- Email: [email protected]
Lipase activity in third instar larvae of the blowfly, Calliphora erythrocephala
Insect Biochem., 1975, Vol. 5, pp. 53 to 60. Pergamon Press. Printed in Great Britain
LIPASE ACTIVITY IN T H I R D INSTAR LARVAE OF THE BLOWFLY, C A L L I P H O R A ER Y T H R O C E P H A L A GARETH M. PRICE Unit of Invertebrate Chemistry and Physiology, Agricultural Research Council, University of Sussex, Brighton BN1 9QJ, England
(Received 23 May 1974) A b s t r a c t - - T h e level of lipase activity in tissues of third-instar larvae of the blowfly, Calliphora erythrocephala has been measured. In salivary glands activity was maximal at pH 6"0, while in fat body, muscle, and plasma it was maximal at pH 7 to 8. Activity in salivary glands, muscle and plasma was maximal in 6-day larvae (wandering stage) after which it declined in 7-day larvae and rounded-off white puparia (RO) while in fat body it rose to a maximum at the RO stage. In 7-day larvae the plasma contained 56% of all the lipase activity present in the larva, the salivary glands 15% and the fat body 11%. A large amount of triglyceride was found in the fat body but none was detectable in the plasma. IN A PREVIOUS paper (PRICE, 1974) the levels of activity of various hydrolytic enzymes (acid phosphatase, amylase, protease) in tissues of third instar larvae of the blowfly, Calliphora erythrocephala were measured. I n the present work the lipase activity of various tissues has been measured and the results are discussed with regard to the changes undergone by the larval tissues at the time of metamorphosis. METHODS
Chemicals Lipase substrate (stabilized olive oil emulsion) was purchased from Sigma Chemical Co., and palmitic acid from Koch-Light Laboratories Ltd. All other chemicals were of Analar grade and glass-distilled water was used throughout.
Breeding of Calliphora Blowfly larvae were bred as previously described (PRICE, 1969)o
Isolation of larval tissues Tissues were isolated under a Ringer's medium as previously described (PRICE, 1972).
Determination of protein Protein was determined as previously described (PRICE, 1974).
Determination of lipase activity (a) In fat body, gut, Malpighian tubules, muscle, cuticle, and salivary glands: The tissues from 10 larvae were transferred to 2 ml of Ringer and subjected to three 15 see periods of sonication on an Ultratec Ultrasonic Dismembrator (Schuco Division, American Caduceus Industries, New York, 10011, U.S.A.) at a dial setting of 60. After each 15 see period the probe of the sonicator was cooled by dipping it in cold water. 53
54
GARETH M. PRICE
When the sonicates were examined by light microscopy no whole cells were discernible. The sonicates were centrifuged at 2700 g for 15 min and their lipase activity was determined essentially according to the method of DOWNER and STEELE (1973) as follows. Sonicate-0"2 ml, lipase substrate--0.6 ml, citrate buffer (pH 2'5-8'0) 0"1 M-0"7 ml, and water-0"5 ml, were shaken vigorously for 5 sec and the mixture was incubated at 30°C for 1 hr. The incubate was well shaken with 10 ml of a mixture of N H a SO4-heptane--propan-2-ol (1 : 10 " 40 by vol.), left to stand for 5 min, and then shaken again with 10 ml of waterheptane (1 : 1"5 by vol.). The two phases were allowed to separate and the fatty acid content of 3 ml samples of the upper phase was determined as described by DOLE and MEINERTZ (1960). The fatty acid content (palmitate equivalent) of zero time control samples was always deducted from that obtained with the incubated samples and blanks contained Ringer in place of the tissue extract. All determinations were carried out at least in duplicate. Palmitic acid was used as standard and lipase activity is expressed as mg of palmitic acid produced per tissue per hour at 30°C. (b) In Plasma: haemolymph was centrifuged at 2700g for 10min to precipitate the haemocytes and lipase activity in 50 ~1 of the supernatant was determined as described above.
Extraction and chromatography of ether-solublecompounds (a) Fat body: ten fat bodies from 7-day larvae were homogenized at room temperature in 2 ml of diethyl ether. The homogenate was centrifuged at 2700 g for 10 min, the supernatant decanted and the residue re-extracted with a further 2 ml of ether. The supernatants were combined, evaporated to dryness under a stream of nitrogen and the residue obtained was dissolved in 0"5 ml of ether. Samples (20 gl) were applied to 20 × 20 cm plates of silica gel G and chromatographed in hexane-diethylether-glacial acetic acid (80 ; 20 : 1 by vol.). After chromatography the plates were air-dried and placed for 1 hr in an atmosphere of iodine vapour. Lipids were observed as yellow spots on a white background. (b) Plasma: haemolymph, isolated as previously described (PRICE, 1972), was centrifuged at 2700 g for 15 min to precipitate the blood cells. The plasma (0"5 ml) was homogenized with 3 ml of ether and centrifuged, the supernatant being decanted and the residue reextracted with 1 ml of ether. The combined supernatants were evaporated to dryness and the residue obtained was dissolved in 0"5 ml of ether. Further procedure was as described in (a). RESULTS
Effect of incubation time W h e n plasma f r o m 6-day Calliphora larvae was incubated at 30°C for various periods with the lipase substrate, e n z y m e activity was linear for virtually the first h o u r after w h i c h the rate declined (Fig. 1). I n s u b s e q u e n t experiments, activity was m e a s u r e d over a 1 hr period.
