Octopamine mobilization of lipids and carbohydrates in the house cricket, Acheta domesticus

J. Insect Physiol.

Vol. 37, No. 3, pp. 193-199, Printed in Great Britain. All rights reserved

1991

0022-1910/91 $3.00 + 0.00 Copyright 0 1991 Pergamoo Press plc

OCTOPAMINE MOBILIZATION OF LIPIDS AND CARBOHYDRATES IN THE HOUSE CRICKET, ACHETA DOMESTICUS P. E. FIELDS and J. P. WOODRING Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70808, U.S.A. (Received 5 January 1990; revised 13 November 1990) Abstract-Injected octopamine elevated both lipids and sugars in the blood of head-ligated adult Achefu domesticus. Head ligation precludes any involvement of peptide hormone release

from the corpus cardiacum and eliminates stress induced hyperglycaemia. A 50% increase was the maximal lipaemic response to either octopamine or synthetic cricket adipokinetic hormone, indicating a feedback inhibition. The time course of the lipid response to injected octopamine was shorter than for injection of the cricket’s own adipokinetic hormone, which may be related to different receptors. The alpha adrenergic antagonist phentolamine blocked both the lipid and sugar mobilization action of octopamine. For the fust time an age-dependent hyperlipaemic response for octopamine is reported, which was very similar to the age-response curve for the cricket’s own adipokinetic hormone. A correlation between ovarial development and the sugar and lipid responsiveness to octopamine indicated a functional relationship of octopamine responsiveness to reproduction. A dibutyryl CAMP-induced elevation of both lipids and sugars indicate that CAMP is involved in both sugar and lipid mobilization by octopamine. The phosphorylase a inhibitor flavin mononucleotide inhibited both the lipid and sugar response to octopamine, which implies that phosphorylase a is involved in the octopamine-induced mobilization of both sugar and lipids. The neurotransmitter effectiveness for the hyperlipaemic response was: octopamine > norepinephrine > dopamine > epinephrine. Key

Word Index:

hyperlipemia;

Acheta;

phosphorylase;

crickets; octopamine; adipokinetic CAMP; fat body; blood

INTRODUCHON

released from the corpus cardiacum cause lipid or carbohydrate mobilization in many insects (see review by Wheeler et al., 1988). A specific adipokinetic hormone may elevate the blood trehalose levels in one species but in other species it elevates the blood lipid levels [see Glide (1990) for review]. The adipokinetic hormones bind to phentolamine-sensitive beta adrenergic receptors (Orchard, 1982) and thereby activate CAMP and phosphorylase a in fat body cells (G&de and Beenakkers, 1977). An increase in CAMP thus leads to either lipid or carbohydrate mobilization (Glide and Holwerda, 1976; Gole and Downer, 1979), depending upon the system. Whether sugar or lipid is mobilized is therefore determined in later steps in the pathway to sugar or lipid release. The neurotransmitter, octopamine, also functions as a neurohormone in insects, producing either a hyperlipaemic or a hyperglycaemic response in uivo and in vitro (Downer, 1979; Orchard et al., 1982). To date all reports indicated that octopamine induced either hyperhpaemia or hyperglycaemia but not both. For example, octopamine injected into Locusta elevated blood lipids but not blood sugars (Orchard et ui., 1981; Orchard and Lang, 1984). Injected into Peptide hormones

hormone;

hyperglycemia;

