- Email: [email protected]
Oxidation of fat during flight of male Douglas-fir beetles, Dendroctonus pseudotsugae
J. Insect Physiot., 1971. Vol. 17, pp. 1555
to 1563. Pmgamon Press. Printed in Great Britain
OXIDATION OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR BEETLES, DlWllROCTOlVUS PSEUDOTSUGAE* S. N. THOMPSON
and R. B. BENNETT
Department of Biological Sciences, Simon Fraser University, Bumaby 2, B.C. (Received 9 November 1970) Abstract-During flight, male beetles lost weight primarily due to oxidation of fatty acids. RQ values of 0.7 and 0.8 were obtained for non-flying and flying insects respectively, indicating that Dendroctonus pseudotsugae is predominantly a fat utilizer, with some carbohydrate utilization during early flight periods. Selective oxidation of fatty acids was evident. The monounsaturates, C16:l and Cl8 :l were oxidized at the greatest rate, followed by the saturates, palmitic (C16:0), stearic (C18:0), and myristic (C14:O) acids, respectively. INTRODUCTION
THE UTILIZATIONof lipid as a major source of energy during flight was reported by FULTONand ROMNEY(1940) in Eu&&x ten&a and extensively documented by WEIS-FOGH (1952) for Schistocerca gregaria. WEIS-FOGH (1952) reports that lipid is an ideal substrate for flying, since hydration of glycogen would make isocaloric quantities eight times heavier than lipid, and result in an exceedingly overweight insect. MEYERet al. (1960) later demonstrated that 5’. gregariu flight muscle was able to oxidize 8 to 18 carbon chain fatty acids, and DOMROESEand GILBERT(1964) demonstrated oxidation of 14C-1-palmitate by flight muscles of Hyalophera cecropia. ATKINS (1966a, b), demonstrated a positive correlation between the fat content of Dendroctonus pseudotsugae and inclination to fly. Moreover, flown beetles had significantly less lipid than non-flown or control beetles (ATKINS, 1969). The purpose of our experiments was to further document the oxidation of fat during flight of D. pseudotsugae. Male beetles were used for convenience and also because males are presently being used in our laboratory for flight behavioural experiments, particularly in regard to their flight response in the presence of pheromone-laden female frass. MATERIALS
AND METHODS
Three separate groups of 30 freshly emerged male beetles were flown on autorecording flight mills for 1, 2, and 5 hr as previously described (BORDEN and BENNETT,1969). After flight the insects were frozen until the analyses were carried * Work supported by National Research Council of Canada grants to Drs. J. S. and J. H. BORDEN. 155.5
BARLOW
1556
S. N.
THOMPSON AND R. B. BENNETT
out. Three non-flown control groups were individually frozen before, during, and after the experimentation period. After initial wet and dry weight determinations, the insects were homogenized in a tissue grinder, the total lipid extracted (BLIGH and DYER, 1959), saponified (LEPPER, 1950), and esterified with diazomethane (SCHLENK and GELLERMAN, 1960). Fatty acid analysis was carried out on a Carlo-Erba gas-liquid chromatograph with a hot wire detector. Two metre glass columns (4 mm i.d.) were packed with 15% diethylene glycol succinate on Chromosorb W(AW), mesh 60/80. The carrier gas was helium. Methylated fatty acid standards containing myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (ClS:l), linoleic (ClS:Z), and linolenic (C18:3) acl‘ds were run with each sample for identification purposes. The unsaturated fatty acids used in the standard mixture, palmitoleic, oleic, linoleic, and linolenic acids, are specific isomers, each of which belongs to a group of isomers represented as C16:1, ClS:l, C18:2, and C18:3 respectively. However, since the chromatographic technique used only separates the mixture according to chain length and degree of saturation, any one of the isomers in a group may serve as a standard for the group. The unsaturated acids were not degraded to determine which isomers were present. In contrast to previous manometric techniques (CHADWICK,1947; WEIS-FOCH, 1967; WILLIAMSet al., 1969), we measured CO, expiration by flying male beetles with an infra-red gas analyser in an open system as described by HAMILTON(1964). Gilson manometric techniques (GILSON, 1963) were used to measure 0s consumption. The rates of change in concentration of these gases were then compared and respiratory quotients (RQ) calculated for non-flying and flying insects. Measurements of CO, concentration were made in a small Plexiglas flight chamber (5.08 x 5.08 x 7.62 cm outside diameter) with a rubber base, a diaphragm pump, and the IR gas analyser. The system was inter-connected with plastic tubing and produced a total volume of 231 cc and a flow rate of 410 cc/min. The analyser was calibrated by adjusting the gain control with a known concentration of prepared gas (250 ppm CO, in NJ so that a deflexion of 50 to 60 per cent on the recorder meter represented a change in concentration of 85 ppm. Because of the rather small size of the Douglas-fir beetle, a closed circulation method was used to give accumulated concentrations rather than an open circulation system as used by HAMILTON(1964). The test insects were suspended inside the Plexiglas chamber by a mount consisting of a short piece of wire partially heat sealed into a piece of glass, which in turn was glued onto the exhaust port and extended 3 cm into the chamber from the base. To this firm base the prothorax of a beetle was attached with a soft silicone glue. After a beetle was attached, the chamber was sealed with Plasticine and allowed to reach equilibrium with the system open. At atmospheric CO, concentration the instrument read 50 per cent on the metre scale. When a test sequence began, the system was closed and the concentration within the Plexiglas cell was measured by the gas analyser and recorded on a paper strip chart recorder.