Effect of pH I n tissues f r o m 7 - d a y larvae lipase activity was maximal at the following p H values; salivary g l a n d s - - 6 . 0 (Fig. 2a), fat b o d y - - 7 . 5 to 8.0 (Fig. 2b), m u s c l e - 7.5 to 8.0 (Fig. 2c), and p l a s m a - - 7 to 8.0 (Fig. 2d). F o r the cuticle, gut and M a l p i g h i a n tubules activity was low over the whole of the p H range 3 to 8 with no observable o p t i m u m . M e a s u r e m e n t at the start and at the end of the incubation period s h o w e d that the p H r e m a i n e d constant d u r i n g this time. I n s u b s e q u e n t experiments the |ipase activity of any one tissue was assayed at the p H o p t i m u m for that tissue.
55
L I P A S E A C T I V I T Y I N B L O W F L Y LARVAE
25
T• m
2c
.~
.c_
0"~ 0
•~_ I0 u -D o "n'~ o
I
I
I
1
I
2
5
4
Incubation time,
hr
FIG. 1. Effect of incubation time on lipase activity of plasma from 6-day Calliphora larvae. Plasma was isolated and enzyme activity was assayed as described in the text.
Effect of age Lipase activity in salivary glands (Fig. 3a) and plasma (Fig. 3d) was maximal in 6-day larvae after which it declined in 7-day larvae and rounded-off white puparia (RO). In fat body(Fig. 3b) there was a slight fall in activity over the period 0"6~
(b) 0'4
(a) 0-21
I
I
i
I
I
4
5
6
7
B
I-2-
3
I
I
I
I
t
4
5
6
7
8
4
5
6
7
8
15
0-8
04
5
3
I
I
I
I
I
4
5
6
7
8
,,=
3
pH of.incubation medium
FIO. 2. Effect of pH on lipase activity of a---salivary glands, b--fat body, c--muscle and d--plasma. Ordinate units, for a, b, and c--mg of palmitate produced• tissue/hr, for d--rag of palmitate produced/ml of plasma/hr× 10 -1. Tissues were isolated from 6-day CaUiphora larvae and enzyme activity was assayed as described in the text.
56
GARETH M.
PRICE
from the 5 to the 6-day larva after which the activity rose in 7-day larvae and RO puparia. It was found that the fatty acid content (palmitate equivalent) of the fat body rose from 0"08 to 0.25 mg per fat body as the larva aged from 4 to 7 days and then fell to 0.1 mg at the RO stage. These 'zero time control' levels were taken into account when calculating the lipase activity of the fat body. In muscle (Fig. 3c) activity increased gradually over the larval period attaining a maximum in 7-day larvae and then falling at the RO stage. For the cuticle, gut, and Malpighian tubules activity was very low over the whole of the age range.
0e
1"5
I0
0
O,z
(b)
0'5
4
1
1
I
1
5
6
7
R.O
0-4--
4
1
I
1
5
6
7
R.O
[
7
R,0
Ic
Cd)
4
]
I
I
1
5
6
7
R,0
~.,
Age of larvae,
4
5
6
days
Fxc. 3. Lipase activity in tissues from third-instar Calliphora larvae of various ages. a--salivary glands, b--fat body, e--muscle, and d--plasma. Ordinate units, for a, b, and c--rag of palmitate produeed]tissue/hr, for d--mg of palmitate produced/m] of plasma/hr×10 -1. Tissues were isolated and enzyme aetivity was assayed as described in the text. RO is the rounded-off white puparial stage.
Secretion of lipase by fat body Groups of 10 fat bodies from 4-day larvae were incubated at 30°C for various periods in 1 ml of Ringer-amino acid mixture at pH 7.4 (PRICE, 1969). At the end of the desired period the flask contents were centrifuged at 600 g for 5 rain to preeipitate the fat body and lipase activity in samples of the supernatant was determined. Lipase was released rapidly during the first 30 rain of incubation (Fig. 4), the rate of release then decreasing considerably such that the level of activity in the medium at 1 hr was similar to that at 4 hr.
LIPASE
ACTIVITY
IN
BLOWFLY
57
LARVAE
2-0--
"~
1.5
E -~
1.0
J
Q.
