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The effect of flight on the composition of haemolymph in the cockroach, Periplaneta americana
J. lnsecr Physiol. Vol. 32, No. 7,
pp.649-655, 1986
OOZZ-1910186 $3.00+ 0.00 Pergamon Journals Ltd
Printed in Great Britain.
THE EFFECT OF FLIGHT ON THE COMPOSITION OF HAEMOLYMPH IN THE COCKROACH, PERIPLANETA AMERICANA L. E. KING, J. E. STEELE* and S. W. BAJURA Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7 (Received 19 August 1985; revised 21 November 1985) Abstract-The composition of the haemolymph in Periplaneta americana is markedly altered during the 6 h followine a IO-min flight. Normal resting conditions are not re-established for at least 24 h. The haemolympcvolume increases 30% between l&d 6 h after flight while the concentration of Na+, remains constant. Within 2 min after the beginning of flight there is a significant increase in the concentration of K+ followed by an increase in Ca2+ and Mg 2+ at the time flight is terminated. The increase in volume of the haemolymph is accompanied by a 25% decrease in osmolarity. Twenty-four hours after flight both haemolymph volume and osmolarity are re-established at the pre-flight values. During the return of fluid volume and osmolarity to normal levels, the ion concentrations, with the possible exception of Na+, are kept constant. The concentration of trehalose increases 10% during the first 2 min of flight, then decreases to the resting level within 5min after flight ceases. Twenty minutes after flight the concentration of trehalose rises again, to 35% above the resting level where it remains for at least a day. At 1 day after flight the total trehalose in the haemolymph pool has returned nearly to the pre-flight level. Key Word Index:
American
cockroach,
Periplaneta
americana,
haemolymph,
osmolarity,
ions,
trehalose, octcspamine
INTRODUCTION
although there is no change in the level of glucose. These changes in the concentration of trehalose during flight cannot be accounted for by a change in haemolymph volume although this factor must sometimes be taken into consideration. For example, the haemolymph volume in Rhodnius prolixus flown to exhaustion is decreased by 50% (Gringorten and Friend, 1979), whereas that in Locusta migratoria does not change during flight (Beenakkers, 1973). These observations show that flight does not produce effects common to all species. In this study we show that short-duration flight in the cockroach, Periplaneta americana has effects different from those previously described. These include a protracted rise in the concentration of haemolymph trehalose during the 6 h following flight coincident with an increase in haemolymph volume and a decrease in its osmolarity.
The act of flight in insects sets in motion a complex
set of interactions between the flight musculature and fat body. This relationship reflects the importance of the fat body as a source of energy needed to sustain flight. The role of the fat body in flight metabolism, particularly with respect to hormonal control, has been extensively investigated in the locust and an exhaustive review of the subject has recently appeared (Goldsworthy, 1983). For obvious reasons most studies on insect flight have employed insects which are capable of sustained flight. In contrast to this situation, very little is known about the endocrine control of metabolism in insects that are capable only of weak or intermittent flight. As a prelude to studies on the possible involvement of hormones in the flight metabolism of cockroaches we investigated the effect of flight on various haemolymph characteristics. Since the haemolymph is interposed between the fat body and flight muscles any change in the composition of the haernolymph due to flight may provide useful information on the activities of the fat body and musculature. Energy reserves in the haemolymph are noticeably affected by flight. During the first 30 min of flight the trehalose concentration in Schistocerca gregariu
MATERIALS AND Insects and materials
tains a steady state value equal to half the resting level. In Phormia regina, flight causes a precipitous decline in trehalose from 58 to 15 mM at which concentration flight ceases (Clegg and Evans, 1961) should be addressed.
male American cockroaches (Periplanetn reared as described previously (Steele and Hall, 1985), were transferred to an incubator at 25°C and 75% r.h. 2 days prior to use. During this period the cockroaches were fed a 10% glucose solution. The cockroaches were flown at room temperature (_ 22°C) by attaching a vacuum line (a short length of 3 mm glass tubing with a rubber cuff at one end) to the pronotum of the cockroach. The use of a small fan to direct a stream of air towards the head of the cockroach usually induced flight when the legs were suspended above the substratum. After flight the cockroaches were allowed to rest in individual plastic vials without access to drinking water. RadioAdult
americana),
(Mayer and Candy, 1969) and Locusta migratoria (Jutsum and Goldsworthy, 1976) falls before it at-
*To whom correspondence
METHODS
649
L. E. KING et al.
650
isotopes were obtained from Amersham Canada Ltd, Oakville, Ont. Collection
of haemolymph
Samples of haemolymph were taken from cockroaches at rest and at the times indicated in the figures. A small puncture was made in the intersegmental membrane posterior to the base of a metathoracic leg with a fine needle. The haemolymph was taken up in a chilled haematocrit tube, the end occluded with medical plasticine, and immediately centrifuged in an haematocrit centrifuge at 327g to sediment the haemocytes and prevent clotting. The end of the tube containing the cells and plasticine was then removed and the plasma expelled onto a chilled (O’C) spot plate. It was necessary to pool the haemolymph from 2-3 cockroaches so that sufficient haemolymph was available for each assay. Haemolymph not used immediately was kept frozen. The number of pooled haemolymph samples used for the determination of each value varied from 6 to 12. Huemolymph volume
The haemolymph volume was determined for all cockroaches used in the experiments. Cockroaches were injected with 10 ~1 of 0.15 M NaCl containing 2.22 x lo5 dpm of [3H]inulin 5 min before the haemolymph sample was taken. Preliminary experiments showed that the label was uniformly distributed within this period. The cockroaches were injected between the 5th and 6th abdominal sclerites. Ten ~1 of the pooled haemolymph sample was transferred to a scintillation vial containing 0.5 ml of NCS tissue solubilizer (Amersham) which had been diluted to contain 10% water. The samples were digested at 45°C for 1 h and 10 ml of scintillation medium (Omnifluor, New England Nuclear) then added. The samples were counted in a Unilux II (Nuclear Chicago) scintillation spectrometer and corrected for quenching using the channels ratio method. Trehalose measurement
Two ~1 of haemolymph was deproteinized by addition of 98 ~1 of 80% ethanol. Seventy-five ~1 of the protein-free supernatant was transferred to a stoppered centrifuge tube and the sample dried at 85°C with a stream of dry nitrogen. The samples were converted to the trimethylsilylimidazole derivative for determination by gas chromatography as described by Steele and Hall (1985). Osmotic pressure determination
