The role of free amino acids in osmoregulation of cricket blood (Acheta domesticus)

J. Insect Physiol.. b.01. 26, pp. 613 to 618. ? Per,qumon Press Ltd. 1980. Printed in Great Britain

0022-1910/80~09014613

If;OZ.OO/O

THE ROLE OF FREE AMINO ACIDS IN OSMOREGULATION OF CRICKET BLOOD (ACHETA

DOMESTICUS)

J. P. WOODRINGand E. W. BLAKENEY,JR. Departments of Zoology-Physiology and Biochemistry, Louisiana State University, Baton Rouge, LA 70808, U.S.A. (Received 2 I January 1980) Abstract-The blood volume increased during normal feeding, and did not decrease during fasting at the end of the stadium. The unexpectedly high blood volume of starved crickets might be an adaptation to increase chances for moulting via stretch receptor stimulation. The amount of blood amino acids was not changed by feeding, but increased with fasting or starvation. Thus amino acid levels in the blood were not directly related to amino acid input from the gut. The blood protein concentration did not change during starvation, but the amino acid concentration was 33y0 higher in starved crickets that drank water as opposed to those given saline to drink. Thus amino acid levels in the blood were not related directly to blood protein concentration. The blood amino acid concentration was 19-22 mM/l in response to salt intake by feeding crickets or starved crickets drinking saline. The concentration was 32-38 mM/l when the crickets were fasting prior to and after ecdysis or when starved with water to drink during the time when they would normally be feeding. The increase of amino acids during fasting was due to a proportional increase in all amino acids augmented by a 3 x increase in tyrosine. The increase during salt depleting starvation was due to a doubling of the two predominant amino acids proline and glycine. Proline and glycine were not increased in starved crickets drinking saline, thus starvation was not the reason for the increase. This is the first instance where specific amino acids have been implicated in osmoregulation. Key Word Index: Cricket blood, blood volume, Acheta domesticus, free amino acids, osmoregulation

INTRODUCTION

THE FREEamino acids in the blood of insects are in a state of dynamic equilibrium; that is, the amount of amino acids in the blood fluctuates only within regulated limits in spite of the rapid turnover due to the influx from feeding or catabolism of cellular proteins and the efflux due to cellular growth or peptide-protein secretion. Blood proteins are basically secretions of the fat body. Homeostatic limits of blood amino acid concentrations are maintained even during starvation where tissue cells, haemocytes or blood peptides act as amino acid pools (COLLETT. 1976b; EVANS and CROSSLEY,1974; JUNNIKKULA, 1976). Fluctuations in blood amino acids are associated with periods of feeding or nonfeeding within stages of the life cycle (HILL and GOLDSWORTHY, 1968; BOSQUET,1977b; WOODRINGet al., 1977, BARRETT,1974; FIRLING, 1977), or they are inversely related to blood protein concentration in some species (HILL, 196 ‘: KULKARNIand MEHROTRA. 1970), and are related to the need for soluble particles for osmoregulation (BEADLE and SHAW, 1950; COLLETT,1976a; BOSQUET,1977a; WOODRINGet al., 1977). Certain amino acids fluctuate more than others (BARRETTand LAI-FOOK, 1976; JEUNIAUXet al., 1961; LEVENBOOK,1966), especially tyrosine which is elevated during moulting (WIRTZ and HOPKINS,1974; BARRETT.1974; JUNNIKKALA,1976). Our purpose was to c : if the feeding or nonfeeding induced fluctuations in blood amino acid 613

concentration in house crickets was related to amino acid input or to the resultant change in dietary salt input. Could the low levels of amino acids (20-35 mM/l) in the blood of crickets be significant in osmoregulation when the normal NaCl concentration was around 150 mM/l If so, were all amino acids fluctuating in equal proportions, or only certain amino acids contributing to osmoregulation?