0"5
o
o
I
I
I
1
I
2
~
4
Incubcfion time,
hr
FIG. 4. Secretion of lipase by fat body isolated from 4-day old (feeding-stage) larvae. Isolation and incubation of the fat body and assay of lipase activity in the medium was carried out as described in the text.
Calliphora
Secretion of lipase by salivary glands Groups of 8 glands from larvae of different ages were incubated in 0.8 ml of Ringer at 30°C for 4 hr after which the glands were removed from the medium and the lipase activity in them and in the medium was determined. After 4 hr incubation the level of activity remaining in the glands was similar to that present
i
t~
I
06
;>
•-.d
o
(3.
2
o.2
E 0 4
t
I
I
I
5
6
7
R.O
Age of larvae,
doys
FIG. 5. Lipase activity in salivary glands ( 0 - - 0 ) and in medium (O--O) after 4 hr incubation. Since the level of activity in the gland was so similar to that in the medium only one curve has been drawn. Isolation and incubation of the salivary glands and assay of lipase activity was carried out as described in the text.
58
GARETH M . PRICE
in the medium. Glands from 6-day larvae released the greatest amount of lipase (Fig. 5) the rate of release subsequently decreasing in glands from 7-day larvae and RO puparia.
Distribution of lipase The distribution of lipase activity between various tissues of 7-day larvae is shown in Table 1. On a tissue basis the plasma contains most of the lipase activity (56.3% of the total) followed in descending order by the salivary glands (16%), fat body (11.3) and muscle (9.6) with the gut (3.4), cuticle (1.7) and Malpighian tubules (1.7) possessing the lowest activities. Lipase accounted for a higher proportion of the protein in the salivary gland than it did of the protein in any other tissue of the larva. TABLE l--DISTRIBUTION OF LIPASE ACTIVITY BETWEEN VARIOUS TISSUES OF 7-DAY BLOWFLY LARVAE
mg of Palmitate prod./tissue/hr
Tissue Cuticle Fat body Plasma Malpighian tubules Gut Muscle Salivary glands
0"1 0-7 3"5 t 0-1 0"2 0"6 1"0
Lipase Activity Activity Protein (percentage (mg) per distribution) larva* 1"7 11.3 56"3 1"7 3"4 9"6 16"0
1"62 1"70 2"99 0"05 0"13 2"04 0"06
mg of Palmitate prod./mg of protein /hr x 10 0-62 4"10 11-70 10"00 15"38 2'94 167"0
* Values from PRICE(1972). t Activity per 25 lal of plasma, this being the amount of haemolymph present in one larva (PaIcE, 1972).
Distribution of glyceride Ether-soluble compounds in fat body and plasma from 7-day larvae were extracted, chromatographed and visualized as described in the Methods section. In the fat body the largest spot had the same chromatographic mobility as that of the triglyceride marker, triolein (R I 0.67), whereas in the plasma no compound with such a mobility was detectable. Both the fat body and plasma contained a compound behaving as diolein (R e 0.23). In 7-day blowfly optimal over the pH pH 6.0. Activity in whole pH range with
DISCUSSION larvae lipase activity in fat body, muscle, and plasma was range 7.5 to 8, whereas in salivary glands it was optimal at the gut, cuticle and Malpighian tubules was low over the no clear optimum. Working with the adult male American
LIPASE ACTIVITY IN BLOWFLY
LARVAE
59
cockroach, Periplaneta americana, DOWNER and STEELE(1973) found that lipase activity in the haemolymph was maximal at pH 4.9 and 7.0. With the gut, dual pH optima were again observed, namely at pH 5.0 and 7.2 these results being identical to those of GILBERTet al. (1965). In the present work the plasma lipase exhibited only one pH optimum at 7 to 8 and the gut lipase activity was low over the pH range 3 to 8. The pH optimum (7.5 to 8) exhibited by Calliphora fat body lipase is the same as that exhibited by Periplaneta fat body lipase (DOWNER and STEELS, 1973; GILBERTet al., 1965). Working with the khapra beetle, Trogoderma granarium, NANDArO2~et al. (1973) found that lipase activity of a homogenate of whole pupae and/or pharate adult was optimal at pH 7.5. No activity was detectable in the gut of larvae, and on the basis of this observation the authors concluded that the activity was due to extra-digestive lipase. In the blowfly larva, salivary-gland lipase activity was maximal in 6-day larvae (Fig. 3a) this also being the age of larvae from which isolated glands released lipase at a maximum rate (Fig. 5). Over the period from the 6-day larva to the RO pupa, lipase activity in the salivary glands, muscle, and plasma fell while over the same period that in the fat body rose. Similar results have previously been obtained regarding the levels of protease activity in these tissues (PRICE, 1974). Since in late third-instar larvae the fat body sequesters protein from the haemolymph (KINNWR et al., 1968; MARTINet al., 1971) then the rise in lipase in the fat body may well be due to its sequestration from the haemolymph. Previous in vitro experiments have shown that fat body from 4-day old (feeding