Approximately 2~1 of haemolymph was drawn into an haematocrit tube which was then sealed with Vaseline and the sample quick frozen on dry ice. The osmotic pressure of the sample was determined cryoscopically by the comparative melting point method (Gross, 1954) using a 25% ethanol bath initially chilled to -3°C. Appropriate standards accompanied each analysis. Values for the unknown samples were obtained by interpolation from standard curves prepared from NaCl solutions in the range of lO&lOOO mOs. Ion determination
Nat,
K+, Ca*+ and Mg?+ were determined
by
atomic absorption spectrophotometry. Ten ~1 of haemolymph plasma was diluted to a final volume of 5 ml with a solution containing 25 mM LaCl,, 4 mM CsCl, and 40 mM H,SO, for the measurement of K +, Ca2+ and Mg’+. For Na +, 1 ml of the diluted plasma was further diluted 1: 10 with the lanthanumcesium solution. The lanthanum-cesium solution was employed to overcome anionic suppression and cationic enhancement of the atomic absorption signal (Sanui and Pace, 1972). The concentration of ions in the haemolymph samples was determined by comparison with a “standard haemolymph” containing 150 mM NaCl, 10mM KCl, 10mM MgCl, and 5mM CaCl, which was treated in the same manner as the unknown haemolymph samples. The ion content of the samples was measured using a Varian-Techtron Model AA-5 atomic absorption spectrophotometer. Octopamine determination
Octopamine was measured by the method of Molinoff et al. (1969) as modified by Harmer (1976) and Danielson et al. (1977). Octopamine was enzymatically methylated by phenylethanolamine-nmethyl transferase (PNMT), using S-adenosylL-[methyl-3H]methionine ([3H]SAM) as the methyl donor, to yield synephrine. PNMT was prepared in our laboratory from bovine adrenal glands obtained from a local abattoir according to the method of Molinoff et al. (1977) as modified by Harmer (1976). Haemolymph samples used for the determination of octopamine were obtained separately from those used for the measurement of other parameters. Haemolymph from 2-7 cockroaches was pooled to provide a minimum of 100 ~1. Five volumes of 0.1 M Tris-HCl buffer, pH 8.6, containing 1 mM pargyline and 100 pg fusaric acid per ml, was added to the haemolymph and the mixture heated for 5 min in a boiling water bath. After centrifugation for 15 min at 12,OOOg, 150~1 aliquots of the supernatant were mixed with 50~1 of the PNMT-[3H]SAM mixture (20 ~1 0.1 M Tris-HCl buffer, pH 8.6, 30 ~1 PNMT and 0.5 ~1 [3H]SAM [0.5 PCi, 0.05 nmol]). The reaction was stopped after 45 mm incubation at 37°C by the addition of 300 ~1 of NaCl saturated 0.5 M Na borate buffer, pH 11.O containing 100 fig synephrine per ml. Five ml of ethyl acetate were added, the samples mixed by vortexing for 1 min and then centrifuged at 1OOg for 5 min. One-half ml samples of the ethyl acetate extract were placed in conical centrifuge tubes and dried at 50°C under a stream of nitrogen. The residue was dissolved in 100~1 methanol containing 2Opg of carrier synephrine and the entire volume applied to the preadsorption region of a Linear-K TLC plate (Whatman LK6D, Clifton, N.J.). The plates were developed for 5 h at 23°C using tert-amyl alcohol-25% aqueous methylamine solution, 4: 1 (v/v), as the developing solvent. The plates were dried at 60°C for 5 min and the synephrine visualized under ultra-violet light. The quenched areas were removed and eluted with 1 ml of methanol for 30 min. Radioactivity was determined as described above (haemolymph volume). Blanks and standards were prepared from 0.1 M Tri-HCl buffer solution (pH 8.6) containing 1 mM pargyline and 1OOpg ml-’ of fusaric acid, and buffer solution containing 1 pg of
651
Effect of flight on haemolymph
rx-octopamine respectively. Internal standards prepared by adding 1 ng of rx-octopamine sample of tissue homogenate.
were to a
Statistical analysis The differences berween controls (0 time) and flown insects for each hacmolymph parameter measured were tested using a. Model I analysis of variance [ANOVA] (Sokal and Rohlf, 1973) and an F-test of significance. A Pearson product momentum analysis of correlation (Sokal and Rohlf, 1973) between haemolymph volume, total trehalose and osmotic pressure was also performed by Minitab on a Cyber 73 computer.
RESULTS
Haemolymph volume The mean volume of haemolymph in the cockroach increases with time after flight (Fig. 1). The increase in haemolymph volume due to 10 min flight only becomes significantly different from the controls (0 time) when the cockroaches have been allowed to rest for 50 min. Neverth’eless, an upward trend is evident 20 min after the completion of flight. The volume of haemolymph found 1 and 6 h after flight are both significantly greater (F = 14.8, P < 0.01) than that found in the unflown controls. Furthermore, unflown controls which have been mounted but not removed from the substratu.m showed no change in haemolymph volume compared to the unmounted (0 time) controls. The absolute increase in haemolymph volume due to flight. (measured 6 h after flight) averaged 33 ~1, an increase of 26%. Twenty-four hours after flight the volume of haemolymph had returned to the resting value. Osmotic pressure During the first 2 min of flight there is a small but significant (P < 0.05) increase in the osmolarity of the
jr Oo
$0
A
I5
50
.A.
”
6h
“24h
Time Cn~i~~
Fig. 2. The decrease in haemolymph osmotic pressure following flight, The wide bar on the abscissa indicates the maximal period of tethered flight. The values shown are the mean + the standard error of the mean (n = 12). The F value of 19.9 indicates that the decrease in osmolarity is significant (P < 0.01). The open circle at 6 h represents the haemolymph osmotic pressure in tethered but unflown cockroaches.
haemolymph (Fig. 2). Thereafter the osmolarity shows a steady decline, reaching a minimum value 6 h after flight is completed (F = 19.9, P < 0.01). At 6 h the osmotic pressure was decreased by 73 mOs but at 24 h had returned to the resting level. The decreasing osmolarity shows a significant correlation (r = -0.46, P < 0.05) with the increasing volume of haemolymph (Fig. 1). Trehalose concentration During the first 2 min of flight the concentration of trehalose increases from 49.1 to 55.3 mM (Fig. 3). This increase is significant (P < 0.05) but of relatively short duration so that 5 min after flight is terminated the level of trehalose has returned to the resting level. It is not known whether the level of trehalose remains high throughout the period of flight or decreases before the cessation of flight. Shortly after flight ceases haemolymph trehalose increases once again
Fig. 1. The change in haemolymph volume due to flight as a function of time. The wide bar on the abscissa between 0 and IO min shows the maximal duration of tethered flight. The values shown are the mean &-the standard error of the mean (n = 12). The Fvalue of 14.8 indicates that the volume of haemolymph at 1 and 6 h after flight is greater (P < 0.01) than that of unflown (0 min) cockroaches. The solid circle at 6 h shows the haemolymph volume in cockroaches treated identically to flown insects except that flight was prevented by maintaining contact of the tarsi with substratum.
1.P. 32.‘7--E
Fig. 3. The effect of short-duration flight on the concentration of haemolymph trehalose. The wide bar on the abscissa indicates the maximal duration of flight. The values shown are the mean & the standard error of the mean (n = 12). The increase in trehalose after 2 min flight is significant (P < 0.05). The increase beginning after 15 min is highly significant (F = 17.6, P < 0.01). The open circle at 6 h shows the trehalose concentration in tethered but unflown cockroaches.
