Adipokinetic hormone and flight metabolism in the locust

J. Insect Physiol., 1976. Vol. 22, pp. 1559 to 1564. Pergarnon Press. Printed in Great Britain.

HORMONE METABOLISM IN

AND LOCUST

N. L. ROBINSON* and G. J. GOLDSWORTHY Department of Zoology, The University. Hull HU67RX. England (Receioed 25 June 1976)

Abstract--In adult male Schistocerca 20min of tethered flight causes a halving of the haemolymph carbohydrate concentration. Injection of a proteinaceous emulsion of diglyceride 30 min before flight reduces both flight speed and carbohydrate utilisation. This effect can be overcome by the injection of trehalose immediately before the flight. If, in addition to the diglyceride, a dilute extract of the glandular lobes of the corpora cardiaca is injected immediately before flight, either with or without additional trehalose, carbohydrate utilisation is drastically reduced whereas flight speed is unaffected. It is argued that diglyceride competes with trehalose as a substrate for the flight muscles and that adipokinetic hormone from the glandular lobes of the corpora cardiaca stimulates the oxidation of diglyceride in these muscles during flight. This brings about a more complete (non-competitive) inhibition of trehalose utilisation by the flight muscles.

WORTHY (1974) and blown into a small volume of chloroform-methanol (1:2, v/v). Before analysis this LOCUSTS are known to use both lipid and carbowas dried down and the lipids extracted in n-hexane hydrate as fuels for flight. Carbohydrate supplies for GLC (gas-liquid-chromatography) or the energy in the early stages of flight but lipid is later measurement of total lipid by the vanillin method used predominantly during long-term flight (KROGH (GOLDSWORTHYet al., 1972). The remainder of the and WEIS-FOGH, 1951; WEIS-FOGH, 1952; MAYER and hexane was blown off and the residue taken up in CANDY, 1969a; JUTSUM and GOLDWORTHY, 1976). trichloracetic acid (5%) for total carbohydrate quantiIn a previous study (ROBINSONand GOLDSWORTHY, fication by the anthrone method (ROE, 1955). 1974) indirect evidence was presented which suggested GLC analysis of diglycerides was carried out as that locust adipokinetic hormone, in addition to described by JUTSUM and GOLDSWORTHY (1974) mobilising fat body lipid (BEENAKKERS,1969; MAYER except that Dexsil 400 was used as the stationary and CANDY, 1969b; GOLDSWORTHYet al., 1972), prophase and cholesterol propionate as an internal stanmotes also the utilisation of lipid by the flight dard. muscles. The present paper is concerned with a study Initial experiments used emulsions of crude synin the flying locust of the effects of adipokinetic horthetic dipalmitin (Sigma Chemical Co. Ltd.) in 4% mone and substrate availability on flight speed and albumin prepared as described by ROBINSON and substrate utilisation in an attempt to obtain more diGOLDSWORTHY(1974). In later experiments, in which rect confirmation of the above hypothesis. GLC analyses of haemolymph diglycerides were performed, the crude dipalmitin was purified before use on a 7% hydrated florisil column (CARROL and SERMATERIALS AND METHODS DAREVICH,1967). Adult male Schistocerca gregaria were reared under Incorporation of dipalmitin into haemolymph lipoconditions described previously (GOLDSWORTHYet al., proteins was measured after injecting locusts with 1972). Males were separated from females immedi50 ~1 of the emulsion of pure dipalmitin. After 30 min ately after the imaginal moult and maintained under a sample of haemolymph was blown.into 50%-saturcrowded conditions with males of a similar (& 3 days) ated ammonium sulphate and the supematant passed age. through a Millipore filter (0.22 p) to remove free Preparation of extracts of corpora cardiaca and dipalmitin. Dipalmitin in the supernatant (attached measurement of flight speed were as described by JUTto lipoprotein) was determined by GLC after extracSUM and GOLDSWORTHY(1976). tion with chloroform-methanol as described above. Samples of haemolymph (usually 5 ~1) were taken from locusts by the method of JUTSUM and GOLDSRESULTS INTRODUCTION

* Present address: Department of Nottingham.

of Zoology. University

In adult male Schistocerca (18 to 20 days old) the changes in haemolymph carbohydrate during flight 1559