stage) Calliphora larvae, synthesizes proteins rapidly and releases them into the incubation medium (PRICE, 1966). The similarity between the electrophoretic pattern of the proteins in the medium and that of the proteins in the haemolymph indicated that in vivo also the fat body releases proteins into the haemolymph (PRICE and BOSMAN, 1966). The present work has shown that in vitro lipase is released by the fat body indicating that this organ may well be the origin of much of the lipase present in the haemolymph. In 7-day larvae the plasma contained 56% of the lipase activity present in all the tissues (Table 1), the salivary glands containing 16% and the fat body 11%. Lipase accounted for a greater proportion of the protein in the salivary glands than it did of the protein in any other tissue. The fact that lipase activity in salivary glands was at its highest level in 6-day (wandering stage) larvae, these having finished feeding twenty four hours previously, indicates that the enzyme is playing a rble other than that in the digestion of food. It is unlikely that this rble is one concerned solely with the lysis of the gland at metamorphosis for by that time the lipase level in the gland has fallen considerably. It is possible that the lipase is secreted internally into the haemolymph, a possibility which has previously been suggested for protease synthesized in the gland (PRICE, 1974). CHINO and GILBERT(1965) showed that diglyceride was released from the fat body of adult male Hyalophora cecropia but that triglyceride release was negligible. In the present work a large amount of triglyceride was found in the fat body but none was detectable in the plasma, indicating that in Calliphora also, only negligible amounts of triglyceride could be released by the fat body.
60
GARETH M. PRICE
In late third instar blowfly larvae the fat body contains numerous lipid droplets (PRICE, 1969, 1973) much of the lipid probably being in the form of triglyceride. As both triglyceride and lipase are present at high levels in fat body of late third instar larvae then it may be that at that stage the substrate is not available to its enzyme. Working with the blood-sucking bug, Rhodnius prolixus, WmCLESWORTH (1958) showed that in the fat body of this insect, each lipid droplet possessed a 'cap' of esterase. Whether such an arrangement is also present in the blowfly larval fat body is not known. Another possibility is that lipase is stored in the lysosomes and that not until metamorphosis, when the fat body undergoes lysis, do these fuse with the lipid droplets so bringing the enzyme into contact with its substrate.
Acknowledgements--I thank my colleague, Mr. S. E. LEwis for critically reading the manuscript and I am indebted to Mrs. ELIZABETHHUCHESfor skilled technical assistance. REFERENCES CHINO H. and GILBERTL. I. (1965) Lipid release and transport in insects. Biochim. biophys. Acta. 98, 94-110. DOLEV. P. and MEINERTZH. (1960) Microdetermination of long chain fatty acids in plasma and tissues ft. biol. Chem. 235, 2595-2599. DOWNER R. G. H. and STEELEJ. E. (1973) Haemolymph lipase activity in the American cockroach, Periplaneta americana. J. Insect Physiol. 19, 523-532. GILBERT L. E., CHINO H. and DOMROESEK. A. (1965) Lipolytic activity of insect tissues and its significance in lipid transport. J. Insect Physiol. 11, 1057-1070. KINNEAR J. F., MARTINM. D., THOMSONJ. A., and NEUFELDG. J. (1968) Developmental changes in the late larva of Calliphora stygia. Aust.J. biol. Sci. 21, 1033-1045. MARTIN M. D., KINNEARJ. F. and THOMSONJ. A. (1971) Developmental changes in the late larva of Calliphora stygia--IV. Uptake of plasma protein by the fat body. Aust. J. biol. Sci. 24, 291-299. NANDANAN M. D., SEHGALS. S., and ACARWALH. C. (1973) Properties and distribution of extra-digestive lipase in the beetle, Trogoderma. Insect Biochem. 3, 223-230. PRICE G. M. (1966) 'The in vitro incorporation of [U-14C]-valine into fat body protein of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 12, 731-740. PRICE G. M. (1969) Protein synthesis and nucleic acid metabolism in the fat body of the larva of the blowfly, CaUiphora erythrocephala. J. Insect Physiol. 15, 931-944. PRICE G. M. (1972) Tyrosine metabolism in the larva of the blowfly, Calliphora erythrocephala. Insect Biochem. 2, 175-185. PRICE G. M. (1973) Protein and nucleic acid metabolism in insect fat body. Biol. Rev. 48, 333-375. PRICE G. M. (1974) Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 20, 329-357. PRICE G. M. and BOSMANT. (1966) The electrophoretic separation of proteins isolated from the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 12, 741-745. WIGGLESWORTHV. B. (1958) The distribution of esterase in the nervous system and other tissues of the insect Rhodnius prolixus. Quart..7. micr. Sci. 99, 441-450. Key Word Index--Blowfly, larva, lipase.