652
L. E.
KING
al.
et
i
‘2-
I
IO-
%
6.
p-+,.
L
%
6-8
E4 A
15
30 Time
60 (mid
A 2O-.
.A.
”
6hr
“24hr
Fig. 4. The effect of short duration flight on total haemolymph trehalose. The wide bar on the abscissa indicates the maximal duration of flight. The mean values for the total trehalose in the haemolymph are shown but without standard error bars since all standard errors lie within the confines of the symbols (n = 12). The increase in total trehalose following flight is significant (F = 11.8, P < 0.01). The open circle at 6 h shows the trehalose content in tethered but unflown cockroaches.
but this time attains a considerably ensuing 15 min and then remains
higher
level in the
constant for the next 24 h (Fig. 3). This increase is highly significant (F = 17.6, P < 0.01). The constant level of trehalose in the haemolymph between 30 min and 6 h after flight does not reflect a constant pool of trehalose during this period since the volume of haemolymph is gradually increasing. Thus the total trehalose pool continues to increase despite the fact that the concentration remains constant from 30 min onward following flight. This is more clearly seen in Fig. 4 where the size of the trehalose pool is plotted against time. When compared to the resting level the increase in total trehalose is significant (F = 11.8, P < 0.01). Furthermore, a highly significant correlation (r = 0.804, P < 0.01)exists between the increasing volume of haemolymph and the increasing amount of trehalose contained in it. Twenty-four hours after flight the concentration of trehalose is still high, however, the total amount of trehalose is
0
K+ n 15
30
’ Time
IAS 6hr v24hr
I
bnin6p
Fig. 6. The K+ concentration of haemolymph during and following a short period of flight. The maximal duration of flight is indicated by the wide bar on the abscissa. The values shown are the means k the standard error of the means and the numbers adjacent to the symbols indicates the number of samples.
down although cockroach.
not to the level found
in the resting
Ion concentrations Interestingly, the concentrations of Na+, K +, Ca’+ and Mg2+, the four major ions in haemolymph, appear to be altered by flight although the changes do not occur simultaneously. The concentration of Na+ does not change significantly until 24 h after flight when it increases (Fig. 5) in concert with the decrease in haemolymph volume (Fig. 1). Very shortly after flight begins the K+ concentration rises and is then held constant (Fig. 6). Ca2+ and Mg’+ on the other hand do not increase until shortly after flight has ceased (Figs 7-8). Octopamine Octopamine was measured in haemolymph drawn from cockroaches after different periods of flight and flight plus rest (Fig. 9). The data show that the octopamine concentration is initially high and declines not only during the 10 min flight period but also during the period of rest which follows flight. In
+
I5
n
30 Time
60 (mid
”
,A* 6hr
Fig. 5. The Na+ concentration of haemolymph folIowing a short period of flight. The maximal flight is indicated by the wide bar on the abscissa. shown are the means f the standard error of the the numbers adjacent to the symbols indicates of samples.
A
v24hr Tim*
during and duration of The values means and the number
60 (min)
”
IA,
6hr
“24hc
Fig. 7. The Ca2+ concentration of haemolymph during and following a short period of flight. The maximal duration of flight is indicated by the wide bar on the abscissa. The values shown are the means k the standard error of the means and the numbers adjacent to the symbols indicates the number of samples.
Effect of
flight on haemolymph
# ““.”
Fig. 8. The Mg*+conszentration of haemolymph during and following a short period of flight. The maximal duration of flight is indicated by the wide bar on the abscissa. The values shown are the means k the standard error of the means and the numbers adjacent to the symbols indicates the number of samples.
a parallel experiment the change in haemolymph volume due to 10 nun of flight followed by 50 min of rest was determined. At 0 time (unflown) the haemolymph volume was 118.6 f 8.0 ~1 increasing to 176.6 + 4.7 ~1 after 50min rest. This represents a 49% increase (P < 0.001) which is considerably greater than that shown in Fig. 1. DISCUSSION
This study describes some effects of flight on the composition of cockroach haemolymph that are markedly different from those described for other species. These effects include an increase in haemolymph volume and an increase in the concentration of trehalos#e. Since the cockroach is not a strong flier and was induced to fly for a longer than normal period it is possible that the effects noted represent a response to stress rather than a normal physiological condition associated with flight.
SOL-7
fp,I e
3701. B
e500
.\-\ t
Tim@ (mlnl
Fig. 9. The effect 01‘ flight on octopamine concentration in the haemolymph. Haemolymph was obtained from cockroaches which had flown for 2. 10 or IO min followed by 50min rest. Zero time values were obtained from cockroaches which had not been flown. Octopamine was measured as describeId in the Materials and Methods. Each point represents the mean of not less than 5 samples, each sample consisting of haemolymph pooled from at least 5 cockroaches. The values shown are the mean k standard error of the mean.
653
The increase in haemolymph volume resulting from flight differs from the findings reported by Gringorten and Friend (1979) who showed that Rhodnius prolixus flown to exhaustion lost fluid from the haemolymph. Locusta migratoria on the other hand, which is a strong flier, maintains a constant volume of haemolymph during flight (Beenakkers, 1973). The maximal increase in haemolymph volume in the cockroach after flight was 26% in the first series of experiments, and 49% in the second series used for the measurement of haemolymph octopamine. This may reflect a larger initial haemolymph volume in the first group due to a greater hydration of those cockroaches before use. The explanation for the increase in haemolymph volume is unclear but it is not unreasonable to suggest that it may be hormonal in origin. Indeed, it is possible that the release of such a hormone might be triggered by the increase in osmolarity that occurs shortly after flight begins. Alternatively, the increase in K+ might cause release of the hormone by depolarizing the membrane of the cells containing the hormone. Both Wall (1967) and Steele and Tolman (1980) have demonstrated the presence of antidiuretic factors in the neuroendocrine system of Periplaneta americana although the mechanisms controlling the release of the hormone have not been described. Extracts of the corpora cardiaca which contain the antidiuretic hormone have been shown to stimulate the reabsorption of water from the rectum (Wall, 1967; Steele and Tolman, 1980). However, water uptake from the rectum alone is unlikely to explain the increase in haemolymph volume since it contains not more than 3 or 4 ~1 of fluid. Nevertheless, uptake of water from the rectum cannot be ruled out since it may be replenished by water from the anterior region of the gut. Another possible source of water is the salivary glands. Salivary glands in hydrated cockroaches may contain up to 100 ~1 of fluid and this can be reabsorbed during dehydration (Sutherland and Chillseyzn, 1968). Whether this is hormonally regulated and can occur in the short period necessary to explain the present results is not known. Despite the increase in the concentration of trehalose following flight the decreasing osmolarity during this period clearly shows that the increase in fluid volume cannot be attributed to an increase in solute concentration. The question is whether the increase in fluid volume signifies a temporary failure in osmoregulation or a useful adaptation of importance to the recovery from flight. The latter possibility seems more likely since it would allow the accumulation of greater food reserves in the haemolymph and facilitate exchange between the musculature, fat body and gut. In contrast to the changing fluid volume of the haemolymph the concentration of Na*, K+, Ca*+ and Mg2+ appears to be tightly controlled during the period of increasing haemolymph volume, despite increases in the three latter ions soon after flight begins. This ionic constancy agrees with the findings of Edney (1967), Wall (1970) and Tucker (1977a and b) for dehydrated and rehydrated cockroaches. On balance the data offer a strong argument for hormone-mediated increases in haemolymph volume during flight. This may be a mechanism common to all flying insects but more apparent in weak fliers