OXIDATION OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR
BEETLES
1.557
Non-flying recordings were made by keeping the light intensity fairly low, since as soon as it was increased for any length of time the insects usually started flying steadily. To keep the temperature constant at 26°C a water-bath was used to cool the light source. Individual insects were tested either in the non-flying group or in the flying group, but not in both, and if an insect stopped flying during the flight test period the recordings were not included in the calculations. Groups consisted of 20 to 25 insects each. The non-flying tests finished when the recorder readings were greater than 60 per cent on the metre scale, while flight testing finished whenever the insect stopped flying, usually when readings were well past 60 per cent. In 0, consumption measurements the beetle was attached by the prothorax to a wire mount as previously described, and the wire was inserted into a rubber stopper which acted as a base. The mounted beetle was placed in a 25 ml round bottom flask, which was then attached to the respirometer and allowed to equilibrate for 15 min at 26°C. Usually 10 insects were run simultaneously on a 14 station respirometer leaving 4 stations as control thermo-barometers. When the system had equilibrated, the flasks were closed and readings began. Non-flying values were obtained with one group of insects by leaving the water bath tank lights off. Flight was induced in another group of insects by turning on these lights. Again the insects were continually observed and if any stopped flying during the test period readings were discarded. Readings for groups of flying and nonflying insects were taken every 5 min for 2 hr. RESULTS
AND DISCUSSION
(1959) and BEENAKKERS (1969) h ave categorized three physiological types of flight muscle; the carbohydrate utilizers, the dipterous insects (CHADWICK, 1947); the lipid utilizers, the lepidopterous insects (ZEBE, 1954); and the combination utilizers, the orthopterous insects (KROGH and WEIS-FOGH, 1949). During non-flying or rest periods, D. pseudotszqae maintained an RQ of 0.7 (Table 1) indicating oxidation of fat with no substrate interconversion. D. pseudotsugae, therefore, is predominately a fat utilizer. Absolute and relative changes in wet, dry, lipid, and fatty acid weights during flight of D. pseudotsugae males are evident (Figs. 1,2). All decreased during flight. The loss of water was greatest during the first hour of flight as is indicated by the large increase in the dry weight as a percentage of the wet weight (Fig. 2). The flight RQ of 0.8 determined for the first few hours of flight indicates that carbohydrate is being partially utilized with lipids during this early period (Table 1). Similar results were obtained by KROGH and WEIS-FOGH (1951) who demonstrated that S. gregaria is dependent on fat as the main source of energy during flight, but carbohydrate is used during the early stages. MEYER et al. (1960) suggested that the locust uses carbohydrates during the early period of flight until the body temperature reaches a level at which fat can be efficiently oxidized. The relative rates of CO, production in the test apparatus (Fig. 3) remained very constant for resting insects. Flying insects, however, decreased their CO, ZEBE
1558
S. N. THOMPSONANDR. B. BENNETT
production slightly after 40 min in cases where insects flew for 80 min before stopping. Flight cessation was attributed to too high a CO, concentration, since after the apparatus was ‘flushed’ with atmospheric air, flight usually resumed immediately. It is possible to use a semi-open system of detection whereby the CO, concentration is kept constant by addition of a measured amount of atmospheric air and, using this method, longer periods of flight could be monitored without the CO, concentration becoming too high. The CO, concentration would then be proportional to the amount of air added to the system. It would be TABLE I-UPTAKE
OF 0,
AND EVOLUTIONOF CO, BY FLOWN AND NON-FLOWNMALE D. pseudotsugae
Carbon dioxide evolution Mean deflexion time per beetle (min) ppm/min per beetle pg/min per beetle Oxygen uptake $/min per beetle pg/min per beetle Respiratory quotient
Flying
Non-flying
16.02 + l-48 5.31 240
67.41 f 7.10 1.26 0.57
2.10 * 0.21 3.00 RQ = 0.80
0.57 +_0.03 0.82 RQ = 0.70
. fatty
1
2 HOURS
3
acid
weight
4
OF FLIGHT
FIG. 1. Changes in wet, dry, fat, and fatty acid weights after flight of D. pseudotsugae.
OXIDATION
1559
OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR BEETLES
90
80
qdrywek$t(Xofwetweight) o lipid weight
( %of dry weieht) ( %lipid weight)
. fatty acid weight
20 10 5 I
C
1
2
1
I
3
4
I
5
HOURS OF FLIGHT FIG.
2.
Relative
changes
in wet,
dry,
fat, and
fatty acid weights
after
flight
of
D. pseudotsugae.
I
z 8%
FLYING INSECTS RESTING INSECTS ___
%i= “d IOO?Jz”z’ S” w8 OZ”
I
go8O-
MINUTES OF FLIGHT FIG. 3.
