a developmental analysis of the uptake and release of lipids by the fat-body of the tobacco...

Insect Biochem., 1971 , I, 63-80. [Scientechnica (Publishers) Ltd.] 63

A DEVELOPMENTAL ANALYSIS OF THE UPTAKE A N D

RELEASE OF LIPIDS BY THE FAT-BODY OF THE TOBACCO

HORNWORM, M A N D U C A S E X T A

FRANKLIN CHANG* AND STANLEY FRIEDMAN Department of Entomology, University of Illinois, Urbana, Illinois 618Ol, U.S.A.

(Received i i Aug., 197o)

ABSTRACT

Qualitative analyses of the haemolymph and fat-body of hornworm larvae and pupae reveal that all major lipid classes are present. Quantitative analyses demonstrate that the predominant lipid in fat-body is triglyceride (TGL), and in the haemolymph, diglyceride (DGL).

When pre-labeUed fat-body from pupal or pharate adult hornworms is incubated in haemolymph or buffered bovine serum albumin (BSA), there is an appreciable release of radioactivity into the medium. This does not occur when fat-body is incubated in buffered saline. The greatest portion of the released radioactivity is associated with the free fatty acid (FFA) fraction; dilution of haemolymph causes a decrease in release of FFA label from fat-body tissue without affecting the distribution of radioactivity in the other lipid fractions; release of FFA label from the fat-body is inhibited partially by azide and arsenate, but is almost unaffected by cyanide.

There is no appreciable release of radioactivity from pre-labeUed larval hornworm fat-body into haemolymph or buffered BSA. Pre-labelled fat-body from adult Leucophaea maderae releases radioactivity into both haemolymph and buffered BSA, chiefly as FFA. Pre-labeUed fat-body from adult Schistocerca gregaria releases radioactivity into both haemolymph and buffered BSA, chiefly as DGL. Cecropia pupal fat-body preparations release comparatively low levels of radioactivity into haemolymph in the forms of FFA, DGL, and TGL.

LIPID release and transport in insects has received much attention in recent years (Tietz, 1962 , 1967; Chino and Gilbert, 1965; Wlodawer, Lagwinska, and Baranska, 1966; Cook and Eddington, 1967; Beenakkers and Gilbert, 1968; Bhakthan and Gilbert, 1968; Martin, 1969; Mayer and Candy, 1969) as a result of earlier findings that in many cases fats are the principal energy source for flight (Weis-Fogh, 1952; Clements, 1955; Cockbain, 1961 ).

It has been reported that the adult stage of the migratory locust, Locusta migratoria (Tietz, i967) , the desert locust, Schistocerca gregaria (Mayer and Candy, i969) , the differential grasshopper, Melanoplus differentialis, American cockroach, Periplaneta americana, and the pupal as well as the adult stage of the American silkmoth, Hyalophora cecropia (Chino and Gilbert, i965) , preferentially release diglycerides (DGL) from fat- body into a haemolymph medium. However, Wlodawer and others (1966) have demonstrated release of free fatty acids (FFA) from the fat-body of the larval wax moth, Galleria mellonella, and similar results have been obtained for 4 genera of cockroaches (Bhakthan and Gilbert, i968), one of which was P. americana. At variance with the latter result has

* Present address: Department of Entomology, College of Tropical Agriculture, University of Hawaii, Honolulu, Hawaii 96822.

6 4 CHANG AND FRIEDMAN Insect Biochem.

been the finding that triglycerides (TGL) and FFA are the predominant lipids released from American cockroach fat-body (Cook and Eddington, i967). Martin (1969) has recently demonstrated that the primary class of lipid released from adult Pyrrhocoris apterus fat-body is triglyceride.

In an effort to reconcile some of the aforementioned results and, perhaps, gain some new insights into the general problem of lipid release and transport, an intensive investi- gation was carried out using the larval, pupal, and pharate adult stages of the tobacco hornworm, Manduca sexta, with'the woodroach, Leucophaea maderae, the American silkmoth, Hyalophora cecropia, and the desert locust, Schistocerca gregaria, serving as secondary sources of data.

MATERIALS AND M E T H O D S

ANIMALS Hornworm adults (M. sexta) captured in the field produced eggs which hatched in 3-4 days at

room temperature (approximately 23 ° C.). The resulting larvae were kept at room temperature under constant illumination and fed field-grown tomato leaves. Fifth-instar larvae and non- diapausing pupae were obtained from this group.

American silkmoths (H. cecropia) used in this study were also progeny from field-trapped adults. Immediately after hatching, i group of cecropia larvae was placed o n sweet gum leaves under constant illumination and allowed to develop at 27 ° C. with a relative humidity of 80 per cent. This group produced non-diapausing pupae. A second group of larvae was transferred to the field in the 5th instar and allowed to complete development under field conditions. Diapausing pupae obtained from this group were stored at 6 ° C. for 3-6 months before use and are referred to as 'chil led' pupae.

Adult woodroaches (L. maderae) came from a stock culture maintained on water and solid dog food.

Adult desert locusts (S. gregaria) were obtained from a commercial source (T. Gerrard and Co. Ltd., Sussex, England) and used immediately.

Adult differential grasshoppers (Melanoplus differentialis) and Carolina grasshoppers (Dissosteira carolina) were caught in the field and also used immediately.

No attempt was made to select individuals on the basis of sex.