MATERIAL Crickets under a

were

reared

AND

METHODS

at 30°C

on

an

oligidic

diet

LD 12:12 hr photoperiod as described by CLIFFORDet al. (1977). At 30°C over 507~ of the last instar larvae moulted by the 9th day, and these were called one-day-old adults. Periodic fasting was part of the normal life cycle of A&eta domesticus. Using dyed food and hourly dissection, we found that last instar larvae and adult female crickets did not eat for 10-12 hr after ecdysis and large amounts of food were not eaten until the end of the first day. Feeding started to decline at midstadium and larvae were completely fasting during the last 24-36 hr of the stadium. Thus, g-day larvae had fasted for at least 1 day, and l-dayold larvae and adults had fasted for at least 2 days. There was an additional period of very reduced feeding (not complete fasting) that started on the 8th day of virgin female life. By day 10, the body became so engorged with eggs, that could not be laid without mating, that the digestive tract was compressed and

0.28 0.24 5.10

0.35 3.76 1.31

0.39 0.55 0.93

1.45 0.45

1.41

2.45 1.10 2.92 22.69

1.88

0.29 0.40 5.75

1.41 5.20 1.09

0.36 0.62 1.45

4.85 0.62

1.89

3.13 1.35 4.04 32.44

2.54

asp glu pro

ala gly val

met ilu leu

tyr phe

his

lys arg mx-4 Total

NH,

0.21 0.55 10.25 0.73 9.57 0.24 0.49 0.26 0.50 1.05 0.18 1.79 2.44 0.69 3.76 32.71 4.09

0.39 4.79 0.42

0.43 0.18 0.29

0.82 0.11

0.80

2.32 0.56 1.64 19.27

2.29

H*O

4d larv star

0.25 0.55 5.72

4d larv star salt

of 18 amino

2.00

1.97 1.01 3.48 31.38

1.78

4.43 0.86

0.39 0.76 1.44

1.31 3.83 2.46

1.22 6.45

8d larv cant fast

1.19 0.77 5.41 38.10 4.11

2.12 1.03 3.37 29.48 2.32

3.34 0.69

4.74 1.04 1.43

0.48 1.05 1.67

0.41 0.63 1.08

1.40

1.38 6.20 3.99

0.43 1.22 8.80

Hz0

8d larv star

of last instar

1.34 3.51 2.58

0.32 0.60 5.32

8d larv star salt

acids in the blood

36.25 5.47

1.74 1.34 4.65

1.37

4.25 0.71

0.35 1.03 1.67

1.13 6.13 3.79

0.30 1.04 6.76

Id virg cant fast

larval

1.11 2.07 0.46 3.52 22.05 1.73

1.24 2.12 1.04 3.72 29.16 2.67

0.29 0.50 0.75 1.32 0.39

0.36 0.66 0.96 2.24 0.64

20.65 2.47

2.56 0.43 2.13

0.80

0.48 0.43 0.63 0.88 0.18

0.39 4.73 1.67

0.18 0.38 4.79

0.11 0.14 5.39 0.64 3.60 1.75

4d virg star salt

4d virg cant feed

female house crickets

1.40 5.40 1.88

0.48 7.08

2d virg cant feed

and adult

0.07 0.15 6.96

0.05 0.41 10.20

27.64 5.68

21.44 1.66 32.88 4.24

1.69 0.89 2.71

1.11 0.56 3.00

3.77 0.73 2.40

1.20

0.29 0.56 0.92 2.08 0.48 0.96

0.15 0.37 0.70 1.63 0.33

1.03 0.59 0.76 1.90 0.49

0.80 5.15 0.43

0.23 0.71 9.48

10d virg cant fast

21.89

1.95 0.61 3.29

1.07

0.15 0.48 0.82 1.37 0.22

0.85 4.30 1.42

0.30 0.56 4.52

10d mated cant feed

conditions

0.95

0.70 3.93 0.81

0.43 8.20 0.96

Hz0

8d virg cant feed

or starved

4d virg star

under control

?

F $ P .z

1.79 0.64 2.62 28.22 5.37

3

z! b .F

3

i

.* ?

1.22

0.29 0.52 0.86 2.07 0.42

0.92 5.79 0.35

0.30 0.84 9.59

10d virg star H,O

Since they eluted together, the peaks of serine. threonine, asparagine and glutamine were combined (mx-4). Starved crickets (star) were given either water (H,O) or saline (salt) to drink. Control crickets (cant) were provided with food and water, but they were feeding (feed) or fasting (fast) depending on the time after moulting (see Materials and Methods). Age is given in days (d) for larvae (larv) or for virgin females (virg) or mated females (mated).