654
L. E. KING et al.
when the hormone release mechanism is stimulated excessively. Trehalose exhibits a biphasic response to flight; a small increase (- 10%) during the early stage of flight which disappears after the first few minutes of rest, to be followed by a greater increase (-30%) which is maintained for the next 24 h. The finding that trehalose levels increase is surprising since flight, in those species where it has been examined, is usually associated with a decrease in the concentration of trehalose (Clegg and Evans, 1961; Jutsum and Goldsworthy, 1976; Van der Horst et al., 1978). The nature of the stimulus which provokes an initial weak increase followed by a second strong increase in haemolymph trehalose is of particular interest. The stress effect, described by Matthews and Downer (1974) and Downer (1979) as being responsible for a modest increase in trehalose, probably is the cause of the increase in trehalose which occurs during the first few minutes of flight since both are of the same magnitude. The evidence suggests that the rise in trehalose which occurs with the onset of flight may be due to the release of octopamine into the haemolymph. When octopamine is injected into cockroaches to yield a final concentration in the haemolymph of 10m6 M-10-j M there is a 100% increase in the level of circulating trehalose (Downer, 1979). Furthermore, octopamine has been shown to stimulate the release of trehalose from fat body in vitro (Gole and Downer, 1979). To complete the cycle of events Bailey et al. (1983) have shown that flight in Periplaneta americana causes an increase in the concentration of octopamine in the haemolymph. These results have, in general, been confirmed by the octopamine measurements obtained in the present study. The secondary increase in haemolymph trehalose differs from the initial increase in two important respects. Firstly, a considerably larger amount of trehalose is released to the haemolymph and secondly, the duration of the hypertrehalocaemic response is many hours longer than that associated with the first few minutes of flight. In all probability the second hypertrehalocaemic response to flight results from the release of hypertrehalocaemic peptides which are present in the corpus cardiacuun (Steele, 1963) although this remains to be proven. Certainly the protracted hypertrehalocaemic state observed after flight is similar to that which follows the injection of corpus cardiacum extract (Steele, 1963). Since flight lasting only 10min causes 75% depletion of glycogen in the flight musculature of Periplaneta americana (Downer and Matthews, 1976) it is possible that the post-flight increase in haemolymph trehalose is important in the resynthesis of muscle glycogen. We suggest that the release of the corpus cardiacum hormones is neurally controlled by mechanisms linked to the expenditure of energy (flight). This view is supported by the finding that trehalose in the haemolymph of Calliphora erythrocephala can only be maintained at a consant level during flight if the nervous connections between the brain and corpus cardiacurn are intact, otherwise trehalose declines to the point where flight is impossible (Vejbjerg and Normann, 1974). The importance of the neural connections for the release of hypertrehalocaemic hormone in CaNiphora erythrocephala
is shown by the fact that hormone release does not occur in response to electrical stimulation of the brain if the nerves are severed (Normann and Dave, 1969). The accumulating evidence, although falling short of proving that the hypertrehalocaemic hormone maintains the level of trehalose in the resting insect, suggests that it may do so in the working insect. Experiments are currently under way to determine whether the titre of hormone in the haemolymph increases as a result of flight. The octopamine measurements, although similar to those obtained by Bailey et al. (1983) as far as the decrease accompanying rest after flight is concerned, differ in one important aspect. Our measurements do not show the low resting (0 time) values obtained by the other workers. This is because our data, which were obtained before the study by Bailey et al. (1983) appeared, did not take into consideration the role of stress in the release of octopamine and precautions were not taken to isolate the cockroaches before use. Our study, like that of Bailey et al. (1983) suggests that efficient mechanisms exist for the clearance of octopamine from the haemolymph although the increase in haemolymph volume also has a role in decreasing the concentration of the amine. Acknowledgement-This study was supported, in part, by the award of a grant to J.E.S. from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES Bailey B. A., Martin R. J. and Downer R. G. H. (1983) Haemolymph octopamine levels during and following flight in the American cockroach, Periplaneta americana. Can. J. Zool. 62, 19-22. Beenakkers A, M. T. (1973) Influence of flight on lipid metabolism in Locusta migratoria. Insect Biochem. 3, 303-308. Clegg J. S. and Evans D. R. (1961) The physiology of blood trehalose and its function during flight in the blowfly. J. exp. Biol. 38, 771-792. Danielson T. J., Boulton A. A. and Robertson H. A. (1977) m-Octopamine. p-octopamine and phenylethanolamine in rat brain: a sensitive, specific assay and the effects of some drugs. J. Neurochem. 29, 1131-1135. Downer R. G. H. (1979) Induction of hypertrehalosemia by excitation in Periplaneta americana. J. Insect Physiol. 25, 59-63. Downer R. G. H. and Matthews J. R. (1976) Glycogen depletion of thoracic musculature during flight in the American cockroach, Periplaneta americana L. Comp. Biochem. Physiol. SSB, 501-502. Ednev E. B. (1968) ~ , The effect of water loss on the haemolymph of Arenivaga sp. and Periplaneta americana. Comp. Biochem. Physiol. 25, 149-158. Goldsworthy G. J. (1983) The endocrine control of flight metabolism in locusts. Adv. Insect Physiol. 17, 149-204. Gole J. W. D. and Downer R. G. H. (1979) Elevation of adenosine 3’,5’-monophosphate by octopamine in fat body of the American cockroach, Periplaneta americana L. Camp. Biochem. Physiol. 64C, 223-226. Gringorton J. L. and Friend W. G. (1979) Haemolymph volume changes in Rhodnius prolixus during flight. J. exp. Bioi. 83, 325-333. Gross W. J. (1954) Osmotic responses in the sipunculid Dendrostomum zoskricolum. J. exp. Biol. 31, 402423. Harmer A. J. (1976) The Neurochemistry and Pharmacology of Octopamine. Ph.D. Thesis, University of Cambridge, England.