LIPASE ACTIVITY IN T H I R D INSTAR LARVAE OF THE BLOWFLY, C A L L I P H O R A ER Y T H R O C E P H A L A GARETH M. PRICE Unit of Invertebrate Chemistry and Physiology, Agricultural Research Council, University of Sussex, Brighton BN1 9QJ, England
(Received 23 May 1974) A b s t r a c t - - T h e level of lipase activity in tissues of third-instar larvae of the blowfly, Calliphora erythrocephala has been measured. In salivary glands activity was maximal at pH 6"0, while in fat body, muscle, and plasma it was maximal at pH 7 to 8. Activity in salivary glands, muscle and plasma was maximal in 6-day larvae (wandering stage) after which it declined in 7-day larvae and rounded-off white puparia (RO) while in fat body it rose to a maximum at the RO stage. In 7-day larvae the plasma contained 56% of all the lipase activity present in the larva, the salivary glands 15% and the fat body 11%. A large amount of triglyceride was found in the fat body but none was detectable in the plasma. IN A PREVIOUS paper (PRICE, 1974) the levels of activity of various hydrolytic enzymes (acid phosphatase, amylase, protease) in tissues of third instar larvae of the blowfly, Calliphora erythrocephala were measured. I n the present work the lipase activity of various tissues has been measured and the results are discussed with regard to the changes undergone by the larval tissues at the time of metamorphosis. METHODS
Chemicals Lipase substrate (stabilized olive oil emulsion) was purchased from Sigma Chemical Co., and palmitic acid from Koch-Light Laboratories Ltd. All other chemicals were of Analar grade and glass-distilled water was used throughout.
Breeding of Calliphora Blowfly larvae were bred as previously described (PRICE, 1969)o
Isolation of larval tissues Tissues were isolated under a Ringer's medium as previously described (PRICE, 1972).
Determination of protein Protein was determined as previously described (PRICE, 1974).
Determination of lipase activity (a) In fat body, gut, Malpighian tubules, muscle, cuticle, and salivary glands: The tissues from 10 larvae were transferred to 2 ml of Ringer and subjected to three 15 see periods of sonication on an Ultratec Ultrasonic Dismembrator (Schuco Division, American Caduceus Industries, New York, 10011, U.S.A.) at a dial setting of 60. After each 15 see period the probe of the sonicator was cooled by dipping it in cold water. 53
54
GARETH M. PRICE
When the sonicates were examined by light microscopy no whole cells were discernible. The sonicates were centrifuged at 2700 g for 15 min and their lipase activity was determined essentially according to the method of DOWNER and STEELE (1973) as follows. Sonicate-0"2 ml, lipase substrate--0.6 ml, citrate buffer (pH 2'5-8'0) 0"1 M-0"7 ml, and water-0"5 ml, were shaken vigorously for 5 sec and the mixture was incubated at 30°C for 1 hr. The incubate was well shaken with 10 ml of a mixture of N H a SO4-heptane--propan-2-ol (1 : 10 " 40 by vol.), left to stand for 5 min, and then shaken again with 10 ml of waterheptane (1 : 1"5 by vol.). The two phases were allowed to separate and the fatty acid content of 3 ml samples of the upper phase was determined as described by DOLE and MEINERTZ (1960). The fatty acid content (palmitate equivalent) of zero time control samples was always deducted from that obtained with the incubated samples and blanks contained Ringer in place of the tissue extract. All determinations were carried out at least in duplicate. Palmitic acid was used as standard and lipase activity is expressed as mg of palmitic acid produced per tissue per hour at 30°C. (b) In Plasma: haemolymph was centrifuged at 2700g for 10min to precipitate the haemocytes and lipase activity in 50 ~1 of the supernatant was determined as described above.
Extraction and chromatography of ether-solublecompounds (a) Fat body: ten fat bodies from 7-day larvae were homogenized at room temperature in 2 ml of diethyl ether. The homogenate was centrifuged at 2700 g for 10 min, the supernatant decanted and the residue re-extracted with a further 2 ml of ether. The supernatants were combined, evaporated to dryness under a stream of nitrogen and the residue obtained was dissolved in 0"5 ml of ether. Samples (20 gl) were applied to 20 × 20 cm plates of silica gel G and chromatographed in hexane-diethylether-glacial acetic acid (80 ; 20 : 1 by vol.). After chromatography the plates were air-dried and placed for 1 hr in an atmosphere of iodine vapour. Lipids were observed as yellow spots on a white background. (b) Plasma: haemolymph, isolated as previously described (PRICE, 1972), was centrifuged at 2700 g for 15 min to precipitate the blood cells. The plasma (0"5 ml) was homogenized with 3 ml of ether and centrifuged, the supernatant being decanted and the residue reextracted with 1 ml of ether. The combined supernatants were evaporated to dryness and the residue obtained was dissolved in 0"5 ml of ether. Further procedure was as described in (a). RESULTS
Effect of incubation time W h e n plasma f r o m 6-day Calliphora larvae was incubated at 30°C for various periods with the lipase substrate, e n z y m e activity was linear for virtually the first h o u r after w h i c h the rate declined (Fig. 1). I n s u b s e q u e n t experiments, activity was m e a s u r e d over a 1 hr period.