Expiration
of CO,
by flying
and non-flying
D. pseudotsugae
male beetles.
1560
S. N. THOMPSON
AND R. B. BENNETT
important
to see if any metabolic changes occur after 2 hr of flight as male D. and secondary attractants much more strongly after this time (R. B. Bennett, unpublished data). The fatty acid pattern (Fig. 4) and percentage composition (Table 2) are, in general, consistent with that previously reported for coleopterous insects (BARLOW, 1964). However, the high C16:l content is unique, and is generally characteristic of dipterous insects (BARLOW, 1964) and a few lepidopterous insects (BRACKEN and HARRIS, 1969).Changesinthe percentage compositionandweightofindividual fattyacids during flightare shown in Table 2 and Fig. 5. In general, the weight of all fatty acids decreased during flight. Some selective oxidation is evident. The
pseudotsugue appear to react to primary
FIG.
4. The fatty acid pattern of non-flown male D. pseudotsugae beetles.
TABLE 2--CHANGEIN PERCENTAGECOMPOSITIONANDwEIGHTOF D. pseudotsugae AFTER FLIGHT
INDIVIDUALFATTYACIDS OF
-~ Flown
Acid* c14:o 16:O 16:l 18:O 18:l IS:2
Control (non-flown) 0.77 13.23 23.67 I.83 59.80 1.10
+ 0.16 f 0.58 + 0.65 f 0.14 of:0.24 rt 0.21
1 hr Wt. (mgx 10e4) 59*14 994 + 40 1983 f 85 137+12 4506_tl93 58+16
“/b 0.7 11.9 26.3 1.8 57,2 2.1
5 hr
2 hr Wt.
y0
Wt.
%
wt.
49 833 1841 126 4004 147
0.6 13.4 23.5 I.2 61.3 0
37 817 1434 73 3739 0
0.4 19.7 19.7 4.8 55.3 0
5 236 236 58 64 0
* The first number represents the number of carbon atoms, the second, the number of double bonds.
OXIDATION OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR
BEETLES
1561
monounsaturates, Cl8 :l and C16: 1 fatty acids respectively, are oxidized at the greatest rate, followed by the saturates pahnitic (C16:0), stearic (C18:0), and myristic (C14:O) acids respectively (Fig. 5). These results are contrary to the supposition of BEJZNAKKERS (1965) who contended that palmitate and oleate are preferentially oxidized. Although the per cent composition of fatty acids does 10,000
1
2
3
4
5
HOURS OF FLIGHT
FIG. 5. Change in weight of individual fatty acids of male D. pseudotsugae during flight.
change with flight time (Table Z), in vitro isolation and characterization of the oxidative enzyme system itself would be necessary to determine conclusively if there is a chain length or saturation specificity. It would appear, in the present case, that selective oxidation of monounsaturates is due primarily to their high levels. Random selection of molecules would result in the greatest use of those in the highest concentration. DUPONT and MATHIAS (1970) however have demonstrated that rats oxidize unsaturates more rapidly than saturates via the methylmalonyl-Co A pathway. 50
1562
S. N. THOMPSONANDR. B. BENNETT
During the analyses some oxidation of Cl8 :2 fatty acids occurred as is indicated by the presence of many short chain fatty acids evident in Fig. 4. The values obtained for C18:2 (Table 2, Fig. 5) are, therefore, questionable. However, this acid is found in very small or trace amounts in this species. ATKINS (1966b) reported that young female adults of D. pseudotsugae which initially responded negatively to host material, showed a positive response to the same material after varying amounts of flight exercise. Beetles with more than 20 per cent of their dry weight composed of fat usually rejected suitable host material and displayed a strong inclination to fly and disperse and those with less than 10 per cent fat usually failed to fly continuously for more than a few minutes. On the other hand, beetles with between 10 and 20 per cent fat, although capable of sustained flight, usually responded readily to host material. In our experiments, the fat of freshly emerged male control beetles was 14.79 2 0.82 per cent of the dry weight (Fig. 2). This decreased during flight until after 5 hr the fat was only 5.21 per cent of the dry weight. Most of the beetles flew continuously as described in the Materials and Methods section until they were stopped and frozen. Male beetles, therefore, appear to lack the relationship between lipid content and inclination to fly. Males have been shown previously not to exhibit a precise response on fresh host material (ATKINS, 1966), and not to be involved in host selection Females orient themselves primarily to (MCMULLEN and ATKINS, 1962). host material, while males orient themselves to the boring females (ATKINS, 1966a, b). Also, males appear to have less lipid than females. In general, this supports the findings of various workers for a great variety of insects as reviewed by GILBERT (1967). Females bark beetles, Ips paraconfusus, have been shown to accumulate and utilize fat for oijgenesis (PENNER, 1970). Also D. pseudotsugae females probably require more lipid for flight than males, since they are the ‘pioneers’, while the males usually follow directly to the new host. ATKINS (1969) concludes that behavioural changes may not be due to fat combustion alone but that accumulation of metabolic by-products or selective combustion of different types of lipids may be responsible. Our results support such a supposition, since there appears to be selective oxidation of monounsaturated fatty acids (Fig. 5). Although unlikely, it is possible that some dimorphic differences in fatty acid metabolism occur, since previous workers have demonstrated distinct behavioural sex differences in scolytid beetles as described above. However, ATKINS (1966) reports that ‘. . . The loss of weight and fat by males was similar to that found in females, i.e. in both sexes, the stored fat was utilized to maintain life processes.’
