proline can enter the tricarboxylic acid cycle. It can also be formed from arginine. The addition of proline to bee rations failed to make an artificial diet the nutritive equivalent of pollen (Barker, 1971). Honey bees have low titers of proline dehydrogenase (Crabtree & Newsholme, 1970). This should limit proline oxidation for the provision of energy in muscle. Now we can add that in bees proline does supply energy but as a minor source. These findings support the contention of Sacktor (1965) and Weiss-Fogh (1967) that the availability of carbohydrate influences the portion of the metabolic fuel that is supplied by amino acids. Bees evidently have sufficient carbohydrate available from nectar and honey to fill most of their flight energy requirements. In the customary separation techniques, ,a-alanine contaminates a-amino acids. Although ,a-alanine responds to similar reagents, its metabolic role differs; it terminates peptide chains and does not form proteins. ,a-alanine can arise from metabolism of pyrimidines by several possible pathways. If ,a-alanine is considered as a fragment of the pantothenic acid moiety of coenzyme A or as a degradent of uracil, then its increase with flight is not surprising. When von Frisch (1967) correlated distance flown vs. tempo of dancing, he obtained a nonlinear relationship. He discussed the correction factors which had been used to obtain a linear relationship. Heran (von Frisch, 1967) introduced two constants, but neither had a physiological basis. Von Frisch accepted one factor which he termed a constant of forgetfulness. But he also wrote, "When bees flying from the hive to the feeding station meet with a headwind, then its hindrance is balanced by a tailwind on the return." In terms of energy expended, this balance needs a correction. More time is spent in the headwind than in the tailwind. Perhaps the tempo of dancing is related to sugar utilization, and the two constants of Reran represent windage correction and proline metabolism to energy. At
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least we have new insight for a physiological basis for a nonlinear polyfactorial relationship between dance tempo and flight range. Acknowledgements-John J. Corrigan of Indiana State University was a wellspring of information during our preliminary trials. John R. Wharrie produced the photographs from color slides; John Edwards suggested the use of drones; Judson U. McGuire, Jr., analyzed the data; and George D. Butler, Jr., helped calculate correlations. All four are members of the Agricultural Research Service of the U.S.D.A., and their support is deeply appreciated. REFERENCES AUCLAIR J. L. & JAMIESON C. H. (1948) A qualitative analysis of amino acids in pollen collected by bees. Science 108, 357-358. BARKER R. J. (1972) Whether the superiority of pollen in diet of honey bees is attributable to its high content of free proline. Ann. ent. Soc. Am. 65, 270 BARKER R. J., ICE K. K., SPRATT G. & MCCAUGlmY w. F. (1972) Unusually high proline in royal jelly of honey bees. Ann. ent. Soc. Am. (Submitted for publication.) BERGMAN I. & LoXLEY R. (1970) New spectrophotometric method for the determination of proline in tissue hydrolysates. Analyt. Chem. 42, 702-706. BROSEMER R. W. & VEERABHADRAPPA P. S. (1965) Pathway of proline oxidation in insect flight muscle. Biochim. biophys. Acta 110, 102-112. BURSELL E. (1963) Aspects of the metabolism of amino acids in the tsetse fly, Glossina (Diptera). J. Insect Physiol. 9, 439-452. BURSELL E. (1966) Aspects of the flight metabolism of tsetse flies (Glossina). Compo Biochem. Physiol. 19, 809-818. CRABTREE B. & NEWSHOLME E. A. (1970) The activities of proline dehydrogenase, glutamic dehydrogenase, aspartate-oxoglutarate aminotransferase and alanine-oxoglutarate aminotransferase in some insect flight muscles. Biochem.J. 117, 1019-1021. FLORKIN M. & JEUNIAUX C. (1965) Hemolymph: composition. In The Physiology of Insecta (Edited by ROCKSTEIN M.), Vol. 3, pp. 110-152. Academic Press, New York. VON FRISCH K. (1967) The Dance Language and Orientation of Bees, pp. 114-129. Harvard University Press, Cambridge. FuJll N., KAWANO I., KUWAHARA H. & IWAMURA I. (1962) Studies on the free amino acids in honey bees at different stages of development-I. Observations on the free acids present in adults and nymphs of drones. Bull. Miyazaki Univ. Fac. Agr. 7, 1-4. GILLIAM M. & MCCAUGHEY W. F. (1972) Total amino acids in developing worker honey bees (Apis mellifera L.). Experientia. (In press.) GILMOUR D. (1965) The Metabolism of Insects, pp. 16-17. Oliver & Boyd, Edinburgh. HARRIS C. K., TlGANE E. & HANES C. S. (1961) Quantitative chromatographic methods-7. Isolation of amino acids from serum and other fluids. Can. J. Biochem. Physiol. 39, 439-451. KEMPTHORNE O. (1952) Design and Analysis of Experiments, pp. 79-87. Wiley, New York. KIRSTEN E., KIRSTEN R. & ORESE P. (1963) Das Verhalten von freien Aminosiiuren, energiereichen Phosphorsaure-Verbindungen und einigen Glykolyse- und Tricarbonsiiure-cyclus-Substaten in Muskeln von Locusta migratoria bei der Arbeit. Biochem. Z. 337, 167-178. LUE P. F. & DIXON S. E. (1967) Studies in the mode of action of royal jelly in honeybee development-VII. The free amino acids in the haemolymph of developing larvae. Can.J. Zool. 45, 205-214. MAYER R. J. & CANDY D. J. (1969) Changes in energy reserves during flight of the desert locust, Schistocerca gregaria. Compo Biochem. Physiol. 31, 409-418.
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PATAK I G. (1969) Techniques of Thin-layer Chromatography in Amino Acid and Peptide Chemistry, 2nd English edn (revised), p. 90. Humphrey Science, Ann Arbor, Michigan. PRATT J. J., JR. (1950) A qualitative analysis of the free amino acids in insect blood. Ann. ent. Soc. Am. 43, 573-580. PRATT J. J., JR. & HOUSE H. L. (1949) A qualitative analysis of the amino acids in royal jelly. Science 110, 9-10. REMBOLD H. (1965) Biologically active substances in royal jelly. Vitams Horm. 23, 359-382. SACKTOR B. (1965) Metabolism of amino acids in muscle. In The Physiology of Insecta (Edited by ROCKSTEIN M.), Vol. 2, pp. 525-528. Academic Press, New York. SACKTOR B. & CHILDRESS C. C. (1967) Metabolism of proline in insect flight muscle and its significance in stimulating the oxidation of pyruvate. Archs Biochem. Biophys. 120, 583-588. SOTAVALTA O. (1954) On the fuel consumption of the honeybee (Apis mellifica) in flight experiments. Ann. Zool. Soc. Zool.-botan. Fenn. Vanamo 16, 1-27. WEIS-FoGH T. (1967) Metabolism and weight economy in migrating animals, particularly birds and insects. In Insects and Physiology (Edited by BEAMENT J. W. L. & TREHERNE J. E.), pp. 143-159. Academic Press, New York.
Key Word Index-Apis mellifera L.; proline; flight; muscle; energy; ammo acid; ,a-alanine; drones; honey bee.