PREPARATION OF HAEMOLYMPH Haemolymph was removed from hornworm larvae according to the procedure of Wlodawer and

others (I966), from hornworm and cecropia pupae and pharate adults by a method similar to that described by Chino and Gilbert (x965), from cockroach adults by the method of Sternburg and Corrigan (I959) , and from locust adults as described by Tietz (I962). The method for removal of haemocytes consisted of centrifugation at ioo g for 5 minutes in the cold. In all cases, glutathione (2 ~tmoles per ml. haemolymph) or phenylthiourea (2-3 crystals per ml. haemolymph) were added to inhibit tyrosinase activity.

PRE-LABELLING OF FAT-BODY Fat-body from all animals was carefully dissected out and washed with cold o'oI M phosphate/

saline buffer (o'9 per cent NaC1 in o'oI M K2HPO,/KHIPO~, pH 7"4) to remove haemolymph. It (i5o-2oo rag. wet weight) was then incubated for x-2 hours at 320 C. in 2 ml. of o'oI M phosphate/saline buffer, pH 7"4, containing sodium palmitate-i .14 C (specific activity, 53 me. per mmole; radiopurity greater than 99 per cent) as the free salt or as an albumin complex prepared according to Masironi and Depocas (x96x). Fat-body treated in this manner is referred to as 'pre-labelled', and details of the labelling are described in the tables.

INCUBATION PROCEDURES Varying amounts (I5o-2oo rag. wet weight) of pre-labelled fat-body were typically incubated

for I hour at 32 ° C. in I ml. of each of the following media: (x) haemolymph (with or without

1971, I UPTAKE AND RELEASE OF FAT-BODY LIPIDS 6 5

haemocytes), either whole or diluted with phosphate/saline buffer, (2) phosphate/saline buffer, and (3) phosphate/saline buffer containing x per cent BSA.

After incubation, the mixture was very gently filtered with suction through Whatman No. 41 filter paper and the fat-body washed 3 times with 3-ml. portions of phosphate/saline buffer. The buffer washes were pooled with the initial filtrate (approximately 9 ml. total) and, with the fat-body, were retained for further analysis.

EXTRACTION AND SEPARATION PROCEDURES

Lipids were extracted from the filtrate with 25 ml. Dole's mixture (isopropanol/heptane/x N HzSO4, 4 ° : lO : I, by volume). The Dole's mixture was washed with two 5-mL portions of distilled water and the organic solvent layer containing the lipid was concentrated in vacuo.

Fat-body tissue was homogenized in an all-glass Potter Elvehjem homogenizer with 7 ml. of Dole's mixture and the mixture washed as above. The fat-body was then re-extracted with 7 ml. of fresh Dole's mixture, the mixture washed with water, and the 2 portions of organic solvent pooled and concentrated in vacuo. The lipid extracts were separated by thin-layer chromatography on activated silica gel G plates (3oo microns thick) according to the procedure described by Freeman and West (1966), and visualized by spraying the plates with a 0.2 per cent solution of 2', 7'- dichlorofluorescein in ethanol. The plates were then dried overnight at 60 ° C., and the spots detected under ultra-violet irradiation. For permanent records, spots were made visible by charring with a saturated solution of potassium dichromate in 8o per cent (by weight) HsSO4 for 12 hours at I2O ° C.

RADIOCHROMATOGRAPHY AND QLIANTITATION OF LIPIDS

Each of the areas on the plate corresponding to an appropriate lipid standard was scraped off with a single-edge razor blade into scintillation vials for counting or into 13 x Ioo-mm. culture tubes for quantitation via the sulphuric acid-dichromate method of Amenta (1964). For counting, 15 ml. of scintillation fluid (4 g. 2,5-diphenyloxa zole (PPO) per 1. toluene) were added to each vial, previous work having revealed that quenching in this system was negligible up to 18o mg. of silica gel G. Aqueous samples were counted in Bray's mixture (Bray, I96O). Samples were counted in a Beckman LS-25o Liquid Scintillation System with an external standard and all counts con- verted to disintegrations per minute (d.p.m.).

RESULTS QUALITATIVE AND QUANTITATIVE ANALYSES OF HAEMOLYMPH AND FAT-BODY LIPIDS FROM HORNWORM LARVAE AND PUPAE

Free fatty acids, cholesterol, 1,2- and 1 ,3-DGL , cholesterol esters, T G L , and mono- glycerides ( M G L ) are evident in larval and pupal haemolymph when resolved by the Freeman-West (1966) procedure. Phospholipids arc probably present, but not well resolved, as indicated by a spot very close to the origin in several chromatograms. A typical chromatogram with no attempt to depict intensity is illustrated in Fig. I . Chromatograms of larval and pupal fat-body lipids show the same general qualitative picture.

T h e size and colour-intensity of the resolved spots give some indication of the relative proportion of lipids present in a given sample. T h e striking difference in lipid constitu- tion between fat-body and haemolymph lies in the large percentage of D G L , both 1,2 and 1, 3 isomers, present in the haemolymph, and the dominance of T G L in the fat-body. These findings are in accordance with the observations of other workers (Tietz, 1962; Chino and Gilbert, 1965; Wlodawer and others, 1966; Cook and Eddington, 1967) using insects from other families and orders.

Quantitative analyses of larval and pupal fat-body and haemolymph lipids bear out the above observations. In a typical experiment, i ml. of whole haemolymph from samples pooled from io larvae or 5 pupae was used. As seen in Table I , D G L accounts

66 CHANG AND FRIEDMAN Insect Biochem.

for 53 per cent of the total lipid present in I ml. of larval haemolymph and 50 per cent in I ml. of pupal haemolymph. Moreover, the total lipid content per ml. of pupal haemolymph is tenfold greater than larval haemolymph. TGL comprises greater than 84 per cent of the lipids in the larval fat-body and 79 per cent in the pupal fat-body

Cholerteryl palmkate

moo==

OTripalmkin QO_D

0 Cholesterol

0000

4 Pahnltlc acid

o.hOuo

0 Monopalmltln

Phosphollplds . .