4d larv cant feed

Id larv cant fast

1. The concentration

Age Stage Cond

Table

Role of free amino acids in osmoregulation

feeding impeded. ‘Starvation’, as distinct from ‘fasting’, was induced by removal of food for three days at periods when the crickets would normally have been feeding. Four-day-old starved (st) crickets (larvae and adults), had their food removed at the end of day 1, and 8-day-old starved crickets had their food removed on day 5. When the food was removed, a fresh watering device was added that contained either distilled water or saline. The saline was made up to match the cation concentration of the blood; 155 mM NaCl, 7 mM K, 8 mM CaCl, and 4 M MgCl,. Thus, there were two kinds of starved crickets, a salt depleting starvation (water) and a non salt depleting starvation (saline). All blood samples were collected in capillary tubes as it flowed from clipped cerci in response to gentle squeezing of the body. The blood was allowed to clot and the resultant serum after for 10 min., centrifugation was used for all analyses. The chloride ion concentration was determined with a chloridometer (5 ~1 sample), and the blood volume determined using the 14C-inulin dilution method. A Beckman scintillation counter was used to determine the dilution of counts/min in a 5 ~1 serum sample 1 hr after injecting 1 ~1 of r4C-insulin. Serum samples for amino acid analyses were pooled (50 ~1) and an equal volume of 25x-sulphosalicyclic acid added (final SSA concentration was 10%) in order to deproteinate the serum. After centrifugation and lypophilization, the samples were dissolved in 100 ~1 of 0.2 N sodium citrate buffer (pH 2.2) and recentrifuged. Aliquotes of the supernatant were analyzed on a Beckman Model 119 Analyzer using a one-column methodology as described in Beckman Bulletin A-7B-116. Under these conditions asparagine, glutamine, threonine, and serine elute as one broad peak. This peak was calculated using the response factor for serine, allowing detection of relative changes in the total of these 4 amino acids. A general increase in all amino acids was observed after acid hydrolysis, which indicated the presence of soluble peptides in the blood.

615 RESULTS

The blood volume of fed-controls increased, though the volume as a percentage of total cricket weight decreased (Table 2). Starvation induced an increase in blood volume with little or no increase in intracellular water. When larval crickets were starved for 5 days, the average (n =60) total body weight changed as follows: 209 mg (ld)-214 mg-217 mg-211 mg-201 mg (5d). After 3 days of starvation (Table 2) there was an 8 mg dry body weight loss and a 10 mg gain in total body water, and all of the water gain was in the blood (13 ~1). There was no change in the blood volume with fasting, following feeding, until after ecdysis when the blood volume decreased. Table 1 is a compilation of the concentration of 18 free amino acids during the last larval stadium and the first 10 days of the adult female. The amino acids serine, threonine, asparagine, and glutamine were not sufficiently separated on the column used, and are given as an unseparated mixture (MX-4). The total concentration of amino acids declined after 3 days of feeding (Tables 1 and 2) though the amount did not change in spite of the large influx of amino acids from the digestive tract. The decreased amino acid concentration resulting from feeding was gradual, as can be seen by comparing adults after one day of feeding (2d-virg) to those after 3 days of feeding (Table 1). The concentration decreased only because of blood dilution. Three days of starvation (with water to drink) resulted in increased concentration and amount of amino acids, the blood volume having increased, with no amino acid input from the gut. When starved larvae were given saline to drink the amount of amino acids remained constant, but as with the fed crickets the blood dilution caused a concentration decrease. Fasting at the end of the larval stadium increased the amount of amino acids, the blood volume having not changed but the concentration increased. Three days of starvation (with water) at the end of the stadium elevated the amino acid concentration above fasting levels resulting from 1.5 days of nonfeeding.

Table 2. Total body water. dry body weight, blood volume, osmolality, chloride ion concentration, concentration in larval house crickets

Wet weight (mg)* Dry weight (mg)* Percentage body water (“,,I Total body water* (~1) Percent blood volume (“,,I Blood volume (~1) Blood osmolality (mOs Kg) Blood chloride cont. (mM:LI Blood amino acid cont. (mM/L)

and total free amino acid

1 day old Control

4 day old Control

4 day old Star-H,0

4 day old Star-Saline

8 day old Control

209 + S(12) 52 + 2(S) 15 157 f 3(8) 19 40 + 2(17) 352 + 4 (8) 125 + 4(10) 32

338 + 4(12) 108 + 3(8) 68 230 + 2(8) 15 51 + 2(18) 370 + 2 (6) 154 + 3(10) 22

211 * 6(6) 44 f 3 (6) 79 167 i 3 (6) 25 53 & 2(17) 348 + 5(8) 137 * 3(10) 33

220 * 5(4) 45 * 3(4) 19 174) 3(6) 27 60 + 4(7) 367 f 5 (8) 152 + 3(10) 19

370 i 6(10) 115 f 3(8) 69 255 * 3 (8) 15 56 + 3 (8) 345 If: 4(8) I22 & 3 (8) 31

The insects comprised control one, four, and eight-day-old last instar larvae and four-day-old larvae star&( star) for three days but given water (H,O or saline (saline) to drink. Control larvae were provided with food and water, but one and eightday-old controls were f&rrrg. *These numbers (n) are the number of cartons containing IO-12 crickets each. All other numbers (n) are of indrvidual crickets. X f S.E.