Effect of pH I n tissues f r o m 7 - d a y larvae lipase activity was maximal at the following p H values; salivary g l a n d s - - 6 . 0 (Fig. 2a), fat b o d y - - 7 . 5 to 8.0 (Fig. 2b), m u s c l e - 7.5 to 8.0 (Fig. 2c), and p l a s m a - - 7 to 8.0 (Fig. 2d). F o r the cuticle, gut and M a l p i g h i a n tubules activity was low over the whole of the p H range 3 to 8 with no observable o p t i m u m . M e a s u r e m e n t at the start and at the end of the incubation period s h o w e d that the p H r e m a i n e d constant d u r i n g this time. I n s u b s e q u e n t experiments the |ipase activity of any one tissue was assayed at the p H o p t i m u m for that tissue.
55
L I P A S E A C T I V I T Y I N B L O W F L Y LARVAE
25
T• m
2c
.~
.c_
0"~ 0
•~_ I0 u -D o "n'~ o
I
I
I
1
I
2
5
4
Incubation time,
hr
FIG. 1. Effect of incubation time on lipase activity of plasma from 6-day Calliphora larvae. Plasma was isolated and enzyme activity was assayed as described in the text.
Effect of age Lipase activity in salivary glands (Fig. 3a) and plasma (Fig. 3d) was maximal in 6-day larvae after which it declined in 7-day larvae and rounded-off white puparia (RO). In fat body(Fig. 3b) there was a slight fall in activity over the period 0"6~
(b) 0'4
(a) 0-21
I
I
i
I
I
4
5
6
7
B
I-2-
3
I
I
I
I
t
4
5
6
7
8
4
5
6
7
8
15
0-8
04
5
3
I
I
I
I
I
4
5
6
7
8
,,=
3
pH of.incubation medium
FIO. 2. Effect of pH on lipase activity of a---salivary glands, b--fat body, c--muscle and d--plasma. Ordinate units, for a, b, and c--mg of palmitate produced• tissue/hr, for d--rag of palmitate produced/ml of plasma/hr× 10 -1. Tissues were isolated from 6-day CaUiphora larvae and enzyme activity was assayed as described in the text.
56
GARETH M.
PRICE
from the 5 to the 6-day larva after which the activity rose in 7-day larvae and RO puparia. It was found that the fatty acid content (palmitate equivalent) of the fat body rose from 0"08 to 0.25 mg per fat body as the larva aged from 4 to 7 days and then fell to 0.1 mg at the RO stage. These 'zero time control' levels were taken into account when calculating the lipase activity of the fat body. In muscle (Fig. 3c) activity increased gradually over the larval period attaining a maximum in 7-day larvae and then falling at the RO stage. For the cuticle, gut, and Malpighian tubules activity was very low over the whole of the age range.
0e
1"5
I0
0
O,z
(b)
0'5
4
1
1
I
1
5
6
7
R.O
0-4--
4
1
I
1
5
6
7
R.O
[
7
R,0
Ic
Cd)
4
]
I
I
1
5
6
7
R,0
~.,
Age of larvae,
4
5
6
days
Fxc. 3. Lipase activity in tissues from third-instar Calliphora larvae of various ages. a--salivary glands, b--fat body, e--muscle, and d--plasma. Ordinate units, for a, b, and c--rag of palmitate produeed]tissue/hr, for d--mg of palmitate produced/m] of plasma/hr×10 -1. Tissues were isolated and enzyme aetivity was assayed as described in the text. RO is the rounded-off white puparial stage.
Secretion of lipase by fat body Groups of 10 fat bodies from 4-day larvae were incubated at 30°C for various periods in 1 ml of Ringer-amino acid mixture at pH 7.4 (PRICE, 1969). At the end of the desired period the flask contents were centrifuged at 600 g for 5 rain to preeipitate the fat body and lipase activity in samples of the supernatant was determined. Lipase was released rapidly during the first 30 rain of incubation (Fig. 4), the rate of release then decreasing considerably such that the level of activity in the medium at 1 hr was similar to that at 4 hr.
LIPASE
ACTIVITY
IN
BLOWFLY
57
LARVAE
2-0--
"~
1.5
E -~
1.0
J
Q.