REFERENCES ATKINS M. D. (1966a) Studies on the fat content of the Douglas-fir beetle. Bi-Monthly Research Notes, Can. Dept. Forest. 22, 3. ATKINSM. D. (196613) Laboratory studies on the behavior of the Douglas-fir beetle, Dendroctonus pseudotsugae Hopkins. Can. Ent. 98, 953-991. ATKINS M. D. (1969) Lipid loss with flight in the Douglas-fir beetle. Can. Ent. 101, 164-165.
OXIDATIONOF FAT DURINGFLIGHTOF MALEDOUGLAS-FIR BEETLES
1563
BARLOW J. S. (1964) Fatty acids in some insect and spider fats. Cmt. J. Biochem. 42, 1365-1374. BEENAKKERS A. M. (1965) Transport of fatty acids in Locusta migratoria during sustained flight. J. Insect Physiol. 11, 879-888. BEENAKKERS A. M. (1969) Carbohydrate and fat as a fuel for insect flight. A comparative study. J. Insect Physiol. 15, 353-361. BLIGH E. G. and DYERW. J. (1959) A rapid method of total lipid extraction and purification. Can.J. Biochem. Physiol. 37, 911-917. BORDENJ. H. and BENNETTR. B. (1969) A continuously recording flight mill for investigating the effect of volatile substances on the flight of tethered insects. J. econ. Ent. 62, 782-785. BRACKENG. K. and HARRISP. (1969) High palmitoleic acid in Lepidoptera. Nature, Lond. 224, 84-85. CHADWICKL. E. (1947) The respiratory quotient of Drosophila in flight. Biol. Bull., Woods Hole 93, 229-239. DOMROESEK. A. and GILBERT L. I. (1964) The role of lipid in adult development and flight muscle development in Hyalophora cecropia. J. exp. Biol. 41, 573-590. DUPONT J. and MATHIAS M. M. (1970) Bio-oxidation of linoleic acid via methylamalonyl-Co A. Lipids 4, 478-483. FULTONR. A. and ROMNEYV. E. (1940) The chloroform soluble components of beet leafhoppers as an indication of the distance they move in the spring. J. agric. Res. 61, 737743. GILBERT L. I. (1967) Lipids and their metabolism in insects. A. Rev. Biochem. 10, 141160. GILSONW. E. (1963) Differential respirometer of simplified and improved design. Science, Wash. 141, 531-532. HAMILTON G. A. (1964) The occurrence of periodic or continuous discharge of carbon dioxide by male desert locust (Schistocerca gregaria Forskal) measured by an infra-red gas analyser. Proc. R. Sot. (B) 160, 373-395. KROGH A. and WEIS-FOGH T. (1951) Respiratory exchange of the desert locust before, during and after flight. r. exp. Biol. 28, 244-357. LEPPERH. A., ed. (1950) Methods of Analysis. Association of Official Agricultural Chemists, Washington, D-C., U.S.A. MCMULLENL. H. and ATKINSM. D. (1962) On the flight and host selection of the Douglasfir beetle Dendroctonus pseudotsugae. Can. Ent. 94, 1309-1325. MEWR H., PREISSB., and BAUERS. (1960) The oxidation of fatty acids by a particulate fraction from the desert locust (Schistocerca gregaria) thorax tissues. Biochem. J. 76, 27-53. PENNERK. R. (1970) Metabolism of fatty acids in Ips paraconfusus (Lanier): In vivo synthesis of fatty acids from acetate-l -l*C in freshly emerged females. M.Sc. Thesis, Simon Fraser University, Bumaby 2, B.C. SCHLENKH. and GELLERMANJ. L. (1960) Esterification of fatty acids with diazomethane on a small scale. Analyt. Chen. 32, 1412-1414. WEIS-FOGH T. (1952) Fat combustion and metabolic rate of flying locusts Schistocerca gregaria Forskhl. Phil. Trans. R. Sot. (B) 237, l-36. WEIS-FOGH T. (1967) Respiration and tracheal ventilation in locusts and other flying insects. J. exp. Biol. 47, 561-587. WILLIAMSM. W., WILLIAMSC. S., GUNNINGB. F., and TO~NX J. C. (1969) Oxygen consumption of the western horse lubber grasshopper. Ann. ent. Sot. Am. 62, 297. ZEBE E. (1954) uber den Stoffwechsel der Lepidopteran. 2. vergl. Physiol. 36, 290-317. ZEBEE. (1959) Die Verteilung von Enzymen des Fettstuffwechsels im Heusschreckenkiirper. Verh. dtsch. zool. in Miinster/Westf. 309-314.