J. P.

616

WOODRING

AND

l PRO :’

AGE

IN DAYS

Fig. 1. Changes in the concentration of proline, glycine, mixture of serine-threonineunseparated tyrosine, asparagine-glutamine (mx-4) and others (summed remaining 11 amino acids) during feeding and fasting of the last instar larvae and adult virgin females. Fasting occurred on the days before and after the moult (M), and when virgin females became distended with eggs (8-10 days old). Figure 1 shows the normal fluctuations of proline, glycine, tyrosine, the serine-threonine-asparagineglutamine mixture and the sum of the other 11 amino acids during the alternating periods of fasting and feeding. Except for tyrosine, all amino acids changed in relatively equal proportions, depending upon their concentration, with the concentration higher during fasting and lower during feeding. The amino acid concentration during starvation with saline to drink was basically the same as during feeding (Table l), but during starvation (with water) the greatly elevated concentrations of proline and glycine caused the total amino acid concentration to reach the fasting levels. However, proline and glycine were not elevated during fasting. The significant difference between the blood of starved crickets with water or saline was about 10 mIv&/l of proline-glycine. Tyrosine concentrations were three times higher during fasting because that moulting occurred. The tyrosine was when concentration remained as low as fed-controls when the crickets were starved. The reduced feeding of loday-old virgin females resulted in an increased concentration of proline but not glycine. Laying loday-old female crickets, which ate voraciously, had a reduced blood amino acid concentration. The blood osmolality and chloride concentration was lower during fasting than during feeding (Table 2). When starved (with water) the osmolality and chloride concentration were as low as in fasting crickets, but when starved crickets were given saline to drink, the osmolality and chloride concentration of the blood was equal to that of fed controls. Salt intake, from food or saline, caused an increase in blood

E. W. BLAKENEY, JR. osmolality, which decreased without the salt intake. In general, the amino acid concentration was higher whenever the chloride concentration was low and lower when the chloride concentration was high. The acid concentration did not increased amino compensate completely for the reduced chloride concentration resulting from fasting or starvation (water), thus the osmolality was reduced. The blood protein concentration in fed controls increased after ecdysis to a midstadium high of 6.7 + 0.2% (n=20), then decreased slightly to 5.8 +_ 0.2% (18) with the normal fasting at the end of the stadium. A further decrease occurred after ecdysis before feeding induced an increase in the adult females. There was no change in blood protein concentration when larvae were starved. Starved from days 1 to 4 the protein concentration went from 3.4 + 0.1% (18) to 3.8 _t 0.2 (9), and starved from days 4 to 7 the protein concentration went from 5.2 f 0.2 (20) to 5.9 f 0.2 (16). With the increased blood volume of starved larvae (Table 2) from days 1 to 4, the amount of blood protein apparently increased. There was an indication that feeding crickets, or those given saline when starved, generally had a lower blood ammonia concentration than those fasting or given water when starved, particularly in the adult females (Table 1). In the case of starved crickets given water or fasting crickets the amount of ammonia in the blood increased. DISCUSSION Because we used serum, the possible contribution of free amino acids released from ruptured haemocytes must be addressed. We found that the total haemocyte count increased by 40% during the last larval stadium and that 3 days of starvation significantly reduced the haemocyte count (unpublished). It was unlikely that haemocytes were responsible for the observed change in amino acids levels because there was a 50% difference in amino acid concentration in starved crickets given water or saline to drink but no difference ‘in the haemocyte count. EVANS and CROSSLEY(1974) found that only 6% of the total blood amino acids was in the haemocytes of Calliphora, though 65”/, of the glutamate-aspartate was cell bound. The increased blood volume in starved crickets was unusual, because in other insects the blood volume decreased or remained stable after starvation (HILL and GOLDSWORTHY, 1970; BOSQUET, 1977a). There was a distinct blood dilution with starvation but not with fasting. Why would an insect gain water weight, when starved during a period when they normally would be feeding but not during a period of fasting? We can not explain how, but we can speculate on a possible adaptive advantage. In several insects, it has been shown that a critical mass must be achieved, rather early in the stadium, in order to stimulate stretch receptors to cause the brain to programme the next moult, and that moulting could be induced in larvae that were just below critical weight by increasing the blood volume with saline injections (NIJHOUT, 1979). Thus, in a population of mixed ages, the number of crickets capable of moulting when food was depleted would be increased through increased blood volume.