1560

N. L. ROBINSON AND Table

1. Changes

No. of observations

Length of flight 10 min 20 min

in the concentration

Distance flown Mean revs f SE

6 6

G. J. GOLDSWORTHY

of carbohydrate

773 + 102 2041 i 196

and changes

None Albumin Saline Albumin DGL DGL + DGL + DGL + Trehalose Trehalose GLL

+ saline GLL trehalose trehalose

+ GLL

+ GLL

19.4 * 6.6 7.4 f 2.8

before the test flight (Table 2). In this case, however, flight speed was increased (P < 0.05) relative to those animals receiving only the injection of dipalmitin (Table 2: Fig. 1A). Locusts injected only with the extract of corpora cardiaca exhibited both reduced flight speed and carbohydrate utilisation (P < 0.05 in each case) when compared with control locusts (Table 2; Fig. 1A). When locusts which had been injected with dipalmitin 30min previously were injected with trehalose (6 mg in 10 ~1 of saline) and flown immediately, carbohydrate utilisation was increased (P < 0.01) in com-

in haemolymph carbohydrate and diglyceride flight in adult male Schistocrrcu

Number of observations

Injection*

Relative rate of decrease in haemolymph carbohydrate Mean + SE (pg/pl/rev x t03)

12.8 f 1.8 15.1 f 3.4

brought about an approximate halving of the blood total carbohydrate (Tables 1 and 3). When 50~1 of an emulsion of dipalmitin (3 mg) were injected 30 min before the test flight, both carbohydrate utilisation and flight speed were significantly (P < 0.05) reduced (Tables 2 and 3). Utilisation of carbohydrate was suppressed further (P < 0.01) when an extract of corpora cardiaca (0.02 pair of glandular lobes in 10 ~1 of saline) was injected 30 min after the dipalmitin emulsion, i.e. immediately performance

flight in Schistocrrca

Decrease in haemolymph carbohydrate @g/PI) Mean + SE

were similar to those described previously for Locustu (JUTSUM and GOLDSWORTHY, 1976) and a 20 min flight

Table 2. Flight

during

Change in haemolymph carbohydrate Actual decrease Relative decrease Mean + SE Mean + SE kg/&rev x 103) kg//d)

Revolutions flown mean k SE 2041 2010 2051 2056 1572 2019 1819 2091 2314 2068 1668

6 6 6 6 12 12 12 12 6 6 6

k * * + k k i. * * & *

concentrations

196 97 109 144 163 136 69 188 97 73 97

15.1 15.6 16.3 14.6 x.4 3.9 17.2 4.3 38.4 11.8 5.2

f + f f + * + f f + f

3.4 3.0 3.0 3.0 2.3 1.3 2.6 1.4 3.3 1.5 2.2

7.4 7.9 8.5 7.1 5.5 2.1 9.4 2.1 16.6 5.8 3.8

+ * + f + + + + * + *

2.8 2.1 3.3 1.8 1.1 0.7 1.4 0.3 1.0 0.5 0.8

during

a 20 min

Total diglyceride increase Mean + SE (Pcg/Pl) 3.1 2.2 2.3 2.6 4.7 15.5 1.3 25.6 0.6 17.4 3.8

+ 0.8 k 0.6 + 0.6 k 0.9 * 2.0 +_ 1.5 & 0.3 k 2.5 k 0.2 * 1.3 + 1.0

* Albumin (4”“) and DGL (Dipalmitin, 3 mg) were injected in 50 ~1 of saline 30 min before flight; Trehalose (6 mg). Saline and GLL (0.02 pair of glandular lobes of the corpora cardiaca) were injected in 10 ~1 immediately before flight, See text for further details. Table 3. The effect of 20mm

Number of observations

Treatment Control DGL DGL + GLL DGL + trehalose DGL + trehalose + GLL Trehalose Trehalose + GLL GLL * For explanation

flight on the concentration

of abbreviations

‘4 I2 12 12 12 6 6 6

of haemolymph

Lipid concentration pg/pl Final Initial mean k SE mean k SE 3.1 17.3 15.4 16.0 15.5 3.4 5.3 3.3

see text and Table 2.

+ * t f + & + &

1.1 4.4 4.1 2.3 2.9 2.3 2.2 1.0

5.7 22.1 30.9 17.4 41.0 4.0 22.7 7.1

* + I + f * f +

1.4 1.7 2.1 0.8 3.0 0.7 2.9 1.5

metabolites

in Schistocerca

Carbohydrate concentration fig/p1 Final Initial mean + SE mean f SE 32.4 29.1 29.8 53.1 56.5 57.1 51.3 31.3

f f i + + f * +

4.2 5.4 3.5 6.3 3.6 5.8 4.2 5.1

17.0 20.8 25.8 36.0 52.2 18.8 39.5 26.7

f 3.1 k 4.8 + 5.1 rf: 6.4 2 1.3 * 3.5 k 4.8 + 3.4

1561

Adipokinetic hormone and flight metabolism in the locust Fl!ghl

performance

15

10

of flrghl

lmrnl

Fig. 1. Flight speed in adult male Schistocerca after various injections. For explanation of abbreviation see text or Table 2. Standard errors have been omitted for clarity