Llpld rtkdard: . _094c

Llpid &act fro; larvai hornworm haemolymph

FIG. I.-Qualitative lipid analysis of larval haemolymph by thin-layer chromatography on silica gel G. Details of the procedure are described in Materials and Methods.

Table I.-THE LIPID CONTENT OF LARVAL AND PUPAL HAEMOLYMPH

LARVA PUPA CONSTITUENT

Haemolymph Percentage Haemolymph Percentage (mg. per ml.) of Total (mg. per ml.) of Total

Triglyceride Diglyceride Monoglyceride Free fatty acid

Total lipid content

0’15 0’77 0.15 0.38

I’45

10.3 3.00 28.8 53-I 5.28 50.8 10.3 0’01 0’1 26.3 2’11 20.3

100’0 10.40 100’0

Haemolymph,(inclusive of haemocytes) used in each analysis was pooled from IO larval or 5 pupal homworms. After extraction with Dole’s mixture, the lipids were separated on silica gel G TLC according to Freeman and West (1966) and the lipid classes quantitated via the procedure of Amenta (1964). Other lipids are present in amounts too small to be measured accurately.

1971, i UPTAKE AND RELEASE OF FAT-BODY LIPIDS 67

(Table H). The total amount of lipid per mg. wet weight of pupal fat-body is twofold greater than larval fat-body. These results are in agreement with the findings of other workers using different insects (Tietz, 1962, 1967; Chino and Gilbert, 1965; Wlodawer and others, 1966; Cook and Eddington, 1967).

EXPERIMENTS WITH Manduca sexta: TIME-COURSE OF in vitro INC()RPORATION OF SODIUM

PALMITATE-I-14C INTO THE FAT-BODY LIPIDS OF THE LARVA

The rate of uptake and esterification of palmitate-i-t4C by hornworm larval fat-body over a 4-hour time period was studied by incubating 2 g. (wet weight) fat-body from seven 5th-instar larvae in phosphate/saline buffer in the presence of labelled palmitate.

Table / / . - - T H E LIPID CONTENT OF LARVAL AND PUPAL FAT-BODY

CONSTITUENT

Triglyceride Diglyceride Monoglyeeride Free fatty acid

Total lipid content

LARVA

Fat-body (mg. per 200 rag.

wet weight)

X7"O 2"0 O'6 0"6

20"2

Percentage of Total

84"2 I0"0 2"9 2"9

IOO'O

PUPA

Fat-body (mg. per 2oo mg.

wet weight)

38"0 6'0 2"0

2"0

48"0

Percentage of Total

79"1 x2"5 4"2 4"2

I00"O

Fat-body used in each analysis was pooled from io larvae or 5 pupae. From this pooled sample 2oo rag. (wet weight) were taken and extracted with Dole's mixture. The extracts were chromatographed as outlined under Table L

The data showing the changes in radioactivity in each lipid class throughout the incubation period are contained in Table I I I and more clearly depicted in Fig. 2. It can be seen that labelled palmitate is very rapidly taken up from the medium in the first 5 minutes of incubation and then 'shut off' in some manner. The radioactivity in the FFA fraction immediately starts to fall, levels off within 60 minutes, and remains almost constant over the rest of the incubation period. Concomitant with the decrease in labelled FFA is a sharp rise in T G L counts in the first 5 minutes of incubation. The increase in T G L radioactivity continues for approximately 3 hours, peaks, then starts to fall. The DGL and MGL radioactivity, after slight increases in the first 5 minutes of incubation, level off, and remain relatively constant, but at low levels, over the rest of the incubation period. The fall in FFA radioactivity suggests a rapid turnover into T G L perhaps via MGL and DGL, whose levels of radioactivity remain low at all times. The reason for the cessation of palmitate uptake after 5 minutes by the larval fat-body remains unexplained. Wlodawer and others (1966) demonstrated a similar incorporation pattern in their experiments with wax moth larval fat-body and showed that diffusion played a major role in palmitate movement into their fat-body preparations.

A calculation made from Table 111 shows that before uptake stops, more than 26 per cent of the label in the medium is incorporated by fat-body lipids. This compares


favourably with the percentage incorporation of label demonstrated by Tietz (1962) over a period of 6o minutes in Locusta (lO-2O per cent) and Cook and Eddington (1967) in P. americana (3 ° per cent).

Table IlL--TIME-COURSE OF In Vitro INCORPORATION OF PALMITATE-I-14C INTO THE NEUTRAL LIPIDS OF THE LARVAL FAT-BODY OF Manduca sexta

TIME DISINTEGRATIONS PER MINUTE (minutes)

FFA MGL DGL TGL CHOL CE+HYDR

5 15 30 60

1 2 0

18o 24 °

57,391 2 ,521 6,783 41 ,391 1,3o4 o 37,403 2 ,435 11,739 47,478 435 435 25,826 1,478 I 1,478 54,956 783 o 11,391 956 8,956 67,3o5 453 o 6,783 1,3o4 11,913 lol,O43 6o9 348 9,391 1,3o4 13,391 III,I3O 609 1,739 4,435 609 lO,348 83,748 o o

Two g. (wet weight) of fat-body from seven 5th-instar larvae were divided into 2oo-mg. portions and incubated at 320 C. in I'5 ml. phosphate/saline buffer (o'9 per cent NaC1 in O.Ol M KgHPOJ KHaPO4, pH 7"4) containing sodium palmitate-x-14C (4"2 × IO ~ d.p.m.) for the above time intervals. After incubation, the tissues were removed, washed thoroughly with cold phosphate/saline buffer, and extracted by homogenization in Dole's mixture with an all-glass Potter-Elvehjem homogenizer.

x

6.