Role of free amino acids in osmoregulation The amount of blood amino acids remained constant (32 mM x 40 &51 ~1 = 25 mM) in 4-day fed controls when compared to l-day fasting control crickets. Therefore, with feeding, the amino acid concentration decreased in response to blood dilution in spite of massive amino acid input from the gut. During growth the removal of amino acids from the blood was balanced exactly by input from the digestive tract. The amount of blood amino acids in fasting or in starved crickets given water to drink increased, and these additional amino acids could not have come from food. Thus, the total blood amino acid concentration was not directly related to dietary amino acid availability. The blood amino acid levels in adult Cdiphora also was little altered in response to feeding or starvation (COLLETT, 1976a). The pattern of high blood amino acid concentration during fasting and low concentrations during feeding was found in crickets and locusts (WOODRINGet al., 1977; HILL and GOLDSWORTHY, 1968), but was not observed in other insects (BARRETTand LAI-FOOK, 1976; PRABHUand NAYER,1971; TERRAet al., 1973). In larval Chironomus the pattern was reversed; amino acid concentrations were low on the first and last day of the stadium and highest at midstadium (FIRLING, 1977). In some insects the amino acid titre may be directly related to feeding or the lack of it, but not in house crickets. In some insects an inverse correlation of blood protein concentration to amino acid concentration was evident (HILL, 1962; KULKARNIand MEHROTRA, 1970; WCNJDRING et al., 1977), but in other species such a relationship was not indicated (BEADLEand SHAW, 1950; HILL and GOLDSWORTHY,1970). Blood amino acids increased markedly in starved crickets given water to drink, but the protein content remained unchanged. Therefore, a direct interrelationship of blood amino acids and protein levels was not evident. Blood proteins in Locusta were synthesized from blood amino acids (TOBEand LOUGHTON,1969), and the rates of protein synthesis could theoretically influence the amount of amino acids in the blood. The blood amino acid level in Calliphora was thought to be in equilibrium with the larger intracellular pool (COLLETT, 1976a, b) rather than to blood protein levels. Some blood amino acids were derived from blood peptides (BOSQUET,1976b) in Philosamiu and others from the haemocytes of Culliphoru (EVANSand CROSSLEY,1974). BOSQUET(1976b) suggested that a decrease in protein synthesis during starvation was not due to nonavailability of amino acids, because the concentration increased during starvation, but due to the decreased pumping of amino acids into cells. Thus, though blood protein are made from blood amino acids and catabolism of blood protein may contribute to blood amino acid levels, the overall concentrations of amino acids and proteins in the blood are not directly related in many insects. It has been generally accepted that blood amino acids play a role in osmoregulation, but the extent of the contribution of amino acids in osmoregulation and whether all amino acids were involved was uncertain. Starved Sialis larvae given tap water showed an increase in total blood NPS and a decrease in chlorides, and when given saline to drink the NPS decreased and chlorides increased (BEADLEand SHAW, 1950). COLLETT(1976b) suggested that the amino acids