Effect of flight on haemolymph Jutsum A. R. and Goldsworthy G. J. (I 976) Fuels for flight in Locusta. J. Inseci Physioi. 22, 243-249. Matthews J. R. and Downer R. G. H. (1974) Origin of trehalose in stmss-induced hyperglycaemia in the American cockroach, Periplanela americana. Can. J. Zool. 52, 1005-1010. Mayer R. J. and Candy D. J. (1969) Control of haemolymph hnid concentration ~durina locust flight: an adipokinetic hormone from the corpora cardiaca. 7. Insect Piysiol. 15, 61 l-620. Molinoff P. B., Landsberg L. and Axelrod J. (1969) An enzymatic assay for octopamine and other /I-hydroxylated phenylethylamines. J. Pharmac. exp. Ther. 170, 253-261. Molinoff P. B., Weinshilboum R. and Axelrod J. (1971) A sensitive enzymatic assay for doaamine-P-hydroxylase. J. Pharmac. exp. Ther. 178, 425431. Normann T. C. and Dave H. (1969) Experimentally induced release of a neurohormone influencing haemolymph trehalose level in ICalliphora erythrocephala (Diptera). Gen. camp. Endow. 12, 449459. Sanui H. and Pace N. (1972) An atomic absorption method for cation measurements in kjeldahl digests of biological materials. Analyr. Biochem. 47, 57-66. Sokal R. R. and Rohlf F. J. (1973) Infroduction to Biosratistics. W. H. Freeman, San Francisco. Steele J. E. (1963) The site of action of insect hyperglycaemic hormone. Gen. camp. Endocr. 3, 4c-52. Steele J. E. and Hall S. (1985) Trehalose synthesis and
655
glycogenolysis as sites of action for the corpus cardiacum in Periplaneta americana. Insect Biochem. 15, 529-536. Steele J. E. and Tolman J. H. (1980) Regulation of water transport in the cockroach rectum by the corpora cardiaca*orpora allata system: The requirement for Na+. J. romp. Physiol. 138, 357-365. Sutherland D. J. and Chillseyzn J. M. (1968) Function and operation of the cockroach salivary reservoir. J. Insect Physiol. 14, 2 l-3 I. Tucker L. E. (1977a) Effect of dehydration and rehydration on the water content and Na+ and K+ balance in adult male Periplaneta amerieana. J. exp. Biol. 71, 49-66. Tucker L. E. (1977b) Regulation of ions in the haemolymph of the cockroach Periplaneta americana during dehydration and rehydration. J. exp. Biol. 71, 95-110. Van Dcr Horst D. J., Van Doorn J. M. and Beenakkers A. M. T. (1978) Dynamics in the haemolymph trehalose pool during flight of the locust, Locusta migratoria. Insect Biochem. 8, 413416. Vejbjerg K. and Normann T. C. (1974) Secretion of hyperglycaemic hormone from the corpus cardiacum of flying blowflies, Calliphora erythrocephala. J. Insecr Physiol. 20, 1189-l 192. Wall B. J. (1967) Evidence for anti-diuretic control of rectal water absorption in the cockroach, Periplaneta americana. L. J. Insect Physiol. 13, 565-578. Wall B. J. (1970) Effects of dehydration and rehydration on Periplaneta americana. J. Insect Physiol. 16, 1027-1042.
pp.649-655, 1986
OOZZ-1910186 $3.00+ 0.00 Pergamon Journals Ltd
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THE EFFECT OF FLIGHT ON THE COMPOSITION OF HAEMOLYMPH IN THE COCKROACH, PERIPLANETA AMERICANA L. E. KING, J. E. STEELE* and S. W. BAJURA Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7 (Received 19 August 1985; revised 21 November 1985) Abstract-The composition of the haemolymph in Periplaneta americana is markedly altered during the 6 h followine a IO-min flight. Normal resting conditions are not re-established for at least 24 h. The haemolympcvolume increases 30% between l&d 6 h after flight while the concentration of Na+, remains constant. Within 2 min after the beginning of flight there is a significant increase in the concentration of K+ followed by an increase in Ca2+ and Mg 2+ at the time flight is terminated. The increase in volume of the haemolymph is accompanied by a 25% decrease in osmolarity. Twenty-four hours after flight both haemolymph volume and osmolarity are re-established at the pre-flight values. During the return of fluid volume and osmolarity to normal levels, the ion concentrations, with the possible exception of Na+, are kept constant. The concentration of trehalose increases 10% during the first 2 min of flight, then decreases to the resting level within 5min after flight ceases. Twenty minutes after flight the concentration of trehalose rises again, to 35% above the resting level where it remains for at least a day. At 1 day after flight the total trehalose in the haemolymph pool has returned nearly to the pre-flight level. Key Word Index:
American
cockroach,
Periplaneta
americana,
haemolymph,
osmolarity,
ions,
trehalose, octcspamine
INTRODUCTION
although there is no change in the level of glucose. These changes in the concentration of trehalose during flight cannot be accounted for by a change in haemolymph volume although this factor must sometimes be taken into consideration. For example, the haemolymph volume in Rhodnius prolixus flown to exhaustion is decreased by 50% (Gringorten and Friend, 1979), whereas that in Locusta migratoria does not change during flight (Beenakkers, 1973). These observations show that flight does not produce effects common to all species. In this study we show that short-duration flight in the cockroach, Periplaneta americana has effects different from those previously described. These include a protracted rise in the concentration of haemolymph trehalose during the 6 h following flight coincident with an increase in haemolymph volume and a decrease in its osmolarity.
The act of flight in insects sets in motion a complex
set of interactions between the flight musculature and fat body. This relationship reflects the importance of the fat body as a source of energy needed to sustain flight. The role of the fat body in flight metabolism, particularly with respect to hormonal control, has been extensively investigated in the locust and an exhaustive review of the subject has recently appeared (Goldsworthy, 1983). For obvious reasons most studies on insect flight have employed insects which are capable of sustained flight. In contrast to this situation, very little is known about the endocrine control of metabolism in insects that are capable only of weak or intermittent flight. As a prelude to studies on the possible involvement of hormones in the flight metabolism of cockroaches we investigated the effect of flight on various haemolymph characteristics. Since the haemolymph is interposed between the fat body and flight muscles any change in the composition of the haernolymph due to flight may provide useful information on the activities of the fat body and musculature. Energy reserves in the haemolymph are noticeably affected by flight. During the first 30 min of flight the trehalose concentration in Schistocerca gregariu
MATERIALS AND Insects and materials
tains a steady state value equal to half the resting level. In Phormia regina, flight causes a precipitous decline in trehalose from 58 to 15 mM at which concentration flight ceases (Clegg and Evans, 1961) should be addressed.