to 1563. Pmgamon Press. Printed in Great Britain
OXIDATION OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR BEETLES, DlWllROCTOlVUS PSEUDOTSUGAE* S. N. THOMPSON
and R. B. BENNETT
Department of Biological Sciences, Simon Fraser University, Bumaby 2, B.C. (Received 9 November 1970) Abstract-During flight, male beetles lost weight primarily due to oxidation of fatty acids. RQ values of 0.7 and 0.8 were obtained for non-flying and flying insects respectively, indicating that Dendroctonus pseudotsugae is predominantly a fat utilizer, with some carbohydrate utilization during early flight periods. Selective oxidation of fatty acids was evident. The monounsaturates, C16:l and Cl8 :l were oxidized at the greatest rate, followed by the saturates, palmitic (C16:0), stearic (C18:0), and myristic (C14:O) acids, respectively. INTRODUCTION
THE UTILIZATIONof lipid as a major source of energy during flight was reported by FULTONand ROMNEY(1940) in Eu&&x ten&a and extensively documented by WEIS-FOGH (1952) for Schistocerca gregaria. WEIS-FOGH (1952) reports that lipid is an ideal substrate for flying, since hydration of glycogen would make isocaloric quantities eight times heavier than lipid, and result in an exceedingly overweight insect. MEYERet al. (1960) later demonstrated that 5’. gregariu flight muscle was able to oxidize 8 to 18 carbon chain fatty acids, and DOMROESEand GILBERT(1964) demonstrated oxidation of 14C-1-palmitate by flight muscles of Hyalophera cecropia. ATKINS (1966a, b), demonstrated a positive correlation between the fat content of Dendroctonus pseudotsugae and inclination to fly. Moreover, flown beetles had significantly less lipid than non-flown or control beetles (ATKINS, 1969). The purpose of our experiments was to further document the oxidation of fat during flight of D. pseudotsugae. Male beetles were used for convenience and also because males are presently being used in our laboratory for flight behavioural experiments, particularly in regard to their flight response in the presence of pheromone-laden female frass. MATERIALS
AND METHODS
Three separate groups of 30 freshly emerged male beetles were flown on autorecording flight mills for 1, 2, and 5 hr as previously described (BORDEN and BENNETT,1969). After flight the insects were frozen until the analyses were carried * Work supported by National Research Council of Canada grants to Drs. J. S. and J. H. BORDEN. 155.5
BARLOW
1556
S. N.
THOMPSON AND R. B. BENNETT
out. Three non-flown control groups were individually frozen before, during, and after the experimentation period. After initial wet and dry weight determinations, the insects were homogenized in a tissue grinder, the total lipid extracted (BLIGH and DYER, 1959), saponified (LEPPER, 1950), and esterified with diazomethane (SCHLENK and GELLERMAN, 1960). Fatty acid analysis was carried out on a Carlo-Erba gas-liquid chromatograph with a hot wire detector. Two metre glass columns (4 mm i.d.) were packed with 15% diethylene glycol succinate on Chromosorb W(AW), mesh 60/80. The carrier gas was helium. Methylated fatty acid standards containing myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (ClS:l), linoleic (ClS:Z), and linolenic (C18:3) acl‘ds were run with each sample for identification purposes. The unsaturated fatty acids used in the standard mixture, palmitoleic, oleic, linoleic, and linolenic acids, are specific isomers, each of which belongs to a group of isomers represented as C16:1, ClS:l, C18:2, and C18:3 respectively. However, since the chromatographic technique used only separates the mixture according to chain length and degree of saturation, any one of the isomers in a group may serve as a standard for the group. The unsaturated acids were not degraded to determine which isomers were present. In contrast to previous manometric techniques (CHADWICK,1947; WEIS-FOCH, 1967; WILLIAMSet al., 1969), we measured CO, expiration by flying male beetles with an infra-red gas analyser in an open system as described by HAMILTON(1964). Gilson manometric techniques (GILSON, 1963) were used to measure 0s consumption. The rates of change in concentration of these gases were then compared and respiratory quotients (RQ) calculated for non-flying and flying insects. Measurements of CO, concentration were made in a small Plexiglas flight chamber (5.08 x 5.08 x 7.62 cm outside diameter) with a rubber base, a diaphragm pump, and the IR gas analyser. The system was inter-connected with plastic tubing and produced a total volume of 231 cc and a flow rate of 410 cc/min. The analyser was calibrated by adjusting the gain control with a known concentration of prepared gas (250 ppm CO, in NJ so that a deflexion of 50 to 60 per cent on the recorder meter represented a change in concentration of 85 ppm. Because of the rather small size of the Douglas-fir beetle, a closed circulation method was used to give accumulated concentrations rather than an open circulation system as used by HAMILTON(1964). The test insects were suspended inside the Plexiglas chamber by a mount consisting of a short piece of wire partially heat sealed into a piece of glass, which in turn was glued onto the exhaust port and extended 3 cm into the chamber from the base. To this firm base the prothorax of a beetle was attached with a soft silicone glue. After a beetle was attached, the chamber was sealed with Plasticine and allowed to reach equilibrium with the system open. At atmospheric CO, concentration the instrument read 50 per cent on the metre scale. When a test sequence began, the system was closed and the concentration within the Plexiglas cell was measured by the gas analyser and recorded on a paper strip chart recorder.