Periplunetu octopamine elevated blood sugars (Downer, 1980). Octopamine released in the corpus cardiacum not only enters the blood but it also mediates the release of adipokinetic hormone from the glandular portion of the corpus cardiacum (Pannabecker and Orchard, 1986). Octopamine binds to alpha adrenergic receptors, leading to a rapid elevation of CAMP in the fat body of Periplunetu (Gole and Downer, 1979) and in Locustu (Orchard et al., 1982). The next step in the octopamine-induced release of lipids and sugars is the activation of the enzyme, glycogen phosphorylase a. Van Marrewijk et al. (1983) showed that injection of octopamine into Periplunetu induced a significant elevation of fat body phosphorylase a, which was associated with elevation of blood sugars. Treatment of fat body with exogenous CAMP caused a significant elevation of the active form of the enzyme (Steele, 1964; McClure and Steele, 1981). This study was undertaken to quantify the effects of octopamine on both lipid and sugar mobilization in Achetu domesticus. Lipid mobilization in Achetu was clearly mediated by adipokinetic hormone (Woodring et al., 1989a), but the importance of octopamine for both lipid and sugar mobilization and

P. E. FIELDSand J. P.

194

the characterization of the response in this species was not clear. Since it appeared that both adipokinetic hormone and octopamine were involved with the mobilization of lipids, the effect of octopamine on the activation of fat body phosphorylase in this species was also examined. METHODS

AND MATERIALS

Rearing and handling of crickets

House crickets (A. domesticus) were reared according to the methods of Clifford and Woodring (1990). Only adult, unmated females were used in the following studies. Crickets were anaesthetized by a 5-10s exposure to carbon dioxide prior to blood sampling. The right prothoracic leg was cut off and 2 ~1 of blood was collected, the left leg was cut off for the second sample. Both plasma lipids and sugars increased in response to handling in this species (Woodring et al., 1989b), which was eliminated by neck ligation (Table 1). The handling response was caused by octopamine release from the corpora cardiaca (Woodring et al., 1989b). Throughout this study only neck-ligated adult females were used. Octopamine injections

The various concentrations of octopamine (Sigma) were injected in a volume of 1 ~1 with a Hamilton syringe. A response time of 60 min was selected Table 1. Effects of handling* on the blood lipid and sugar titres (mg/ml) in non-ligated (control) and ligated 4-6-day-

old females Treatment Control Ligated Control Ligated

sugars sugars lipids lipids

Initial [t = 0)

n

Increase (t+30min)

n

5.38 * 0.15 5.41 +0.13 20.5 & 0.42 18.7f 0.73

30 23 32 15

0.86&0.14p 0.10 f 0.10 0.64*0.21p 0.00+0.21

8 12 I1 8

*Crickets were anesthetized 5-10 s with carbon dioxide and injected with water. Ligated crickets were tied off between the head and thorax 5 h prior to the first blood sample (t = 0). “Indicates a significant increase (ANOVA, P < 0.05).

WOODRING

because this was when the maximal response occurred. The injected concentration of octopamine used was high because of the dilution by the blood (volume about 100 ~1 in an adult female) and the reported short in z&u half-life (2-3 tin) of octopamine in insects (Goosey and Candy, 1982). We estimated the concentration of octopamine actually reaching the fat body to be about 10-7-10-6M, which is about the effective concentration for octopamine that was found for isolated fat body tissue in Locusta (Orchard et al., 1982). Antagonists and agonists

The adrenergic receptor antagonists, phentolamine (alpha blocker) and propranolol (beta blocker) were injected 10 min prior to octopamine injection. A potent allosteric inhibitor of phosphorylase a (Sprang et al., 1982), flavin mononucleotide, was injected 10min prior to octopamine. Dibutryl CAMP and dibutyryl cGMP are lipophilic agonists of CAMP and cGMP, that are known to elicit responses mediated by these cyclic nucleotides in relatively low doses (Hadley, 1985). One-hundred nmol of each were injected into 4-6-day-old females to test their effects on lipid/sugar mobilization. One pmol of caffeine, an octopamine mimic that stimulates the second messenger CAMP (Sutherland, 1972), was injected to see if lipids and/or sugars were mobilized. Metabolic assays

The haemolymph lipid titre was assayed using the colourimetric sulphophosphovanillin procedure (Barnes and Blackstock, 1973). The carbohydrate titre (total blood sugar) was determined using the phenol-sulphuric acid method (Montgomery, 1957). Activation of fat -body phosphorylase