0"5
o
o
I
I
I
1
I
2
~
4
Incubcfion time,
hr
FIG. 4. Secretion of lipase by fat body isolated from 4-day old (feeding-stage) larvae. Isolation and incubation of the fat body and assay of lipase activity in the medium was carried out as described in the text.
Calliphora
Secretion of lipase by salivary glands Groups of 8 glands from larvae of different ages were incubated in 0.8 ml of Ringer at 30°C for 4 hr after which the glands were removed from the medium and the lipase activity in them and in the medium was determined. After 4 hr incubation the level of activity remaining in the glands was similar to that present
i
t~
I
06
;>
•-.d
o
(3.
2
o.2
E 0 4
t
I
I
I
5
6
7
R.O
Age of larvae,
doys
FIG. 5. Lipase activity in salivary glands ( 0 - - 0 ) and in medium (O--O) after 4 hr incubation. Since the level of activity in the gland was so similar to that in the medium only one curve has been drawn. Isolation and incubation of the salivary glands and assay of lipase activity was carried out as described in the text.
58
GARETH M . PRICE
in the medium. Glands from 6-day larvae released the greatest amount of lipase (Fig. 5) the rate of release subsequently decreasing in glands from 7-day larvae and RO puparia.
Distribution of lipase The distribution of lipase activity between various tissues of 7-day larvae is shown in Table 1. On a tissue basis the plasma contains most of the lipase activity (56.3% of the total) followed in descending order by the salivary glands (16%), fat body (11.3) and muscle (9.6) with the gut (3.4), cuticle (1.7) and Malpighian tubules (1.7) possessing the lowest activities. Lipase accounted for a higher proportion of the protein in the salivary gland than it did of the protein in any other tissue of the larva. TABLE l--DISTRIBUTION OF LIPASE ACTIVITY BETWEEN VARIOUS TISSUES OF 7-DAY BLOWFLY LARVAE
mg of Palmitate prod./tissue/hr
Tissue Cuticle Fat body Plasma Malpighian tubules Gut Muscle Salivary glands
0"1 0-7 3"5 t 0-1 0"2 0"6 1"0
Lipase Activity Activity Protein (percentage (mg) per distribution) larva* 1"7 11.3 56"3 1"7 3"4 9"6 16"0
1"62 1"70 2"99 0"05 0"13 2"04 0"06
mg of Palmitate prod./mg of protein /hr x 10 0-62 4"10 11-70 10"00 15"38 2'94 167"0
* Values from PRICE(1972). t Activity per 25 lal of plasma, this being the amount of haemolymph present in one larva (PaIcE, 1972).
Distribution of glyceride Ether-soluble compounds in fat body and plasma from 7-day larvae were extracted, chromatographed and visualized as described in the Methods section. In the fat body the largest spot had the same chromatographic mobility as that of the triglyceride marker, triolein (R I 0.67), whereas in the plasma no compound with such a mobility was detectable. Both the fat body and plasma contained a compound behaving as diolein (R e 0.23). In 7-day blowfly optimal over the pH pH 6.0. Activity in whole pH range with
DISCUSSION larvae lipase activity in fat body, muscle, and plasma was range 7.5 to 8, whereas in salivary glands it was optimal at the gut, cuticle and Malpighian tubules was low over the no clear optimum. Working with the adult male American
LIPASE ACTIVITY IN BLOWFLY
LARVAE
59
cockroach, Periplaneta americana, DOWNER and STEELE(1973) found that lipase activity in the haemolymph was maximal at pH 4.9 and 7.0. With the gut, dual pH optima were again observed, namely at pH 5.0 and 7.2 these results being identical to those of GILBERTet al. (1965). In the present work the plasma lipase exhibited only one pH optimum at 7 to 8 and the gut lipase activity was low over the pH range 3 to 8. The pH optimum (7.5 to 8) exhibited by Calliphora fat body lipase is the same as that exhibited by Periplaneta fat body lipase (DOWNER and STEELS, 1973; GILBERTet al., 1965). Working with the khapra beetle, Trogoderma granarium, NANDArO2~et al. (1973) found that lipase activity of a homogenate of whole pupae and/or pharate adult was optimal at pH 7.5. No activity was detectable in the gut of larvae, and on the basis of this observation the authors concluded that the activity was due to extra-digestive lipase. In the blowfly larva, salivary-gland lipase activity was maximal in 6-day larvae (Fig. 3a) this also being the age of larvae from which isolated glands released lipase at a maximum rate (Fig. 5). Over the period from the 6-day larva to the RO pupa, lipase activity in the salivary glands, muscle, and plasma fell while over the same period that in the fat body rose. Similar results have previously been obtained regarding the levels of protease activity in these tissues (PRICE, 1974). Since in late third-instar larvae the fat body sequesters protein from the haemolymph (KINNWR et al., 1968; MARTINet al., 1971) then the rise in lipase in the fat body may well be due to its sequestration from the haemolymph. Previous in vitro experiments have shown that fat body from 4-day old (feeding