male American cockroaches (Periplanetn reared as described previously (Steele and Hall, 1985), were transferred to an incubator at 25°C and 75% r.h. 2 days prior to use. During this period the cockroaches were fed a 10% glucose solution. The cockroaches were flown at room temperature (_ 22°C) by attaching a vacuum line (a short length of 3 mm glass tubing with a rubber cuff at one end) to the pronotum of the cockroach. The use of a small fan to direct a stream of air towards the head of the cockroach usually induced flight when the legs were suspended above the substratum. After flight the cockroaches were allowed to rest in individual plastic vials without access to drinking water. RadioAdult
americana),
(Mayer and Candy, 1969) and Locusta migratoria (Jutsum and Goldsworthy, 1976) falls before it at-
*To whom correspondence
METHODS
649
L. E. KING et al.
650
isotopes were obtained from Amersham Canada Ltd, Oakville, Ont. Collection
of haemolymph
Samples of haemolymph were taken from cockroaches at rest and at the times indicated in the figures. A small puncture was made in the intersegmental membrane posterior to the base of a metathoracic leg with a fine needle. The haemolymph was taken up in a chilled haematocrit tube, the end occluded with medical plasticine, and immediately centrifuged in an haematocrit centrifuge at 327g to sediment the haemocytes and prevent clotting. The end of the tube containing the cells and plasticine was then removed and the plasma expelled onto a chilled (O’C) spot plate. It was necessary to pool the haemolymph from 2-3 cockroaches so that sufficient haemolymph was available for each assay. Haemolymph not used immediately was kept frozen. The number of pooled haemolymph samples used for the determination of each value varied from 6 to 12. Huemolymph volume
The haemolymph volume was determined for all cockroaches used in the experiments. Cockroaches were injected with 10 ~1 of 0.15 M NaCl containing 2.22 x lo5 dpm of [3H]inulin 5 min before the haemolymph sample was taken. Preliminary experiments showed that the label was uniformly distributed within this period. The cockroaches were injected between the 5th and 6th abdominal sclerites. Ten ~1 of the pooled haemolymph sample was transferred to a scintillation vial containing 0.5 ml of NCS tissue solubilizer (Amersham) which had been diluted to contain 10% water. The samples were digested at 45°C for 1 h and 10 ml of scintillation medium (Omnifluor, New England Nuclear) then added. The samples were counted in a Unilux II (Nuclear Chicago) scintillation spectrometer and corrected for quenching using the channels ratio method. Trehalose measurement
Two ~1 of haemolymph was deproteinized by addition of 98 ~1 of 80% ethanol. Seventy-five ~1 of the protein-free supernatant was transferred to a stoppered centrifuge tube and the sample dried at 85°C with a stream of dry nitrogen. The samples were converted to the trimethylsilylimidazole derivative for determination by gas chromatography as described by Steele and Hall (1985). Osmotic pressure determination
Approximately 2~1 of haemolymph was drawn into an haematocrit tube which was then sealed with Vaseline and the sample quick frozen on dry ice. The osmotic pressure of the sample was determined cryoscopically by the comparative melting point method (Gross, 1954) using a 25% ethanol bath initially chilled to -3°C. Appropriate standards accompanied each analysis. Values for the unknown samples were obtained by interpolation from standard curves prepared from NaCl solutions in the range of lO&lOOO mOs. Ion determination
Nat,
K+, Ca*+ and Mg?+ were determined
by
atomic absorption spectrophotometry. Ten ~1 of haemolymph plasma was diluted to a final volume of 5 ml with a solution containing 25 mM LaCl,, 4 mM CsCl, and 40 mM H,SO, for the measurement of K +, Ca2+ and Mg’+. For Na +, 1 ml of the diluted plasma was further diluted 1: 10 with the lanthanumcesium solution. The lanthanum-cesium solution was employed to overcome anionic suppression and cationic enhancement of the atomic absorption signal (Sanui and Pace, 1972). The concentration of ions in the haemolymph samples was determined by comparison with a “standard haemolymph” containing 150 mM NaCl, 10mM KCl, 10mM MgCl, and 5mM CaCl, which was treated in the same manner as the unknown haemolymph samples. The ion content of the samples was measured using a Varian-Techtron Model AA-5 atomic absorption spectrophotometer. Octopamine determination
Octopamine was measured by the method of Molinoff et al. (1969) as modified by Harmer (1976) and Danielson et al. (1977). Octopamine was enzymatically methylated by phenylethanolamine-nmethyl transferase (PNMT), using S-adenosylL-[methyl-3H]methionine ([3H]SAM) as the methyl donor, to yield synephrine. PNMT was prepared in our laboratory from bovine adrenal glands obtained from a local abattoir according to the method of Molinoff et al. (1977) as modified by Harmer (1976). Haemolymph samples used for the determination of octopamine were obtained separately from those used for the measurement of other parameters. Haemolymph from 2-7 cockroaches was pooled to provide a minimum of 100 ~1. Five volumes of 0.1 M Tris-HCl buffer, pH 8.6, containing 1 mM pargyline and 100 pg fusaric acid per ml, was added to the haemolymph and the mixture heated for 5 min in a boiling water bath. After centrifugation for 15 min at 12,OOOg, 150~1 aliquots of the supernatant were mixed with 50~1 of the PNMT-[3H]SAM mixture (20 ~1 0.1 M Tris-HCl buffer, pH 8.6, 30 ~1 PNMT and 0.5 ~1 [3H]SAM [0.5 PCi, 0.05 nmol]). The reaction was stopped after 45 mm incubation at 37°C by the addition of 300 ~1 of NaCl saturated 0.5 M Na borate buffer, pH 11.O containing 100 fig synephrine per ml. Five ml of ethyl acetate were added, the samples mixed by vortexing for 1 min and then centrifuged at 1OOg for 5 min. One-half ml samples of the ethyl acetate extract were placed in conical centrifuge tubes and dried at 50°C under a stream of nitrogen. The residue was dissolved in 100~1 methanol containing 2Opg of carrier synephrine and the entire volume applied to the preadsorption region of a Linear-K TLC plate (Whatman LK6D, Clifton, N.J.). The plates were developed for 5 h at 23°C using tert-amyl alcohol-25% aqueous methylamine solution, 4: 1 (v/v), as the developing solvent. The plates were dried at 60°C for 5 min and the synephrine visualized under ultra-violet light. The quenched areas were removed and eluted with 1 ml of methanol for 30 min. Radioactivity was determined as described above (haemolymph volume). Blanks and standards were prepared from 0.1 M Tri-HCl buffer solution (pH 8.6) containing 1 mM pargyline and 1OOpg ml-’ of fusaric acid, and buffer solution containing 1 pg of
651
Effect of flight on haemolymph
rx-octopamine respectively. Internal standards prepared by adding 1 ng of rx-octopamine sample of tissue homogenate.
were to a
Statistical analysis The differences berween controls (0 time) and flown insects for each hacmolymph parameter measured were tested using a. Model I analysis of variance [ANOVA] (Sokal and Rohlf, 1973) and an F-test of significance. A Pearson product momentum analysis of correlation (Sokal and Rohlf, 1973) between haemolymph volume, total trehalose and osmotic pressure was also performed by Minitab on a Cyber 73 computer.
RESULTS
Haemolymph volume The mean volume of haemolymph in the cockroach increases with time after flight (Fig. 1). The increase in haemolymph volume due to 10 min flight only becomes significantly different from the controls (0 time) when the cockroaches have been allowed to rest for 50 min. Neverth’eless, an upward trend is evident 20 min after the completion of flight. The volume of haemolymph found 1 and 6 h after flight are both significantly greater (F = 14.8, P < 0.01) than that found in the unflown controls. Furthermore, unflown controls which have been mounted but not removed from the substratu.m showed no change in haemolymph volume compared to the unmounted (0 time) controls. The absolute increase in haemolymph volume due to flight. (measured 6 h after flight) averaged 33 ~1, an increase of 26%. Twenty-four hours after flight the volume of haemolymph had returned to the resting value. Osmotic pressure During the first 2 min of flight there is a small but significant (P < 0.05) increase in the osmolarity of the
jr Oo
$0
A
I5
50
.A.