OXIDATION OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR
BEETLES
1.557
Non-flying recordings were made by keeping the light intensity fairly low, since as soon as it was increased for any length of time the insects usually started flying steadily. To keep the temperature constant at 26°C a water-bath was used to cool the light source. Individual insects were tested either in the non-flying group or in the flying group, but not in both, and if an insect stopped flying during the flight test period the recordings were not included in the calculations. Groups consisted of 20 to 25 insects each. The non-flying tests finished when the recorder readings were greater than 60 per cent on the metre scale, while flight testing finished whenever the insect stopped flying, usually when readings were well past 60 per cent. In 0, consumption measurements the beetle was attached by the prothorax to a wire mount as previously described, and the wire was inserted into a rubber stopper which acted as a base. The mounted beetle was placed in a 25 ml round bottom flask, which was then attached to the respirometer and allowed to equilibrate for 15 min at 26°C. Usually 10 insects were run simultaneously on a 14 station respirometer leaving 4 stations as control thermo-barometers. When the system had equilibrated, the flasks were closed and readings began. Non-flying values were obtained with one group of insects by leaving the water bath tank lights off. Flight was induced in another group of insects by turning on these lights. Again the insects were continually observed and if any stopped flying during the test period readings were discarded. Readings for groups of flying and nonflying insects were taken every 5 min for 2 hr. RESULTS
AND DISCUSSION
(1959) and BEENAKKERS (1969) h ave categorized three physiological types of flight muscle; the carbohydrate utilizers, the dipterous insects (CHADWICK, 1947); the lipid utilizers, the lepidopterous insects (ZEBE, 1954); and the combination utilizers, the orthopterous insects (KROGH and WEIS-FOGH, 1949). During non-flying or rest periods, D. pseudotszqae maintained an RQ of 0.7 (Table 1) indicating oxidation of fat with no substrate interconversion. D. pseudotsugae, therefore, is predominately a fat utilizer. Absolute and relative changes in wet, dry, lipid, and fatty acid weights during flight of D. pseudotsugae males are evident (Figs. 1,2). All decreased during flight. The loss of water was greatest during the first hour of flight as is indicated by the large increase in the dry weight as a percentage of the wet weight (Fig. 2). The flight RQ of 0.8 determined for the first few hours of flight indicates that carbohydrate is being partially utilized with lipids during this early period (Table 1). Similar results were obtained by KROGH and WEIS-FOGH (1951) who demonstrated that S. gregaria is dependent on fat as the main source of energy during flight, but carbohydrate is used during the early stages. MEYER et al. (1960) suggested that the locust uses carbohydrates during the early period of flight until the body temperature reaches a level at which fat can be efficiently oxidized. The relative rates of CO, production in the test apparatus (Fig. 3) remained very constant for resting insects. Flying insects, however, decreased their CO, ZEBE
1558
S. N. THOMPSONANDR. B. BENNETT
production slightly after 40 min in cases where insects flew for 80 min before stopping. Flight cessation was attributed to too high a CO, concentration, since after the apparatus was ‘flushed’ with atmospheric air, flight usually resumed immediately. It is possible to use a semi-open system of detection whereby the CO, concentration is kept constant by addition of a measured amount of atmospheric air and, using this method, longer periods of flight could be monitored without the CO, concentration becoming too high. The CO, concentration would then be proportional to the amount of air added to the system. It would be TABLE I-UPTAKE
OF 0,
AND EVOLUTIONOF CO, BY FLOWN AND NON-FLOWNMALE D. pseudotsugae
Carbon dioxide evolution Mean deflexion time per beetle (min) ppm/min per beetle pg/min per beetle Oxygen uptake $/min per beetle pg/min per beetle Respiratory quotient
Flying
Non-flying
16.02 + l-48 5.31 240
67.41 f 7.10 1.26 0.57
2.10 * 0.21 3.00 RQ = 0.80
0.57 +_0.03 0.82 RQ = 0.70
. fatty
1
2 HOURS
3
acid
weight
4
OF FLIGHT
FIG. 1. Changes in wet, dry, fat, and fatty acid weights after flight of D. pseudotsugae.
OXIDATION
1559
OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR BEETLES
90
80
qdrywek$t(Xofwetweight) o lipid weight
( %of dry weieht) ( %lipid weight)
. fatty acid weight
20 10 5 I
C
1
2
1
I
3
4
I
5
HOURS OF FLIGHT FIG.
2.
Relative
changes
in wet,
dry,
fat, and
fatty acid weights
after
flight
of
D. pseudotsugae.
I
z 8%
FLYING INSECTS RESTING INSECTS ___
%i= “d IOO?Jz”z’ S” w8 OZ”
I
go8O-
MINUTES OF FLIGHT FIG. 3.