~ ,O O 1 •

o ~o ~ ~ 8'o 16o I~o Time (minutes)

FIG. 2.--Time-course of in vitro incorporation of palmitate-z-14C into the neutral lipids of the larval fat-body of Manduca sexta. 0 , TGL; l , FFA; O, MGL; A, DGL.

1971, I UPTAKE AND RELEASE OF FAT-BODY LIPIDS 6 9

Of interest is the finding that the pattern of labelling in the larval fat-body after in vitro incubation with 14C-palmitate for i -2 hours is similar to the labelling pattern in the fat-body of larvae fed tomato leaves coated with 14C-palmitate (Table IV). This leads us to believe that in vitro pre-labelling is a valid method for preparing experiments dealing with release of label.

Table IV.--DISTRIBUTION OF RADIOACTIVITY IN THE LIPID FRACTIONS OF THE FAT-BODY AFTER FEEDING 5TH-INSTAR Manduca sexta LARVAE TOMATO LEAVES

COATED WITH 14C-PALMITATE (SODIUM SALT)

DISINTEGRATIONS PER ~INUTE TREATMENT*

Larva fed as described Larva starved 3 hours after treatment Larva starved 6 hours after treatment Larva starved 9 hours after treatment Larva starved 18 hours after treatment

FFA MGL DGL TGL CE

1,543 429 4,600 155,228 343 1,649 380 4,440 167,577 127

665 370 443 31,105 74 lO6 53 477 16,5 °o 4 121 61 182 43,087 909

* Five 5th-instar hornworm larvae were allowed to feed to completion on single tomato leaves (selected for approximately equal size) coated with a solution of sodium palmitate-i-l~C (radioactivity not determined). The larvae were then allowed to feed o n ' cold' tomato leaves for the next 2 hours. Larvae were sacrificed after the above time periods and their fat-bodies analysed according to the standard procedure outlined in the Methods section.

Table V.--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF PRE-LABELLED LARVAL FAT-BODY OF Manduca sexta IN VARIOUS MEDIA

RE-INCUBATION--MIXTURE CONSTITUENT

Medium Haemolymph Phosphate/saline buffer Phosphate/saline buffer/i per cent BSA

Tissue Fat-body at o time Fat-body (haemolymph) Fat-body (buffer) Fat-body (buffer-BSA)

DISINTEGRATIONS PER MINUTE

FFA MGL DGL TGL

286 86 228 457 542 457 571 1,o28

1,257 371 371 1,428

1,428 857 16,457 81,885 4,971 2,717 32,200 94,o14 3,600 1,o85 20,057 12o,742 2,171 1,828 15,6oo ioo, i i 4

Pre-labelled fat-body: I g. (wet weight) of fat-body from ten 5th-instar larvae was incubated in I'5 ml. phosphate/saline buffer (0"9 per cent NaC1 in O.Ol M K2HPOdKHaPOI , pH 7"4) containing sodium palmitate-i-14C (1-2 × lO 6 d.p.m.) for 2 hours at 32 ° C. Two hundred mg. (wet weight) of pre-labelled fat-body, after thorough washing with cold phosphate/saline buffer, were re-incubated in each of the different media (i ml.) for I hour at 32 ° C.

EXPERIMENTS WITH Manduca sexta: INCUBATION OF PRE-LABELLED LARVAL FAT-BODY IN VARIOUS MEDIA

F a t - b o d y f rom 5th- ins tar h o r n w o r m larvae was pre- label led wi th pa lmi ta te- i -14C (as the sod ium salt) and incuba t ed for i hour in var ious media (see Methods section).

7 ° CHANG AND FRIEDMAN Insect Biochem.

Table V contains the results from a typical experiment. Analysis of the fat-body used in the incubation reveals that the lipid esters and FFA fractions are labelled, with the TGL fraction containing the greatest amount of radioactivity. There is no appreciable release of radioactivity into any of the incubation media.

The possibility of the presence of a releasing factor in haemolymph from a later stage of development was tested by incubating haemolymph from pharate adults (i day prior to pupal-adult ecdysis) with pre-labeUed larval fat-body. The results in Table VI show that there is no significant difference between larval and pharate adult haemolymph in stimulating the release of label from larval fat-body. Table VI further reveals that dilution of the haemolymph has no appreciable effect on the release of radioactivity from the fat-body.

Table V.[.--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF PRE-LABELLED LARVAL FAT-BODY OF I/landuca sexta IN VARIOUS MEDIA

DISINTEGRATIONS PER MINUTE RE-INCUBATION--MIXTURE CONSTITUENT

Medium Haemolymph (larval) Haemolymph (pharate adult) Phosphate/saline buffer Phosphate/saline buffer/1 per cent BSA Haemolymph (5 ° per cent)

Tissue Fat-body at o time Fat-body (larval haemolymph) Fat-body (pharate adult haemolymph) Fat-body (buffer) Fat-body (buffer-BSA)

F F A M G L D G L T G L

600 24 ° 195 480 630 o 375 270 12o 15 15 720

1,785 18o o 285 828 257 o 114

9,800 1,8oo 6,060 62,820 5,040 780 lO,O8O 62,14o

lO,98o 1,83o 12,789 74,520 9,780 78o 1I,I6O 87,480 6,6oo 60o 9,9oo 68,I2O

Pre-labelled fat-body: 2 g. (wet weight) of fat-body from ten 5th-instar larvae were incubated in 2'0 ml. phosphate/saline buffer (0"9 per cent NaCI in O'Ol M K2HPO4/KH~PO4, p H 7"4) containing sodium palmitate-I-t~C (2.1 × lO 6 d.p.m.) for I hour at 32 ° C. Re-incubation proceeded as described for Table V. Undiluted haemolymph from pharate adults (I day prior to pupal-adul t ecdysis) was used inclusive of haemocytes.