The phosphorylase extracts (from fat body) were prepared using similar procedures as Ggde (1981), with a few modifications. Fifteen min after injection of test material or distilled water into ten 4-6-day-old adult female Acheta, the fat bodies were removed and immediately immersed in liquid nitrogen. Pooled fat bodies were homogenized in 1.5 ml of ice-cold 30 mM

Table 2. Effects of injected octopamine, corpus cardiacum extracts, and synthetic adipokinetic hormone on fat body phosphorylase activity and sugar/lipid mobilization in head-ligated 4-&day-old Achefa

Treatment

n

Dist. water Corpus cardiacum extracts Syn. adipokinetic hormone Octonamine

8 8 8 8

Active phosphorylase (%) 15.2 + 2.0 86.4 f 7.6 79.0 + 4.9 78.5 f 5.1

Change in sugar cont. (mg/ml) 0.03 & 0.10’ -0.26 f 0.21” 0.05 * 0.20” 0.96 + 0.20b

Change in lipid cont. (mg/ml) 0.08 * 0.11” 10.63 f 2.40’ 11.77 f l.96c 10.21 _+1.61’

Values sharing the same superscript letter are not significantly different (Duncan’s Multiple Range Test, P c 0.05). Changes in sugar and lipid concentrations were measured 60min after injection. Changes in percent active phosphorylase was measured 15 min after injection. Injected concentrations were: 100 nmol of octopamine, 0.1 corpus cardiacum gland equivalent, 50 pmol of Locusfa adipokinetic hormone I.

Octopamine mobilization of sugars and lipids

195

Table 3. Effects of adrenergic antagonists on the octopamine induced lipaemic and glycaemic response in head-ligated 4-6-day-old females

Treatment

n

Dist. water Octopamine Phen. + Oct. Phentolamine Prop. + Oct. Propranolol Phen. + corpus cardiacum ext Corpus cardiacum extracts

10 10 8 7 8 1

I 8

Change in lipid cont. (mglml)

Change (%)

0.04 f 0.15’ 10.21 + 1.61b 1.31 f l.OOc 0.08 *0.13* 10.09 f 0.49b 0.02 f 0.04” 9.62 f 0.96b 10.63 f 2.40b

0 46 7 0 40 0 41 43

Change in sugar cont.

Change

(ma/ml)

(%)

0.02 f 0.08” 0.96 f 0.21b 0.14 + 0.3p 0.03 + 0.12” 0.89 f 0.20b 0.02 f 0.12” 0.15 f 0.39” -0.12 & 0.18”

0 18 3 0 16 0 3 2

Insects were injected with the antagonist 10min prior to the octopamine injection. Changes were measured 60 min after injection. Values (changes in lipid cont.) sharing the same letters are not significantly different (Duncan’s Multiple Range Test, P < 0.05). Doses were: 1 pmol octopamine; 100 nmol phentolamine; 100 nmol propranolol; 0.1 equivalent corpus cardiacum extracts. buffer (pH 7.0) containing 5 mM EDTA and mM sodium fluoride. After centrifugation for 20 min at 17,500 g the infranatant layer between the pellet and the floating fat layer was removed and assayed for phosphorylase activity according to the methods of Childress and Sacktor (1970) as modified by Ziegler et al. (1979). This assay measured activity in the direction of glycogen degradation. Protein determinations were made using the Biuret method. triethanolamine-acetate

RESULTS

It was necessary to distinguish the response to handling (excitation) from that due to the injected octopamine. Head ligation eliminated any response due to handling, and no increase in either blood lipids or sugars was observed 30 min after handling (Table 1). It was also necessary to distinguish between response to injected octopamine and possible response to cricket adipokinetic hormone induced by the octopamine injection. Since the corpus cardiacum is the only known source of this hormone, head ligation (which we employed) eliminated any possible release of the hormone into circulation. Also, synthetic cricket adipokinetic hormone does not affect blood sugar levels but octopamine does (Table 2). Finally, when injected with propanolol, which blocks adipokinetic hormone receptors (see Orchard, 1982), octopamine still elevated lipid levels (Table 3).