number of revolutions flown for each individual locust some of the variation produced by animals flying different distances could be excluded. When this was done it could be seen that, in comparison with control locusts, injection of diglyceride caused no significant reduction in the relative (to distance) rate of carbohydrate utihsation (Table 2). Injection of both dipalmitin and extracts of corpora cardiaca. however, caused a dramatic reduction in carbohydrate utilisation (P < 0.01). When extracts of corpora cardiaca were administered alone. the decrease in the relative rate of carbohydrate use was less marked (Table 2). In animals injected with trehalose immediately prior to the test flight the relative rate of carbohydrate utilisation was double (P < 0.01) that of control locusts. When both trehalose and extracts of corpora cardiaca were injected together, the relative rate of carbohydrate usage was similar to that of locusts injected with dipalmitin. When trehalose was administered to animals injected previously with dipalmitin, the rate of carbohydrate usage during flight was intermediate between that of control locusts and those receiving trehalose alone. When extracts of corpora cardiaca were injected into locusts which received both dipalmitin and trehalose, however, the relative rate of carbohydrate utilisation was considerably reduced (P < 0.01) (Table 2). It is important for the interpretation of the observations above to realise that, 30 min after injection, 607, of the dipalmitin had been incorporated into the haemolymph lipoproteins (Fig. 2) and was thus truly in solution. ’ Using GLC analysis it was possible to monitor the rate of disappearance from the haemolymph of exogenous dipalmitin (Fig. 3). In estimating the rate of DI

parison with animals receiving only dipalmitin (Table 2). Although flight speed was initially high in these locusts it decreased throughout the test period (Fig. 1B). When extracts of corpora cardiaca were administered simultaneously with trehalose to locusts injected 30 min previously with dipalmitin emulsion, carbohydrate utilisation during flight was significantly reduced (P < 0.01) when compared with locusts which received the trehalose and dipalmitin but were not given the hormone preparation (Table 2). The flight speed of locusts receiving all three injections was similar to that of control locusts (Fig. 1). When only trehalose was administered (immediately before flight) both flight speed and the rate of carbohydrate utilisation were increased (P < 0.05 in each case) relative to that of saline-injected control animals (Table 2; Fig. 1). When extracts of corpora cardica were injected with the trehalose both the flight speed and carbohydrate utilisation were reduced in comparison with those locusts which received only trehalose (Table 2; Fig. 1B). When the measured decrease in haemolymph carbohydrate was expressed as a function of the

Ia

20

Cl

Immediately

after injection 30 m,n

later

75

10

5

0 Total dipalm/tin

d/palm!t!n

Fig. 2. Incorporation of injected dipalmitin into haemolymph lipoproteins. Columns represent the mean and standard error for five observations.

N. L. ROBINSON AND G. J. GOLDSWORTHY

1562

BEFORE

AFTER

FLIGHT

FLIGHT

hJhl 1616

ofpolm/t!n L (16 16)

I

27O’C

I

340-c

I

340°C

Fig. 3. Disappearance of dipalmitin from the haemolymph during flight. Typical GLC traces are shown for one animal from each of the three groups. For explanation of the abbreviations wee text and Table 2.

disappearance of dipalmitin it has been assumed that the three major diglycerides (of which dipalmitin is the least prevalent) maintain a constant ratio to each other (see SPENCER and CANDY, 1974) and hence, by measuring the changes in 16:18 and 18:18 diglycerides (see JUTSUMand GOLDSWORTHY.1976). allowance was made for any changes in endogenous dipalmitin. It can be seen from Fig. 4 that only in some flown animals was there any appreciable disappearance of the injected dipalmitin. Injection of trehalose suppressed dipalmitin disappearance in flown animals but extracts of corpora cardiaca. although without effect in resting locusts, had a marked stimulatory effect on the rate of disappearance (Fig. 4).

DISCUSSION JUTSUM and GOLDSWORTHY (1975, 1976) have shown that during short flights in Locusta there is no rapid mobilisation of stored carbohydrate and the observed decrease in blood sugar concentration represents directly, therefore, the utilisation of carbohydrate by the flight muscles. After the early phase of rapid utilisation, the haemolymph carbohydrate concentration stabilises after 30 min so that no further major change in concentration occurs during a subsequent 2.5 hr of flight (JUTSUM and GOLDSWORTHY. 1976). This does not mean. however, that carbohydrate utilisation is nil since during this period there

Adipokinetic

All

locusts

hormone

rece!ved 30 mm

and flight metabolism

3 mg before

in the locust

1563

drpalm!tln experiment

1 4.