617

used for osmoregulation in Drosophila and Calliphora were stored as peptides in tissue, and these peptides were released and hydrolyzed in the blood. The blood osmolality was lower in fasting or starved crickets drinking water than in fed crickets, which meant that the increased amino acids concentration was insufficient to balance salt loss. However, without the increased amino acids the osmolality could have been depressed even more. BOUQUET (1976a) also noted that the blood osmolality of starved Philosumiu larvae decreased even with increased amino acid concentration. In the case of starved crickets drinking water, the increased blood volume (40-53 ~1) would have diluted the chloride concentration to 94 mM (40153 x 125 mM), but that the chloride concentration did not decrease meant that some of the osmoregulation of the blood was due to diffusion of salt ions from the tissue in addition to the increased amount of amino acids. When given saline to drink the blood osmolality of starved larvae increased to equal that of fed larvae, but the amount of amino acids was unchanged (the concentration was l/3 lower), thus with a dietary salt intake, from food or saline, the amount of blood amino acids did not increase and the blood osmolality only increased slightly. Even when amino acids and salts were entering the blood during feeding, the amount of blood amino acids did not change. We conclude that blood amino acid concentrations are regulated in order to hold the fluctuation of blood osmolality to only 20 mOsm in response to varied salt input. All amino acids increased in equal proportions during fasting (Fig. l), and because fasting occurred at the time of moulting the increase was augmented by elevated tyrosine. However, the increased total amino acid concentration in starved crickets drinking water was due to the specific doubling of proline and glycine (Table 1). This represents the first indication that specific amino acids are utilized for blood osmoregulation. Osmotic compensation for reduced salt input during fasting would be programmed as a part of the life cycle and accomplished by a general increase in all amino acids and a tripling of tyrosine concentration. A different compensatory mechanism had to evolve for salt depletion during a time in the life cycle programmed for feeding, which could involve the specific increase ofjust two amino acids. Proline is a predominant amino acid in many insects (BARRETT. 1974; TERRA et al., 1973; COLLETT,1976b; BUHSELL. 1977; FIRLING, 1977), but not in all insects (BARRETT and LAI-FOOK, 1976; DIAJAKUSUMAHand MILES, 1966). At first we thought that increased proline in starved crickets would offer an energy source. because BURSELL(1977) showed that proline was a fuel for flight muscles in Glossinu. Osmoregulation rather than an emergency fuel is a more likely reason for doubled proline concentration in starved crickets, because there was no elevation of proline when starved crickets were given saline to drink. What could be the source of the general amino acid increase during fasting, and could it be the same source for proline-glycine during salt depleting starvation’? COLLETT(1976b) demonstrated the interconversion of glycine, proline and glutamate. and EVANS and CROSSLEY (1974) demonstrated that 65”~ of the whole blood glutamate-aspartate was in the haemocytes.

J. P.

61%

AND

One could postulate that decreasing osmolahty would

trigger the conversion of haemocyte glutamate to proline and its release into the blood. With increasing osmolality (feeding) proline would be converted to glutamate, and be taken up by the haemocytes. Such a glutamate uptake by haemocytes has been demonstrated (EVANSand CROSLEY, 1974). However, there was no evidence of any change in asparagine-glutamine in cricket blood beyond the general increase associated with .fasting. Another source of prolineglycine could be blood peptides, analogous to the tyrosine rich peptides in the blood of Pieris larvae (JU~~IKKA~A, 1976). The biood tyrosine concentration peaked at ecdysis when the crickets were fasting, but lack of feeding was not the reason, because the tryosine concentration was not altered by starvation. The tyrosine peak at ecdysis is assumed to be associated with cuticular tanning (VINCENTand HILLERTON,1979), and such peaks have been found in all insects examined (BARRETT,1974; EVANS and CROSSLEY,1974; WIRTZ and HOPKINS, was no 1974; JUNNIKKALA, 1976). There corresponding change in phenylalanine concentration in cricket blood as tyrosine increased, which WIRTZ and HOPKINS(1974) also noted in Periphetu. They found that tyrosine also peaked in the fat body and integument at ecdysis, and were unable to identify a source for all the tyrosine. JUNNIKKALA (1976) identified a tyrosine rich peptide in the blood of Pieris larvae, which was more soluble than free tyrosine, that he suggested was the pool for high tyrosine levels at ecdysis. The blood peptides of crickets should be examined as a potential pool of tyrosine as well as proline and glycine.

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BEADLEL. C. and SHAWJ. S. (1950) The rentention of salt and regulation of nonprotein fractions in the blood of the aquatic larva, Sialis &aria. J. exp. Biol. 27, 96109. Bos~unr G. (1977a) Hemoiymph modifications during starvation in Philosumiu cvnthiu. I. Volume, osmotic pressure, pH; relation to &racellular water content. Comp. Biochem. Physiol. %A, 373-376. BOUQUETG. (1977b) Hemolymph modifications during starvation in Philos~ia Cynthia. II. Amino acids and peptides. Comp. Biochem. Physiol. %%A,377-382. BURSELLE. (1977) Synthesis of proline by fat body of the tsetse fly (Glossina morsitans): metabolic pathways. Insect Biochem. 7, 427434.

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