stage) Calliphora larvae, synthesizes proteins rapidly and releases them into the incubation medium (PRICE, 1966). The similarity between the electrophoretic pattern of the proteins in the medium and that of the proteins in the haemolymph indicated that in vivo also the fat body releases proteins into the haemolymph (PRICE and BOSMAN, 1966). The present work has shown that in vitro lipase is released by the fat body indicating that this organ may well be the origin of much of the lipase present in the haemolymph. In 7-day larvae the plasma contained 56% of the lipase activity present in all the tissues (Table 1), the salivary glands containing 16% and the fat body 11%. Lipase accounted for a greater proportion of the protein in the salivary glands than it did of the protein in any other tissue. The fact that lipase activity in salivary glands was at its highest level in 6-day (wandering stage) larvae, these having finished feeding twenty four hours previously, indicates that the enzyme is playing a rble other than that in the digestion of food. It is unlikely that this rble is one concerned solely with the lysis of the gland at metamorphosis for by that time the lipase level in the gland has fallen considerably. It is possible that the lipase is secreted internally into the haemolymph, a possibility which has previously been suggested for protease synthesized in the gland (PRICE, 1974). CHINO and GILBERT(1965) showed that diglyceride was released from the fat body of adult male Hyalophora cecropia but that triglyceride release was negligible. In the present work a large amount of triglyceride was found in the fat body but none was detectable in the plasma, indicating that in Calliphora also, only negligible amounts of triglyceride could be released by the fat body.
60
GARETH M. PRICE
In late third instar blowfly larvae the fat body contains numerous lipid droplets (PRICE, 1969, 1973) much of the lipid probably being in the form of triglyceride. As both triglyceride and lipase are present at high levels in fat body of late third instar larvae then it may be that at that stage the substrate is not available to its enzyme. Working with the blood-sucking bug, Rhodnius prolixus, WmCLESWORTH (1958) showed that in the fat body of this insect, each lipid droplet possessed a 'cap' of esterase. Whether such an arrangement is also present in the blowfly larval fat body is not known. Another possibility is that lipase is stored in the lysosomes and that not until metamorphosis, when the fat body undergoes lysis, do these fuse with the lipid droplets so bringing the enzyme into contact with its substrate.
Acknowledgements--I thank my colleague, Mr. S. E. LEwis for critically reading the manuscript and I am indebted to Mrs. ELIZABETHHUCHESfor skilled technical assistance. REFERENCES CHINO H. and GILBERTL. I. (1965) Lipid release and transport in insects. Biochim. biophys. Acta. 98, 94-110. DOLEV. P. and MEINERTZH. (1960) Microdetermination of long chain fatty acids in plasma and tissues ft. biol. Chem. 235, 2595-2599. DOWNER R. G. H. and STEELEJ. E. (1973) Haemolymph lipase activity in the American cockroach, Periplaneta americana. J. Insect Physiol. 19, 523-532. GILBERT L. E., CHINO H. and DOMROESEK. A. (1965) Lipolytic activity of insect tissues and its significance in lipid transport. J. Insect Physiol. 11, 1057-1070. KINNEAR J. F., MARTINM. D., THOMSONJ. A., and NEUFELDG. J. (1968) Developmental changes in the late larva of Calliphora stygia. Aust.J. biol. Sci. 21, 1033-1045. MARTIN M. D., KINNEARJ. F. and THOMSONJ. A. (1971) Developmental changes in the late larva of Calliphora stygia--IV. Uptake of plasma protein by the fat body. Aust. J. biol. Sci. 24, 291-299. NANDANAN M. D., SEHGALS. S., and ACARWALH. C. (1973) Properties and distribution of extra-digestive lipase in the beetle, Trogoderma. Insect Biochem. 3, 223-230. PRICE G. M. (1966) 'The in vitro incorporation of [U-14C]-valine into fat body protein of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 12, 731-740. PRICE G. M. (1969) Protein synthesis and nucleic acid metabolism in the fat body of the larva of the blowfly, CaUiphora erythrocephala. J. Insect Physiol. 15, 931-944. PRICE G. M. (1972) Tyrosine metabolism in the larva of the blowfly, Calliphora erythrocephala. Insect Biochem. 2, 175-185. PRICE G. M. (1973) Protein and nucleic acid metabolism in insect fat body. Biol. Rev. 48, 333-375. PRICE G. M. (1974) Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 20, 329-357. PRICE G. M. and BOSMANT. (1966) The electrophoretic separation of proteins isolated from the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 12, 741-745. WIGGLESWORTHV. B. (1958) The distribution of esterase in the nervous system and other tissues of the insect Rhodnius prolixus. Quart..7. micr. Sci. 99, 441-450. Key Word Index--Blowfly, larva, lipase.