Information contained in Table VII indicates that the total quantity of lipid of any class released from pre-labelled fat-body is negligible.

EXPERIMENTS WITH Manduca sexta: INCUBATION OF PRE-LABELLED PUPAL FAT-BODY IN VARIOUS MEDIA

Table VII I contains the results from a typical experiment in which pupal fat-body (pre-labelled) was incubated in various media. Analysis of the radioactivity released into the medium reveals that most of the label appears in the FFA fraction in both haemolymph and buffered BSA. The effect of BSA in enhancing FFA release from the fat- body is strikingly seen in these pupal experiments. FFA radioactivity found in the buffer controls is, in all cases, much lower than in haemolymph or buffered BSA.

1971, I UPTAKE AND RELEASE OF FAT-BODY LIPIDS 71

Table I X reveals that undiluted haemolymph is more effective in enhancing F F A release f rom pre-labelled fat-body than is either 3 ° or 5o per cent haemolymph diluted with buffer/saline. T h e saline buffer used in this experiment was essentially the one used by Chino and Gilbert (i965), but regardless of the buffers used, radioactivity

Table V I I . - - ' N E T G A I N ' * OF LIPIDS IN THE MEDIUM AFTER INCUBATION OF PRE- LABELLED LARVAL FAT-BODY OF manduca sexta WITH HAEMOLYMPH OR BUFFERED

BSA (I per cent)t

MEDIUM

Experiment z Haemolymph Haemolymph (pharate adult) Phosphate/saline/i per cent BSA

Experiment 2 Haemolymph Phosphate/saline/i per cent BSA

Experiment 3 Haemolymph Haemolymph (50 per cent) Phosphate/saline/I per cent BSA

LIPIDS (mg. per ml. medium)

FFA M G L DGL T G L

0"020 0"075 0"059 o'ooo 0"031 O'OOO O" 1 2 0 O'OOO o ' 1 o 2 0"055 o'ooo o'ooo

0 " 0 0 0 0 ' 0 0 0 0 " 0 0 0 0 " 0 0 0

0'3oo o'ooo o'ooo 0"083

O'OOO O'OOO 0 " 0 0 9 O'OOO

0 " 0 7 2 O'OOO O'OOO O'OOO

0 " 2 8 0 o . o o o 0 " 0 0 0 o ' o o o

* The 'net gain' of lipids in the medium was obtained by subtracting the post-incubation weight of each lipid fraction in the phosphate/saline medium from: (a) the increase in weight of each lipid fraction in the haemolymph upon incubation, or (b) the post-incubation weight of each lipid fraction in the buffered BSA medium. All negative values were considered equivalent to no net gain.

t The above data were compiled from 3 different experiments. Haemolymph used in the experiments was undiluted unless designated otherwise in the table.

Table VIII .--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCU- BATION OF PRE-LABELLED PUPAL FAT-BODY OF Manduca sexta IN VARIOUS MEDIA


Medium Haemolymph Phosphate/saline buffer Phosphate/saline buffer/l per cent

BSA Tissue

Fat-body (haemolymph) Fat-body (buffer) Fat-body (buffer-BSA)


FFA M G L DGL T G L

I64,37o 29,490 17,46o 2,52o 36,900 2,700 5,490 x,44o

211,23o 20,850 19,17o 7,890

25,38o 18,48o 94,8oo 342,96o 17,28o I3,o2o 59,IOO 51o,ooo 26,7oo 34,o8o I81,92o 5o2,26o

Pre-labelled fat-body: I g. (wet weight) of fat-body from 8 post-diapause pupae was incubated for 1 hour as described for Table V with sodium palmitate-i-l~C (7"o × lO 6 d.p.m.). One hundred and fifty mg. of pre-labelled fat body were re- incubated as previously described.

72 CHANG AND FRIEDMAN _Insect Biochem.

appeared in greatest quantity in haemolymph and buffered BSA. It is also interesting to note the non-specificity of the haemolymph in stimulating release of lipids from the fat-body; when 'chil led ' pupal cecropia or grasshopper (M. differentialis or Dissosteira carolina) haemolymph was incubated with hornworm pupal fat-body, F F A was prefer- entiaUy released (Table X). Chino and Gilbert (I965) showed a similar lack of specificity of haemolymph using grasshopper fat-body (M. differentialis) with cecropia pupal haemolymph.

Table-/X.--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF PRE- LABELLED PUPAL FAT-BODY OF Manduca sexta IN VARIOUS MEDIA

EXPERIMENT

I*

2t


Medium Haemolymph Haemolymph (30 per cent) Phosphate/saline buffer

Tissue Fat-body (haemolymph) Fat-body (30 per cent haemolymph) Fat-body (buffer)

Medium Haemolymph Haemolymph (50 per cent) Phosphate/saline buffer Phosphate/saline buffer/x per cent BSA

Tissue Fat-body at o time Fat-body (haemolymph) Fat-body (50 per cent haemolymph) Fat-body (buffer) Fat-body (buffer-BSA)


FFA MGL DGL T G L

11,786 536 257 o 7,671 707 407 o 1,821 17I 257 o

36,257 3,000 2,400 I7,543 39,514 1,8oo 3,771 12,514 35,857 1,714 2,828 19,2oo

17,634 929 648 929 11,549 789 o 84 8,535 84 o o

47,549 1,915 225 o

17,577 6,591 6,591 14,o56 23,915 1,676 I3,211 12,253 41,239 3,634 15,324 I6,I69 46,732 1 ,5 2 1 12,789 11,38o 49,267 1 ,521 14,o56 14,o56

* 1 g. (wet weight) of fat-body from lO post-diapause pupae was incubated for I hour with sodium palmitate-x-l~C (1"65<1o 6 d.p.m.)as described for Table V; 15o mg. of the pre- labelled fat-body were re-incubated as previously described.