Dose response

The normal lipid titre of 4-6-day-old females averaged 20.3 + 0.42 mg/ml (n = 47) and the normal sugar titre averaged 5.40 f 0.14 mg/ml (n = 53) (Table 1). The maximal hyperlipaemic response was about 10 mg/ml (50% increase over control) and the maximal hyperglycaemic response was about 1 mg/ml (18% increase over control) (Tables 1 and 2). The hyperlipaemic response (in terms of mg/ml increase) was therefore about 10 times the hyperglycaemic response. The octopamine ED, for lipids was about 10 nmol compared to about 100 nmol for sugars, indicating that the lipid-mobilizing system was about 10 times more sensitive (Fig. 1). The lowest octopamine dose for a maximal hyperlipaemic response was 25 nmol and the lowest dose for a maximal hyperglycaemic response was 250 nmol (Fig. l), which again indicates a 10 times greater sensitivity of the lipid-mobilizing system. The activation of phosphorylase a was dosedependent, with an ED,, of about 4-5 nmol (Fig. 2). The lowest octopamine dose for maximal activation (7580% activation) was 60 nmol (Fig. 2). The kinetics response (ED,, and K,,,,,) of the phosphorylation were more similar to those of the lipid response than to the sugar response. Time course response

The response to an injection of 1 pmol octopamine was rapid, a 30% hyperlipaemia and a 15%

Table 4. The effects of injection of various neurotranmittors on plasma lipid concentrations in ligated 4-6-day-old female Achera Treatment Dist. water Synephrine Norepinephrine Epinephrine Dopamine

n

Initial lipid cont. (mg/ml)

19 8 I 8 7

17.5 23.4 20.5 23.8 19.3

Change in lipid cont. 0.04*0.15= 1.15 f 1.13ab 6.91 f 0.63’ 0.85 f 0.93p 2.34 f 0.16b

Change (%) 0 5 34 3 12

Values (changes in lipid cone) sharing the same superscript letter are not significantly different (Duncan’s Multiple Range Test, P < 0.05). 100 nmol of each neurotransmittor was injected. Changes were measured 60min after injection.

196

P. E. FIELDSand J. P.

WOODRING

0.6 0.6 0.4 0.2 1

0

2

3

Time after injections -i-4-

0

10 20 30 40 50 60 SO 100 Octopamine dose (nmol)

Fig. 1. Dose response (after 60 min) of plasma lipids for 4-6-day-old adult Acheta to 1~1 injections of 1-lOOnmo1 octopamine. Each point represents the mean f SE (n = 10). Inset: dose response curve of plasma sugars of neck-ligated 4-6-day-old Achera to 1~1 injections of 30-500 nmol octopamine. Each point represents the mean & SE (n = 8). Water injected controls gave 0 increase.

4

5-

y

(h)

Fig. 3. Time course response of the lipaemic response (closed circle-solid line) and the glycaemic response (open circle-dashed line) of 4-6-day-old female A&era to injections of 1 pmol octopamine. Each point represents the mean f SE (n = 8). The solid triangle represent waterinjected controls. The peak was shown to be significant by ANOVA (P < 0.05). Water injected controls gave 0 increase.

Age response curve hyperglycaemia within 30 min (Fig. 3). The maximal hyperlipaemic and hyperglycaemic occurred within 45-60min after injection followed by a decline to control levels within 2.5 h for blood sugars and within

4 h for blood lipids. This indicated a quicker response and recovery time for blood sugars compared to lipids. Water-injected control values showed no change in lipid or sugar titre at any time after injection. Injecting 1 pmol octopamine caused a 50% activation of fat body phosphorylase in only 2.5 min (Fig. 5). A peak activation of about 80% (nonligated) or about 60% (ligated) occurred between 5-15 min after injection (ANOVA, P < 0.05), the percentage activation gradually declined over the next 90min. The phosphorylase activation of untreated animals was 12.0 f 1.7 (n = 6), which was not different (t-test, P > 0.1) from the 13.2 + 2.0 (n = 8) activation with water-injected animals.