”
6h
“24h
Time Cn~i~~
Fig. 2. The decrease in haemolymph osmotic pressure following flight, The wide bar on the abscissa indicates the maximal period of tethered flight. The values shown are the mean + the standard error of the mean (n = 12). The F value of 19.9 indicates that the decrease in osmolarity is significant (P < 0.01). The open circle at 6 h represents the haemolymph osmotic pressure in tethered but unflown cockroaches.
haemolymph (Fig. 2). Thereafter the osmolarity shows a steady decline, reaching a minimum value 6 h after flight is completed (F = 19.9, P < 0.01). At 6 h the osmotic pressure was decreased by 73 mOs but at 24 h had returned to the resting level. The decreasing osmolarity shows a significant correlation (r = -0.46, P < 0.05) with the increasing volume of haemolymph (Fig. 1). Trehalose concentration During the first 2 min of flight the concentration of trehalose increases from 49.1 to 55.3 mM (Fig. 3). This increase is significant (P < 0.05) but of relatively short duration so that 5 min after flight is terminated the level of trehalose has returned to the resting level. It is not known whether the level of trehalose remains high throughout the period of flight or decreases before the cessation of flight. Shortly after flight ceases haemolymph trehalose increases once again
Fig. 1. The change in haemolymph volume due to flight as a function of time. The wide bar on the abscissa between 0 and IO min shows the maximal duration of tethered flight. The values shown are the mean &-the standard error of the mean (n = 12). The Fvalue of 14.8 indicates that the volume of haemolymph at 1 and 6 h after flight is greater (P < 0.01) than that of unflown (0 min) cockroaches. The solid circle at 6 h shows the haemolymph volume in cockroaches treated identically to flown insects except that flight was prevented by maintaining contact of the tarsi with substratum.
1.P. 32.‘7--E
Fig. 3. The effect of short-duration flight on the concentration of haemolymph trehalose. The wide bar on the abscissa indicates the maximal duration of flight. The values shown are the mean & the standard error of the mean (n = 12). The increase in trehalose after 2 min flight is significant (P < 0.05). The increase beginning after 15 min is highly significant (F = 17.6, P < 0.01). The open circle at 6 h shows the trehalose concentration in tethered but unflown cockroaches.
652
L. E.
KING
al.
et
i
‘2-
I
IO-
%
6.
p-+,.
L
%
6-8
E4 A
15
30 Time
60 (mid
A 2O-.
.A.
”
6hr
“24hr
Fig. 4. The effect of short duration flight on total haemolymph trehalose. The wide bar on the abscissa indicates the maximal duration of flight. The mean values for the total trehalose in the haemolymph are shown but without standard error bars since all standard errors lie within the confines of the symbols (n = 12). The increase in total trehalose following flight is significant (F = 11.8, P < 0.01). The open circle at 6 h shows the trehalose content in tethered but unflown cockroaches.
but this time attains a considerably ensuing 15 min and then remains
higher
level in the
constant for the next 24 h (Fig. 3). This increase is highly significant (F = 17.6, P < 0.01). The constant level of trehalose in the haemolymph between 30 min and 6 h after flight does not reflect a constant pool of trehalose during this period since the volume of haemolymph is gradually increasing. Thus the total trehalose pool continues to increase despite the fact that the concentration remains constant from 30 min onward following flight. This is more clearly seen in Fig. 4 where the size of the trehalose pool is plotted against time. When compared to the resting level the increase in total trehalose is significant (F = 11.8, P < 0.01). Furthermore, a highly significant correlation (r = 0.804, P < 0.01)exists between the increasing volume of haemolymph and the increasing amount of trehalose contained in it. Twenty-four hours after flight the concentration of trehalose is still high, however, the total amount of trehalose is
0
K+ n 15
30
’ Time
IAS 6hr v24hr
I
bnin6p
Fig. 6. The K+ concentration of haemolymph during and following a short period of flight. The maximal duration of flight is indicated by the wide bar on the abscissa. The values shown are the means k the standard error of the means and the numbers adjacent to the symbols indicates the number of samples.
down although cockroach.
not to the level found
in the resting
Ion concentrations Interestingly, the concentrations of Na+, K +, Ca’+ and Mg2+, the four major ions in haemolymph, appear to be altered by flight although the changes do not occur simultaneously. The concentration of Na+ does not change significantly until 24 h after flight when it increases (Fig. 5) in concert with the decrease in haemolymph volume (Fig. 1). Very shortly after flight begins the K+ concentration rises and is then held constant (Fig. 6). Ca2+ and Mg’+ on the other hand do not increase until shortly after flight has ceased (Figs 7-8). Octopamine Octopamine was measured in haemolymph drawn from cockroaches after different periods of flight and flight plus rest (Fig. 9). The data show that the octopamine concentration is initially high and declines not only during the 10 min flight period but also during the period of rest which follows flight. In
+
I5
n
30 Time
60 (mid
”
,A* 6hr
Fig. 5. The Na+ concentration of haemolymph folIowing a short period of flight. The maximal flight is indicated by the wide bar on the abscissa. shown are the means f the standard error of the the numbers adjacent to the symbols indicates of samples.
A
v24hr Tim*
during and duration of The values means and the number
60 (min)
”
IA,
6hr
“24hc
Fig. 7. The Ca2+ concentration of haemolymph during and following a short period of flight. The maximal duration of flight is indicated by the wide bar on the abscissa. The values shown are the means k the standard error of the means and the numbers adjacent to the symbols indicates the number of samples.
Effect of
flight on haemolymph
# ““.”
Fig. 8. The Mg*+conszentration of haemolymph during and following a short period of flight. The maximal duration of flight is indicated by the wide bar on the abscissa. The values shown are the means k the standard error of the means and the numbers adjacent to the symbols indicates the number of samples.
a parallel experiment the change in haemolymph volume due to 10 nun of flight followed by 50 min of rest was determined. At 0 time (unflown) the haemolymph volume was 118.6 f 8.0 ~1 increasing to 176.6 + 4.7 ~1 after 50min rest. This represents a 49% increase (P < 0.001) which is considerably greater than that shown in Fig. 1. DISCUSSION
This study describes some effects of flight on the composition of cockroach haemolymph that are markedly different from those described for other species. These effects include an increase in haemolymph volume and an increase in the concentration of trehalos#e. Since the cockroach is not a strong flier and was induced to fly for a longer than normal period it is possible that the effects noted represent a response to stress rather than a normal physiological condition associated with flight.