Jf Pre-labelled fat-body: 2 g. (wet weight) of fat-body from 12 post-diapause pupae were incubated in 1"5 ml. phosphate/saline buffer (o'15 M KC1 in o'o5 M K~HPO,/KH6PO4, pH 6"7) containing sodium palmitate-I-14C (3"3 5< lO 6 d.p.m.) for I hour at 32 ° C. Re-incubation was carried out as described for Table FIlL

Table X I shows the distribution of weight among the lipid classes present in the medium after incubation with pre-labelled fat-body. I t is obvious that most of the increase occurred in the FFA fraction. Specific activities of the lipids in the haemolymph medium and fat-body are contained in Table X l I . Each of the lipid fractions has a lower specific activity in the medium than its counterpart in the fat-body. (The M G L fraction is present in such low concentrations that measurement of change, from which its specific activity is derived, is inaccurate.) Th e results indicate that labelled lipids are released from the fat-body and diluted by ' co ld ' endogenous lipids already present in the haemolymph.


The time-course of lipid release into haemolymph from pre-labeUed pupal fat-body is plotted in Fig. 3. There is a large amount of FFA released during the first 3o minutes

Table X.--DIRTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF PRE- LABELLED PUPAL FAT-BODY OF Manduca sexta IN GRASSHOPPER OR CECROPIA HAEMOLYMPH

INCUBATION MIXTURE

Hornworm pupal fat-body* with cecropia pupal haemolymph

Hornworm pupal fat-bodyt with grasshopper (adult) haemolymph


FFA M G L D G L T G L

20,366 789 1,493 o

88,71o 19,68o 6,990 360

* Incubation conditions as in Table VI I I . t Incubation conditions as in Table IX , experiment 2.

Table X I . - - ' N E T GAIN' OF LIPIDS IN THE MEDIUM AFTER INCUBATION OF PRE-LABELLED PUPAL FAT-BODY OF Manduca sexta with HAEMOLYMPH OR

BUFFERED BSA (I per cent)

MEDIUM

Experiment 1 Haemolymph Phosphate/saline buffer/I per cent BSA

Experiment 2 Haemolymph Phosphate/saline buffer/i per cent BSA

LIPIDS (nag. per ml. medium)


2"555 0"259 o"317 o'157 4"424 o'ooo o'o35 o.ooo

lO"498 0"532 0"243 o'ooo 24"218 o'71o o.ooo o . o o o

The ' net gain' of lipids in the medium was obtained as in Table VII .

Table XII.--DISTRIBUTION OF SPECIFIC ACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF PRE-LABELLED PUPAL FAT-BODY OF Manduca sexta

IN HAEMOLYMPH

COMPONENT

Experiment I Haemolymph Fat-body at o time

Experiment 2 Haemolymph Fat-body at o time

DISINTEGRATIONS PER MINUTE PER rag. LIPID


3,o14 + * 69 36 5,214 281 798 712

8,242 + * 7 0 o 9,65 ° 1,29o 1,526 1,453

* The values for M G L could not be measured with accuracy.

7 4 CHANG AND FRIEDMAN Insect Biochem.

of incubation, after which the rate begins to decrease. The radioactivity levels of the medium glycerides remain low throughout the incubation period.

To reveal whether the release of FFA involves an expenditure of energy by the tissue, the release experiments were carried out in the presence of metabolic inhibitors (sodium 10c

8C

~2 ; 60.

6 -o

40

20.

~:=~--o ? o.. ~. - " o 3'o 60 9b t20 f~o 18o 2io 2,~o

Time (minutes)

FIC. 3 . - -Time-course of lipid release when pre-labelled pupal fat-body of Manduca sexta was incubated in undiluted haemolymph (inclusive of haemoeytes). O, F F A ; m, T G L ; O, D G L ; &, M G L .

Table XIH.--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATING PRE-LABELLED PUPAL FAT-BODY OF Manduca sexta WITH

HAEMOLYMPH CONTAINING VARIOUS INHIBITORS

MEDIUM

Haemolymph (80 per cent) Haemolymph with K C N Haemolymph with Na~HAsO4 Haemolymph with NaN3 Phosphate/saline buffer

D I S I N T E G R A T I O N S PER M I N U T E

F F A M G L D G L T G L

89,879 7,848 1,151 I,ooo 85,795 6,636 1,636 424 45,IZO 4,697 879 667 67,7oo 6,9o9 1,545 7z7 38,454 4,212 I,OOO 969

Pre-labelled fat-body: 2 g. (wet weight) of fat-body from IO post-diapause pupae were incubated as described for Table V I in medium containing sodium palmitate-l-14C as the BSA complex (4"6 x 1o e d.p.m.). Re-incubation in each of the following media proceeded as described for Table V. The media were: haemolymph (80 per cent) (diluted with buffer/saline); haemolymph (80 per cent) containing a final concentration of 40 micromoles per ml. cyanide, azide, or arsenate in phosphate/saline buffer; phosphate/ saline buffer. Haemolymph with cells were used in the experiment.

azide, potassium cyanide, and sodium arsenate) each at a final concentration of 4 ° ~tmoles per ml. haemolymph. In Table X I I I it may be seen that a 3 ° per cent inhibition of FFA release is effected with azide while a 5 ° per cent inhibition is realized with arsenate. The addition of the inhibitors seems to have no effect on any of the other lipid fractions. There is presently no explanation for the ineffectiveness of cyanide.