Expiration
of CO,
by flying
and non-flying
D. pseudotsugae
male beetles.
1560
S. N. THOMPSON
AND R. B. BENNETT
important
to see if any metabolic changes occur after 2 hr of flight as male D. and secondary attractants much more strongly after this time (R. B. Bennett, unpublished data). The fatty acid pattern (Fig. 4) and percentage composition (Table 2) are, in general, consistent with that previously reported for coleopterous insects (BARLOW, 1964). However, the high C16:l content is unique, and is generally characteristic of dipterous insects (BARLOW, 1964) and a few lepidopterous insects (BRACKEN and HARRIS, 1969).Changesinthe percentage compositionandweightofindividual fattyacids during flightare shown in Table 2 and Fig. 5. In general, the weight of all fatty acids decreased during flight. Some selective oxidation is evident. The
pseudotsugue appear to react to primary
FIG.
4. The fatty acid pattern of non-flown male D. pseudotsugae beetles.
TABLE 2--CHANGEIN PERCENTAGECOMPOSITIONANDwEIGHTOF D. pseudotsugae AFTER FLIGHT
INDIVIDUALFATTYACIDS OF
-~ Flown
Acid* c14:o 16:O 16:l 18:O 18:l IS:2
Control (non-flown) 0.77 13.23 23.67 I.83 59.80 1.10
+ 0.16 f 0.58 + 0.65 f 0.14 of:0.24 rt 0.21
1 hr Wt. (mgx 10e4) 59*14 994 + 40 1983 f 85 137+12 4506_tl93 58+16
“/b 0.7 11.9 26.3 1.8 57,2 2.1
5 hr
2 hr Wt.
y0
Wt.
%
wt.
49 833 1841 126 4004 147
0.6 13.4 23.5 I.2 61.3 0
37 817 1434 73 3739 0
0.4 19.7 19.7 4.8 55.3 0
5 236 236 58 64 0
* The first number represents the number of carbon atoms, the second, the number of double bonds.
OXIDATION OF FAT DURING FLIGHT OF MALE DOUGLAS-FIR
BEETLES
1561
monounsaturates, Cl8 :l and C16: 1 fatty acids respectively, are oxidized at the greatest rate, followed by the saturates pahnitic (C16:0), stearic (C18:0), and myristic (C14:O) acids respectively (Fig. 5). These results are contrary to the supposition of BEJZNAKKERS (1965) who contended that palmitate and oleate are preferentially oxidized. Although the per cent composition of fatty acids does 10,000
1
2
3
4
5
HOURS OF FLIGHT
FIG. 5. Change in weight of individual fatty acids of male D. pseudotsugae during flight.
change with flight time (Table Z), in vitro isolation and characterization of the oxidative enzyme system itself would be necessary to determine conclusively if there is a chain length or saturation specificity. It would appear, in the present case, that selective oxidation of monounsaturates is due primarily to their high levels. Random selection of molecules would result in the greatest use of those in the highest concentration. DUPONT and MATHIAS (1970) however have demonstrated that rats oxidize unsaturates more rapidly than saturates via the methylmalonyl-Co A pathway. 50
1562
S. N. THOMPSONANDR. B. BENNETT
During the analyses some oxidation of Cl8 :2 fatty acids occurred as is indicated by the presence of many short chain fatty acids evident in Fig. 4. The values obtained for C18:2 (Table 2, Fig. 5) are, therefore, questionable. However, this acid is found in very small or trace amounts in this species. ATKINS (1966b) reported that young female adults of D. pseudotsugae which initially responded negatively to host material, showed a positive response to the same material after varying amounts of flight exercise. Beetles with more than 20 per cent of their dry weight composed of fat usually rejected suitable host material and displayed a strong inclination to fly and disperse and those with less than 10 per cent fat usually failed to fly continuously for more than a few minutes. On the other hand, beetles with between 10 and 20 per cent fat, although capable of sustained flight, usually responded readily to host material. In our experiments, the fat of freshly emerged male control beetles was 14.79 2 0.82 per cent of the dry weight (Fig. 2). This decreased during flight until after 5 hr the fat was only 5.21 per cent of the dry weight. Most of the beetles flew continuously as described in the Materials and Methods section until they were stopped and frozen. Male beetles, therefore, appear to lack the relationship between lipid content and inclination to fly. Males have been shown previously not to exhibit a precise response on fresh host material (ATKINS, 1966), and not to be involved in host selection Females orient themselves primarily to (MCMULLEN and ATKINS, 1962). host material, while males orient themselves to the boring females (ATKINS, 1966a, b). Also, males appear to have less lipid than females. In general, this supports the findings of various workers for a great variety of insects as reviewed by GILBERT (1967). Females bark beetles, Ips paraconfusus, have been shown to accumulate and utilize fat for oijgenesis (PENNER, 1970). Also D. pseudotsugae females probably require more lipid for flight than males, since they are the ‘pioneers’, while the males usually follow directly to the new host. ATKINS (1969) concludes that behavioural changes may not be due to fat combustion alone but that accumulation of metabolic by-products or selective combustion of different types of lipids may be responsible. Our results support such a supposition, since there appears to be selective oxidation of monounsaturated fatty acids (Fig. 5). Although unlikely, it is possible that some dimorphic differences in fatty acid metabolism occur, since previous workers have demonstrated distinct behavioural sex differences in scolytid beetles as described above. However, ATKINS (1966) reports that ‘. . . The loss of weight and fat by males was similar to that found in females, i.e. in both sexes, the stored fat was utilized to maintain life processes.’