;;

100

a

60

m z

60

6 z 4

40

a-

Newly emerged adults (day 0) did not exhibit a lipaemic or a glycaemic response to octopamine, but a response rapidly developed on the first and second days after the 6nal ecdysis (Fig. 4). The maximal responsiveness of both lipids and sugars remained high and unchanged from days 4-8, then the response to octopamine declined to a lower and unchanging level through day 16 (the last day of the experiment). The responses of 4-8-day-old adults were significantly different from the responses of the IO-16-dayold adults (P < 0.05). Duncan’s Multiple Range Test, which groups points that are not significantly different, associated zero-day responsiveness with that of water-injected controls. Effects of other neurotransmittors on the hyperlipaemic response. Norepinephrine injections elevated

the blood lipids about 65% as much as octopamine injections and dopamine only by 20% (Table 4), whereas neither epinephrine nor synephrine caused

1

T

r

I

ZJI

z ?

z 0.4 0.6 0.2 z.P 1.0 gm 9 cn

20

E oleoo 0

10 20 30 40 50 60 Octopamine dose (nmol)

Fig. 2. Phosphorylase dose-response curve of neck-ligated (closed circle-solid line) and non-ligated (open circledashed line) 4-6-day-old female Acheta to 1~1 of O-106 nmol octopamine. The response was measured as the percentage of phosphorylase a (active form) present in the fat body 15 min after injection. Each point represents the mean + SE of 3-5 replicates of homogenates from 10 treated crickets.

l

0-0

0

2 4 6 6 10 12 14 Female age in day6

16 16

O

Fig. 4. Age-dependent hyperlipaemic response (closed circle-solid line) and hyperglycaemic response (open circledashed line) of female Acheta to iniections of 1 rcmol octopamine.’Water-injected controls gave no response.‘Each point represents the mean + SE (n = 10). The responses for days 4-8 are significantly different from days O-l and days lo-16 (Duncan’s Multiple Range Test, P < 0.05).

Oetopamine mobilization of sugars and lipids

0

5

10

Minutes

15

20

25

30

after injection

Fig. 5. Time course of activation of fat body phosphorylase in neck-ligated (closed circle) and non-ligated (open circle) 4-6-day-old female Achefa to injections of 1 pmol of octopamine. Response was measured as percentage of phosphorylase (I (active form) in the fat body over a 1 h time period. The solid triangles represent water injected controls. Each point represents the mean f SE of 3-5 replicates of homogenates of 10 individuals.

any significant change in haemolymph (Duncan’s Multiple Range Test, P < 0.05).

Injection of the phosphodiesterase inhibitor caffeine caused a small (12%) increase in plasma lipids, also implying a role of CAMP as second messenger in lipid mobilization. Injection of the phosphorylase inhibitor flavin mononucleotide reduced the hyperlipaemic effect of octopamine from 50 to 14% and completely eliminated the hyperglycaemic response (Tables 2 and 5), which implies that phosphorylase a is involved in both lipid and sugar mobilization in Acheta. In Periplaneta octopamine activated fat body phosphorylase a, where there was a hyperglycaemic response (Van Marrewijk et al., 1983) however, in Locusta octopamine did not activate the phosphorylase system even though it did produce a rapid short-term hyperlipaemia (Van Marrewijk et al., 1983).