I971, I UPTAKE AND RELEASE OF FAT-BODY LIPIDS 75

EXPERIMENTS WITH Manduca sexta: INCUBATION OF PRE-LABELLED PHARATE ADULT FAT-BODY IN VARIOUS MEDIA

Difficulties arose in obtaining sufficient quantities of haemolymph and fat-body from hornworm moth adults. Since the results of Beenakkers and Gilbert (i968) with adult cecropia were not altered by the use of pharate adults (i day before adult eclosion), it is assumed in our lipid release experiments that events occurring in the hornworm pharate adult reflect similar events in the adult moth.

Table X I V shows the distribution of radioactivity after the incubation of pre-labeUed pharate adult hornworm fat-body in haemolymph and buffer/saline. Radioactivity

Table XIV.--DISTRIBUTIONS OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF FRE-LABELLED PHARATE ADULT FAT-BODY OF Marldf, lca sexta

IN VARIOUS MEDIA

RE-INCUBATION-MIXTURE CONSTITUENT

Medium Haemolymph Haemolymph (70 per cent) Phosphate/saline buffer

Tissue Fat-body (haemolymph) Fat-body (70 per cent haemolymph) Fat-body (buffer)


FFA MGL DGL TGL

17,o36 21 1,628 21 lO,628 578 1,286 21

Io7 1,5oo 321 193

16,o28 I,o28 6,771 19,o28 13,457 1,457 9,086 13,971 2o,914 6oo 5,743 8,057

The incubation was as described for Table V except that it was continued for x hour. Sodium palmitate-I-t~C content was I'7 × lO 6 d.p.m, and fat-body from 15 pharate adults was used. Re-incubation was as described for Table VIII.

appeared in large amounts in the haemolymph within x hour of incubation whereas no appreciable radioactivity could be found in the buffer/saline medium. Again, as in the pupal hornworm experiments, there was little glyceride-associated radioactivity released.

EXPERIMENTS WITH OTHER INSECTS: Leucophaea maderae, Hyalophora cecropia, AND Schistocerca gregaria

As can be seen from Table X V , the most significant release of lipid from pre-labelled cockroach fat-body into haemolymph was in the FFA fraction. No appreciable radioactivity was found in the buffer/saline medium after incubation. However, as in the hornworm experiments, buffered BSA elicited the release of significant amounts of FFA from the fat-body. Our results with cockroaches are similar to those recently obtained by Bhakthan and Gilbert (i968), and in conflict with earlier results from the same laboratory (Chino and Gilbert, x965).

In contrast to the results of Chino and Gilbert (i965), and Beenakkers and Gilbert (I968), we have only been able to demonstrate a very low level of DGL release from pre-labelled cecropia fat-body into pupal haemolymph. Results from a typical experiment are shown in Table X V I . We have no explanation for this discrepancy and have no reason to believe that our experimental animals (' chilled' pupae) were in any way physiologically different from those used by Chino and Gilbert (i965). It should also be


pointed out that although the release of F F A and T G L was not appreciable, it was always significantly higher than the controls.

In our hands there appear to be differences in the pat tern of isotope incorporation into the fat-body lipids of hornworm and cecropia. Fig. 4 is a picture of the in vitro

Table XV.--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBATION OF PRE-LABELLED ADULT FAT-BODY OF Leuco )haea maderae IN VARIOUS MEDIA

RE-INCUBATION-MIXTURE CONSTITUENT

Medium Haemolymph Phosphate/saline Phosphate/saline buffer/I per cent BSA

Tissue Fat-body at o time Fat-body (haemolymph) Fat-body (buffer) Fat-body (buffer-BSA)

Fat-body from 30 adult cockroaches was


FFA MGL DGL TGL

14,142 1,514 I,OOO 200 570 628 228 942

16,571 2,085 657 971

6,788 1,456 12,879 86,777 4,685 685 13,885 76,514 3,428 1,o28 lO,628 77,657 1,942 1,314 6 ,971 52,574

incubated and re-incubated as described for Table V. The incubation mixture contained sodium palmitate-I-'4C (2"2 × 106 d.p.m.) and incubation was continued for I hour.

Table XVI.--DISTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBA- TION OF PRE-LABELLED PUPAL FAT-BODY OF Hyalophora cecropia IN VARIOUS MEDIA


Medium Haemolymph ttaemolymph (50 per cent) Phosphate/saline buffer

Tissue Fat-body (haemolymph) Fat-body (haemolymph 5o per cent) Fat-body (buffer)


FFA MGL DGL T G L

1,577 394 3,296 1,634 2,704 o 3,070 1,268

780 o 451 158

3,380 1,69o 63,718 338 1,859 2,366 43,775 760 3,296 1,944 59,070 507

Pre-labelled fat-body: 2 g. (wet weight) of fat-body from 5 post-diapause pupae were incubated as described in Table IX, experiment 2. The mixture contained sodium palmitate-x-a4C as the BSA complex (2"9 × lO 6 d.p.m.). Re-incubation was as described for Table V. Haemolyrnph was used with the haemocytes removed.