12

10

t

08

0.6

04

02

0

1 -ill

I--

1

Rested

Control

20 m,n GLL

4

III t cwrtroi

Flown GLL

20

m,n

Treholose

Treholose+GLL

Fig. 4. Rate of disappearance of dipalmitin from the haemolymph during rest and flight. Columns represent the mean and standard errors for five observations. See text or Table 2 for explanation.

is some mobilisation of the limited stores of carbohydrate in the fat body and gut wall. In Locusta, it would appear that after 30 min of flight the mobilisation of carbohydrate matches its utilisation but that only a small amount of sugar is being used (JUTSUM and GOLDSWORTHY,1976). This low rate of utilisation of trehalose, and that measured in the present study in Schistocerca which have been injected with diglyceride and adipokinetic hormone, may represent, therefore, a minimum rate of carbohydrate utilisation necessary for efficient lipid oxidation (Robinson and Goldsworthy, in press). After 20 min of flight, carbohydrate utilisation is thus very low although the haemolymph trehalose concentrations are still relatively high (about 15 pg/pl). This suggests that, after 10min or so of flight, factors other than the concentration of trehalose are acting to reduce carbohydrate utilisation. At this time haemolymph lipid levels are increasing under the action of the adipokinetic hormone (BEENAKKERS, 1969; MAYER and CANDY, 1969b) released from the glandular lobes of the corpora cardiaca (GOLDSWORTHYet al., 1972). During flight it is difficult in normal animals to distinguish between the effects of adipokinetic hormone on flight muscle metabolism and those of the elevated levels of haemolymph diglyceride; neither occurs independently. To overcome this problem the effects of artificially raising the concentration of haemolymph diglyceride, trehalose and hormone levels have been studied. Although the dipalmitin used in these experiments was injected as an emulsion in albumin it is clear that during the

30min pre-flight period a large proportion of the injected dipalmitin is incorporated into haemolymph lipoproteins and can be considered as a ‘physiological’ substrate for the flight muscles. This incorporation process has not been studied further but represents an interesting area for future study. When haemolymph diglyceride concentrations are increased by injection of dipalmitin, carbohydrate utilisation and flight speed are reduced. When the lower flight speed is taken into account. however. the relative rate of carbohydrate utilisation is only slightly less than in control locusts. This suggests that trehalose is still the major fuel but the lower flight speed indicates possibly that metabolic energy production is reduced. The presence of the high lipid levels must. therefore, in some way reduce the capacity of the flight muscles to metabolise the available carbohydrate. When tissue extracts containing adipokinetic hormone are injected with the dipalmitin. flight speed is high but the use of trehalose is greatly reduced. By elevating the titre of adipokinetic hormone the metabolism of the muscle is diverted from carbohydrate oxidation. The high rate of disappearance of dipahnitin in such locusts indicates that the flight muscles are now oxidising lipids to supply their energy requirements. This suggests further that it is probably the oxidation of lipid which is responsible for the marked inhibition of carbohydrate metabolism (ROBINSONand GOLDSWORTHY,1976). When extracts of glandular lobes alone are injected. flight speed and carbohydrate utilisation are reduced. In this situation lipid levels will be increasing under