REFERENCES ATKINS M. D. (1966a) Studies on the fat content of the Douglas-fir beetle. Bi-Monthly Research Notes, Can. Dept. Forest. 22, 3. ATKINSM. D. (196613) Laboratory studies on the behavior of the Douglas-fir beetle, Dendroctonus pseudotsugae Hopkins. Can. Ent. 98, 953-991. ATKINS M. D. (1969) Lipid loss with flight in the Douglas-fir beetle. Can. Ent. 101, 164-165.
OXIDATIONOF FAT DURINGFLIGHTOF MALEDOUGLAS-FIR BEETLES
1563
BARLOW J. S. (1964) Fatty acids in some insect and spider fats. Cmt. J. Biochem. 42, 1365-1374. BEENAKKERS A. M. (1965) Transport of fatty acids in Locusta migratoria during sustained flight. J. Insect Physiol. 11, 879-888. BEENAKKERS A. M. (1969) Carbohydrate and fat as a fuel for insect flight. A comparative study. J. Insect Physiol. 15, 353-361. BLIGH E. G. and DYERW. J. (1959) A rapid method of total lipid extraction and purification. Can.J. Biochem. Physiol. 37, 911-917. BORDENJ. H. and BENNETTR. B. (1969) A continuously recording flight mill for investigating the effect of volatile substances on the flight of tethered insects. J. econ. Ent. 62, 782-785. BRACKENG. K. and HARRISP. (1969) High palmitoleic acid in Lepidoptera. Nature, Lond. 224, 84-85. CHADWICKL. E. (1947) The respiratory quotient of Drosophila in flight. Biol. Bull., Woods Hole 93, 229-239. DOMROESEK. A. and GILBERT L. I. (1964) The role of lipid in adult development and flight muscle development in Hyalophora cecropia. J. exp. Biol. 41, 573-590. DUPONT J. and MATHIAS M. M. (1970) Bio-oxidation of linoleic acid via methylamalonyl-Co A. Lipids 4, 478-483. FULTONR. A. and ROMNEYV. E. (1940) The chloroform soluble components of beet leafhoppers as an indication of the distance they move in the spring. J. agric. Res. 61, 737743. GILBERT L. I. (1967) Lipids and their metabolism in insects. A. Rev. Biochem. 10, 141160. GILSONW. E. (1963) Differential respirometer of simplified and improved design. Science, Wash. 141, 531-532. HAMILTON G. A. (1964) The occurrence of periodic or continuous discharge of carbon dioxide by male desert locust (Schistocerca gregaria Forskal) measured by an infra-red gas analyser. Proc. R. Sot. (B) 160, 373-395. KROGH A. and WEIS-FOGH T. (1951) Respiratory exchange of the desert locust before, during and after flight. r. exp. Biol. 28, 244-357. LEPPERH. A., ed. (1950) Methods of Analysis. Association of Official Agricultural Chemists, Washington, D-C., U.S.A. MCMULLENL. H. and ATKINSM. D. (1962) On the flight and host selection of the Douglasfir beetle Dendroctonus pseudotsugae. Can. Ent. 94, 1309-1325. MEWR H., PREISSB., and BAUERS. (1960) The oxidation of fatty acids by a particulate fraction from the desert locust (Schistocerca gregaria) thorax tissues. Biochem. J. 76, 27-53. PENNERK. R. (1970) Metabolism of fatty acids in Ips paraconfusus (Lanier): In vivo synthesis of fatty acids from acetate-l -l*C in freshly emerged females. M.Sc. Thesis, Simon Fraser University, Bumaby 2, B.C. SCHLENKH. and GELLERMANJ. L. (1960) Esterification of fatty acids with diazomethane on a small scale. Analyt. Chen. 32, 1412-1414. WEIS-FOGH T. (1952) Fat combustion and metabolic rate of flying locusts Schistocerca gregaria Forskhl. Phil. Trans. R. Sot. (B) 237, l-36. WEIS-FOGH T. (1967) Respiration and tracheal ventilation in locusts and other flying insects. J. exp. Biol. 47, 561-587. WILLIAMSM. W., WILLIAMSC. S., GUNNINGB. F., and TO~NX J. C. (1969) Oxygen consumption of the western horse lubber grasshopper. Ann. ent. Sot. Am. 62, 297. ZEBE E. (1954) uber den Stoffwechsel der Lepidopteran. 2. vergl. Physiol. 36, 290-317. ZEBEE. (1959) Die Verteilung von Enzymen des Fettstuffwechsels im Heusschreckenkiirper. Verh. dtsch. zool. in Miinster/Westf. 309-314.