DISCUSSION

lipids

Efects of adrenergic inhibitors on the hyperlipaemic with the alpha-adrenergic response. Pretreatment

receptor antagonist phentolamine before the injection of octopamine inhibited both the hyperlipaemic and hyperglycaemic response to octopamine (Table 3). Pretreatment with the beta-adrenergic receptor antagonist propranolol inhibited neither the hyperlipaemic nor the hyperglycaemic response (Table 3). Acheta octopamine receptors therefore show characteristics similar to octopamine receptors in other insects (Nathanson, 1979; O’Shea and Evans, 1979; Gole and Downer, 1979; Orchard et al., 1982). Egect of cyclic nucleotide analogue and inhibitors.

Injection of dibutyryl CAMP caused an increase in plasma lipids and sugars (Table 5). This suggests that CAMP is involved in the mobilization of both lipids and sugars. These results are consistent with the hyperlipaemia induced by injections of dibutyryl CAMP in Locusta (Gade and Holwerda, 1976) and with the hyperglycaemia induced by injections of theophylline and dibutyryl CAMP in Periplaneta (Hanaoka and Takahashi, 1977). Dibutyryl cGMP caused no change in plasma lipid or sugar titre.

197

By employing head-ligated crickets in this study, any involvement of adipokinetic hormone was eliminated and the effects of injected octopamine could be ascribed to octopamine alone. The head ligation also eliminated the stress induced elevation of blood sugars and lipids that occurs in many insects. The stress response is mediated by octopamine released from the corpus cardiacurn (Downer, 1980; Orchard et al., 1981; Woodring et al., 1989b). Stressed Acheta (unligated) showed a maximal increase in blood sugar of 15%, which was also the maximal increase resulting from injection of octopamine into head-ligated crickets. The 15% blood sugar increase could not be exceeded by any combination of stress or repeated injections of octopamine. This limit of response could indicate a limit on the number of receptor sites or a feedback inhibition of octopamine action on fat body cells induced by a maximal sugar level. The results of this study demonstrate that octopamine injected into head-ligated crickets mobilizes both lipids and sugars by binding to alpha adrenergic type receptors and activating the phosphorylase system via CAMP.

Table 5. Effectsof cyclic nucleotide analogues and inhibitors on plasma lipid and sugar concentrations in head-ligated 4-5-day-old female Acheia

Change in Treatment

n

lipid cont. (mgim))

Dist. water Dibutyryl CAMP Dibutyryl cGMP Caffeine Flavin mononucleotide FMN + OCT

19 8 7 7 7 8

0.04 f 0.15” 5.13 _+0.88b 0.00 * 0.55” 2.56 + 0.74’ 0.03 + 0.18” 3.00 + 0.77’

Change in sugar cont. (mg/ml) 0.03 + 0.09” 0.77 * 0.14b 0.02 * 0.05” 0.33 + 0.12’ 0.06 &-0.07a 0.05 * 0.11”

Values (changes) sharing the same letters are not significantly different (Duncan’s Multiple Range Test, P < 0.05). Changes were measured 60 min after injection. The dose of DcAMP, DcGMP and and Oct. was 100 nmol; caffeine 1 pmol; FMN (flavin mononucleotide) 10 nmol.