1564

N. L. ROBINsONAND G. J. GOLDSWORTHY

the influence of the adipokinetic hormone and any lipid available can be utilised by the flight muscle since the hormone is also present. This seems to account for the failure of lipid to accumulate in the haemolymph as would normally be expected (after injection of the adipokinetic hormone into a resting locust). Under these conditions lipid oxidation is favoured, and hence carbohydrate usage inhibited, much earlier than normal. The reduced flight speed (GOLDSWORTHY rt al., 1973) implies that the total energy production is insufficient for maximum flight speed: presumably the concentration of lipid does not become sufficient for maximum efficient use (Robinson and Goldsworthy, in preparation). The inhibition of trehalose metabolism by injected dipalmitin in the absence of exogenous adipokinetic hormone appears to be, to some extent, competitive; when trehalose concentrations are increased, flight speed is returned to normal initially and sugar utilisation increases. Although the rate of carbohydrate utilisation is high it is less than in those animals injected with only trehalose. In both cases the lipid increase in the blood is less than in control locusts. This suggests that less adipokinetic hormone has been released (HOUBEN and BEENAKKERS.I973 ; JUTSUM and GOLDSWORTHY, 1975:CHEESEMANer al., 1976). It is difficult, therefore. to be certain if the effect of the injected trehalose is due to substrate competition or inhibition of hormone release. When adipokinetic hormone is administered to either of these two experimental groups trehalose disappearance from the haemolymph is reduced. The rate of carbohydrate utilisation in animals receiving all three injections is similar to that in locusts given only dipalmitin and adipokinetic hormone. Thus, when diglycerlde and adipokinetic hormone levels are both high. increasing the trehalose concentration has no effect on its oxidation by the flight muscles. From the results of the experiments described in this paper it is possible to comment on the possible control mechanisms operating on substrate utilisation during flight. The oxidation of trehalose is inhibited by increasing lipid oxidation which is itself maximal only when adipokinetic hormone titres are high and lipid levels sufficient. The present work would suggest that diglyceride levels may need to be in excess of lOpg/pl for maximum utilisation. This figure is in close agreement with that found by SPENCER and CANDY (1974) during prolonged flight in Schistocerctr. Thus carbohydrate utilisation in the flight muscles is reduced dramatically at a time when some 50”” of the total carbohydrate remains in the blood. This conservation of blood sugar may be analagous to that seen in mammals during starvation and represent a need for continued supply of other tissues, especially the nervous system, with carbohydrate.

,4cknowlrdgements_NLR is grateful to the University of Hull for the provision of a research Studentship. This work is supported by grants from the Science Research Council and The Royal Society.

REFERENCES BEENAKKERS A. Ivl. T. (1969) The influence of corpus allaturn and corpus cardiacum on lipid metabolism in Locustamigratoria. Gm. camp. Endocr. 13, Abstract 12. CARROLL K. K. and SERDAREVICH B. (1967) Lipid Chromatographic Analysis 1, 205-237. Dekker. New York. CHEESEMAXP., JLJTSUM A. R.. and GOLDSWORTHYG. J. (19761 Quantitative studies on the release of locust adipokinetic hormone. Physiol. Ent. 1, 115~121. GOLDSWORTHY G. J., MORUUE W.. and GUTHKELCH J. 11972) Studies on insect adipokinetic hormones. Grv. camp Eudocr. 18, 545-551. GOLDSWORTHYG. J.. COUPLANDA. J.. and MORDUE W. (1973) The effects of corpora cardiaca on tethered flight in the locust. J. camp. I%ysio/. 82, 339-346. HOUBENN. M. D. and BEENAKKERSA. M. T. (1975) The influence of haemolymph carbohydrate concentration on the release of adipokinetic hormone during locust flight. J. Endoer. 64, 66P. JUTSUM A. R. and GOLDSWORTHYG. I. (1974) Some effects of mermithid infection on metabolic reserves and Right in Locustu. Int. J. Parasit. 4. 625A30. J~~TSLMA. R. and GOLI>SWORTHYG. J. (1975) Locust adlpokinetic hormone and haemolymph metabolites. J. Elldocr. 64, 65-66P. JUTSUM A. R. and GOLDSWORTHYG. J. (1976) Fuels for flight in Locusta. J. Insect Physiol. 22, 243-249. KROGII A. and WEIS-FOGH T. (1951) The respiratory exchange of the desert locust (Schistocerca gregariu) before. during and after flight J. cup. Biol. 28, 342-357. MAYER R. J. and CANDY D. J. (1969a) Changes in energy reserves during flight of the desert locust, Schistocerca gregaria. Camp. Biochem. Ph!:siol. 31, 409%418. MAYER R. J. and CANDY D. J. (1969b) Control of haemolymph lipid concentration during locust flight: An adipokinetic hormone from the corpora cardiaca. J. Insect Physrol. 15, 611420. ROBINSONN. L. and GOLDSWORTHYG. J. (1974) The effects of locust adipokinetic hormone on flight muscle metabolism 111ciao and it1 citru. J. camp. Physiol. 89, 369-377. ROBINSONN. L. and GOLDSWOR~Y G. J. (1976) Adipokinetic hormone and the regulation of carbohydrate and lipid metabolism in a working flight muscle .preparation. . J: Insect Physiol. (in press). ROE J. H. (1955) The determination of sugar in blood and spinal fluid with the anthrone reagent J. biol. Chem. 212, 335-338. SPENCERI. M. and CANDY D. J. (1974) The effect of flight on the concentrations and composition of haemolymph diacyl glycerols in the desert locust. Biochem. Sot. Trans. 2, 1093-1096. WLIS-FDC;H T. (f952) Fat combustion and metabolic rate of flying locusts (Schistocrrca gregaria Forskal). Phil. True\. R. Sot. Land. (B) 237, l-36.