SOL-7
fp,I e
3701. B
e500
.\-\ t
Tim@ (mlnl
Fig. 9. The effect 01‘ flight on octopamine concentration in the haemolymph. Haemolymph was obtained from cockroaches which had flown for 2. 10 or IO min followed by 50min rest. Zero time values were obtained from cockroaches which had not been flown. Octopamine was measured as describeId in the Materials and Methods. Each point represents the mean of not less than 5 samples, each sample consisting of haemolymph pooled from at least 5 cockroaches. The values shown are the mean k standard error of the mean.
653
The increase in haemolymph volume resulting from flight differs from the findings reported by Gringorten and Friend (1979) who showed that Rhodnius prolixus flown to exhaustion lost fluid from the haemolymph. Locusta migratoria on the other hand, which is a strong flier, maintains a constant volume of haemolymph during flight (Beenakkers, 1973). The maximal increase in haemolymph volume in the cockroach after flight was 26% in the first series of experiments, and 49% in the second series used for the measurement of haemolymph octopamine. This may reflect a larger initial haemolymph volume in the first group due to a greater hydration of those cockroaches before use. The explanation for the increase in haemolymph volume is unclear but it is not unreasonable to suggest that it may be hormonal in origin. Indeed, it is possible that the release of such a hormone might be triggered by the increase in osmolarity that occurs shortly after flight begins. Alternatively, the increase in K+ might cause release of the hormone by depolarizing the membrane of the cells containing the hormone. Both Wall (1967) and Steele and Tolman (1980) have demonstrated the presence of antidiuretic factors in the neuroendocrine system of Periplaneta americana although the mechanisms controlling the release of the hormone have not been described. Extracts of the corpora cardiaca which contain the antidiuretic hormone have been shown to stimulate the reabsorption of water from the rectum (Wall, 1967; Steele and Tolman, 1980). However, water uptake from the rectum alone is unlikely to explain the increase in haemolymph volume since it contains not more than 3 or 4 ~1 of fluid. Nevertheless, uptake of water from the rectum cannot be ruled out since it may be replenished by water from the anterior region of the gut. Another possible source of water is the salivary glands. Salivary glands in hydrated cockroaches may contain up to 100 ~1 of fluid and this can be reabsorbed during dehydration (Sutherland and Chillseyzn, 1968). Whether this is hormonally regulated and can occur in the short period necessary to explain the present results is not known. Despite the increase in the concentration of trehalose following flight the decreasing osmolarity during this period clearly shows that the increase in fluid volume cannot be attributed to an increase in solute concentration. The question is whether the increase in fluid volume signifies a temporary failure in osmoregulation or a useful adaptation of importance to the recovery from flight. The latter possibility seems more likely since it would allow the accumulation of greater food reserves in the haemolymph and facilitate exchange between the musculature, fat body and gut. In contrast to the changing fluid volume of the haemolymph the concentration of Na*, K+, Ca*+ and Mg2+ appears to be tightly controlled during the period of increasing haemolymph volume, despite increases in the three latter ions soon after flight begins. This ionic constancy agrees with the findings of Edney (1967), Wall (1970) and Tucker (1977a and b) for dehydrated and rehydrated cockroaches. On balance the data offer a strong argument for hormone-mediated increases in haemolymph volume during flight. This may be a mechanism common to all flying insects but more apparent in weak fliers
654
L. E. KING et al.
when the hormone release mechanism is stimulated excessively. Trehalose exhibits a biphasic response to flight; a small increase (- 10%) during the early stage of flight which disappears after the first few minutes of rest, to be followed by a greater increase (-30%) which is maintained for the next 24 h. The finding that trehalose levels increase is surprising since flight, in those species where it has been examined, is usually associated with a decrease in the concentration of trehalose (Clegg and Evans, 1961; Jutsum and Goldsworthy, 1976; Van der Horst et al., 1978). The nature of the stimulus which provokes an initial weak increase followed by a second strong increase in haemolymph trehalose is of particular interest. The stress effect, described by Matthews and Downer (1974) and Downer (1979) as being responsible for a modest increase in trehalose, probably is the cause of the increase in trehalose which occurs during the first few minutes of flight since both are of the same magnitude. The evidence suggests that the rise in trehalose which occurs with the onset of flight may be due to the release of octopamine into the haemolymph. When octopamine is injected into cockroaches to yield a final concentration in the haemolymph of 10m6 M-10-j M there is a 100% increase in the level of circulating trehalose (Downer, 1979). Furthermore, octopamine has been shown to stimulate the release of trehalose from fat body in vitro (Gole and Downer, 1979). To complete the cycle of events Bailey et al. (1983) have shown that flight in Periplaneta americana causes an increase in the concentration of octopamine in the haemolymph. These results have, in general, been confirmed by the octopamine measurements obtained in the present study. The secondary increase in haemolymph trehalose differs from the initial increase in two important respects. Firstly, a considerably larger amount of trehalose is released to the haemolymph and secondly, the duration of the hypertrehalocaemic response is many hours longer than that associated with the first few minutes of flight. In all probability the second hypertrehalocaemic response to flight results from the release of hypertrehalocaemic peptides which are present in the corpus cardiacuun (Steele, 1963) although this remains to be proven. Certainly the protracted hypertrehalocaemic state observed after flight is similar to that which follows the injection of corpus cardiacum extract (Steele, 1963). Since flight lasting only 10min causes 75% depletion of glycogen in the flight musculature of Periplaneta americana (Downer and Matthews, 1976) it is possible that the post-flight increase in haemolymph trehalose is important in the resynthesis of muscle glycogen. We suggest that the release of the corpus cardiacum hormones is neurally controlled by mechanisms linked to the expenditure of energy (flight). This view is supported by the finding that trehalose in the haemolymph of Calliphora erythrocephala can only be maintained at a consant level during flight if the nervous connections between the brain and corpus cardiacurn are intact, otherwise trehalose declines to the point where flight is impossible (Vejbjerg and Normann, 1974). The importance of the neural connections for the release of hypertrehalocaemic hormone in CaNiphora erythrocephala
is shown by the fact that hormone release does not occur in response to electrical stimulation of the brain if the nerves are severed (Normann and Dave, 1969). The accumulating evidence, although falling short of proving that the hypertrehalocaemic hormone maintains the level of trehalose in the resting insect, suggests that it may do so in the working insect. Experiments are currently under way to determine whether the titre of hormone in the haemolymph increases as a result of flight. The octopamine measurements, although similar to those obtained by Bailey et al. (1983) as far as the decrease accompanying rest after flight is concerned, differ in one important aspect. Our measurements do not show the low resting (0 time) values obtained by the other workers. This is because our data, which were obtained before the study by Bailey et al. (1983) appeared, did not take into consideration the role of stress in the release of octopamine and precautions were not taken to isolate the cockroaches before use. Our study, like that of Bailey et al. (1983) suggests that efficient mechanisms exist for the clearance of octopamine from the haemolymph although the increase in haemolymph volume also has a role in decreasing the concentration of the amine. Acknowledgement-This study was supported, in part, by the award of a grant to J.E.S. from the Natural Sciences and Engineering Research Council of Canada.
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