198

P. E. FIELDSand J. P. WCODRJNG

Time-dose responses The response of Acheta to octopamine

is unusual because both lipids and sugars are elevated, whereas in other insects injected octopamine produced either hyperlipaemia or hyperglycaemia but not both. The maximal lipid increase (50%) and sugar increase (15%) of Acheta to octopamine was similar to those found in other insects. In Locusta the lipids were elevated in response to octopamine by about 50% (Orchard et al., 1981) and in Periplaneta the sugars were elevated in response to octopamine by about 15% (Downer, 1979, 1980). Not only is 50% the maximal lipid increase for octopamine but 50% is also the maximal response caused by injections of the cricket’s own adipokinetic hormone. This is interesting because octopamine and adipokinetic hormone activate different receptors on the fat body cells (Nathanson, 1979; Orchard, 1982). The same limit of lipid increase (by either adipokinetic hormone or octopamine) indicates an inhibition of lipid mobilization acting at some point in the mobilization pathway beyond the hormonereceptor level that is responding to maximal blood lipid titre. Comparison of the ED, for lipid and sugar release showed that the lipid-mobilizing pathways was about 10 times more sensitive to octopamine than the sugar-mobilizing pathway. Since the same receptor (alpha adrenergic) is being used for lipid and sugar mobilization by octopamine, the differing sensitivity must be based on reactions past the receptor-hormone interaction. The time course of lipid response to octopamine in Acheta was much shorter (1 h; Fig. 3) than the time course of response to the cricket’s own adipokinetic hormone (2 h; Woodring et al., 1990). Such differences could be expected since this hormone and octopamine bind to different receptors. The reason for the quicker hyperglycaemic response to octopamine compared to the hyperlipaemic response however remains unclear. Octopamine induced a rapid activation of fat body phosphorylase a in crickets, which peaked much sooner (which it should) than the peak of lipid/sugar mobilization. Though octopamine can be expected to be present in the corpora cardiaca, as reported in Locusta (Pannabecker and Orchard, 1986), corpora cardiaca extracts did not elevate blood sugars. Probably the amount of octopamine present in the 0.1 corpora cardiaca equivalents we injected was too low to elicit a sugar response. Age dependence

An age-dependent hyperlipaemic response for octopamine has not been previously reported in insects. The age-related response to octopamine is very similar to the synthetic cricket adipokinetic hormone response to age (Woodring et al., 1989a; Woodring et al., 1990) and to the adipokinetic

hormone response in Locusta to age (Van Marrewijk et al., 1984; Mwangi and Goldworthy, 1977). The similarity of age response to octopamine and adipokinetic hormone could indicate that both receptors have a similar pattern of change with age, however it is also possible that the age response pattern could be due to similar age patterns of metabolism or clearance of hormones. A correlation between ovarian development and the sugar and lipid responsiveness to octopamine injections indicated a functional relationship of octopamine responsiveness to reproduction. Accumulation of eggs began at about 2 days after the final ecdysis reaching a peak by day 8-10. By day 12 egg production in virgin females begins to decline as the abdomen filled with eggs (Clifford and Woodring, 1986). The lipid and sugar responsiveness also peaked about day 8 and declined by 50% by day 12 (Fig. 4). An elevated responsiveness of the fat body to hyperlipaemic agents (both octopamine and adipokinetic hormone) during increasing egg production makes sense. As egg production wanes the need for lipid mobilization declines, and a gradual reduction in the responsiveness of the fat body to hyperlipaemic agents also makes sense. It has however never been proven that changed fat body responsiveness to octopamine result from or cause fat body or ovarial tissue changes. Effects of other neurotransmitters

The sequence of neurotransmitter effectiveness for hyperhpemic response in Acheta was: octopamine > norepinephrine > dopamine. Dopamine and norepinephrine were also shown to induce lipid release from the fat body of locusts, but, as in the locust, they were much less effective than octopamine (Orchard et al., 1982). Downer et al., (1984) determined through additivity studies in Periplaneta that dopamine acted through a separate receptor from octopamine located on the corpus cardiacurn. Norepinephrine probably exerts its hyperlipaemic effect in crickets through binding with octopaminergic receptors, as indicated by comparative binding studies with firefly lanterns (Nathanson, 1985). Our observation that epinephrine has no lipid mobilizing capacity in crickets is contrary to evidence of such action on the fat body of the cockroach and silkmoth (Bhakthan and Gilbert, 1968). However this variation in species response is consistent with the variations in binding of octopamine agonists in different species reported by Nathanson (1985). REFERENCES Barnes H. and Blackstock J. (1973) Estimation of lipids in marine animals and tissues: detailed investigation of the sulfophosphovanillin method for total lipids. J. exp. Mar. Eiol. 12, 103-118.

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