incorporation of palmitate-i-14C into pupal cecropia fat-body as a function of time. At 60 minutes after incubation begins, the fat-body shows higher counts in D G L than in any other lipid fraction, a marked contrast to the larval hornworm fat-body in which T G L is heavily labelled within 15 minutes (Fig. 2). Although a similar experiment has not been done with hornworm pupal fat-body, the labelling pat tern after I hour (Tables V I I I and 1X) indicates that it follows the larval pattern, albeit more slowly. A


1-hour pattern similar to the one we have shown in cecropia was demonstrated some time ago by Chino and Gilbert (1965) in the same insect as well as in grasshopper and cockroach. T h e results simply suggest a slower turnover of M G L - ~ D G L - * T G L (if indeed this is t h e correct biosynthetic route)• After 3 hours of incubation, the cecropia

2 t 180

16

30 60 90 120 150 180 240 "lime (minutes)

FIG. 4.--Time-course of in vitro incorporation of sodium palmitate-I-14C into the neutral lipids of the pupal fat-body of Hyalophora cecropia. O, TG L; R, DGL; O, FFA; A, MGL.

Table XVII.--DIsTRIBUTION OF RADIOACTIVITY IN LIPID FRACTIONS AFTER INCUBA- TION OF PRE-LABELLED ADULT FAT-BODY OF Schistocerca gregaria IN VARIOUS MEDIA


Medium Haemolymph (80 per cent) Phosphate/saline buffer Phosphate/saline buffer/I per cent BSA

Tissue Fat-body (haemolymph) Fat-body (buffer) Fat-body (buffer/I per cent BSA)


FFA MGL DGL T G L

20,460 2,940 61,74o 3,12o 2,220 12o 7,740 2,820

19,92o 4,I4O 89,520 22,200

5,94 ° 6,540 I4,52o I I4,48o 5,760 1,86o 9,060 232,48o 3,6oo 1,56o 5,7oo 21o,24o

The incubation was carried out as described for Table VI using approximately o.8 g. (wet weight) of fat-body and sodium palmitate-1-14C as the BSA complex (2"5 × Io 6 d.p.m.). Re-incubation was as described for Table VIII.

pattern resembles that of larval hornworm fat-body incubated for i hour in the radio- active medium. There is at present no evidence to indicate that this is in any way connected with the liberation of lipid as D G L by cecropia.

Using Sehistoeerca gregaria adult fat-body, our experiments showed release of both D G L and F F A label into haemolymph (Table XVII ) . No significant radioactivity was


found associated with any of the other lipid fractions in the medium. Similar results were obtained by Tietz (i967) using Locusta migratoria and Mayer and Candy (1969) with S. gregaria. It is interesting to note that in our experiments with Schistocerca, BSA (i per cent) not only enhanced the release of DGL and FFA but also increased the liberation of TGL.

DISCUSSION It is by now fairly well accepted that lipids play an important role in the flight meta-

bolism of many insects, especially members of the order Lepidoptera (Beall, 1948; Zebe, 1954; Chino and Gilbert, 1965, and others).

Our interest in the problem of lipid release and transport was stimulated by the reported difference between the pathways taken by mammals and insects, and by a critical examination of the analyses and interpretations of the insect experiments in which were revealed several anomalies. Calculations made from the date of Chino and Gilbert (1965) on cecropia showed that although a proportionately larger amount of D G L than T G L was released from fat-body, a larger total amount of the latter may have been released. Examination of the literature revealed a further anomaly with respect to the state of the lipids released from the fat-body of adult Periplaneta americana. Chino and Gilbert (I 965) reported preferential D GL release into haemolymph, whereas experiments done by Cook and Eddington (1967) revealed that T G L and FFA represented the major fractions liberated. To complicate the picture, Bhakthan and Gilbert (I968), examining the effects of several hormones on adipokinetic processes in cockroaches, showed that fat-body of control P. americana liberated an appreciable amount of FFA into the haemolymph, while far less radioactivity was found in the glyceride fractions.

In an effort to determine whether development affects the rate and class of lipid moved, we have examined the larval, pupal, and pharate adult stages of the tobacco hornworm, Manduca sexta. Our in vitro experiments reveal that fat-body from the larval stage of the hornworm does not release appreciable amounts of lipid into a haemolymph medium. (In contrast to this, Wlodawer and others (I966), working with the wax moth (GaUeria mellonella), showed FFA movement from larval fat-body into both haemolymph and buffered saline media.) In the pupal stage of the hornworm, there is appreciable release of FFA from the fat-body into haemolymph while no significant release of any other lipid fractions takes place. Controls consisting of fat-body incubated in buffered saline show a much lower rate of liberation, but the addition of I per cent BSA to this medium greatly enhances FFA release. That the effect of BSA is specific is shown by the fact that casein has little or no effect on this movement (Table XVIII). The BSA effect has also been demonstrated in other insect preparations (Tietz, i962; Chino and Gilbert, 1965 ; Wlodawer and others, 1966 ).

Our findings of preferential FFA release in pupal and pharate adult stages of the tobacco hornworm are consistent with what is known of mobilization of lipid from mammalian adipose tissue. In the latter situation only FFA are released to complex with the serum albumins in the blood (Masoro, 1968 ).

In support of our findings in Manduca sexta, the following points are significant: (x) The in vitro pattern of labelling found in fat-body is similar to that found in fat-body of larvae pulse-fed labelled tomato leaves; (2) the specific activities of the lipid fractions found in the haemolymph medium after incubation with pre-labelled fat-body are lower than their counterparts in the fat-body; (3) an exhaustive evaluation of the methodology


of extraction and quantitation showed very good recoveries of lipid standards (labelled) from haemolymph and other media; (4) FFA release in Leucophaea maderae (as shown by Bhakthan and Gilbert, i968), and D G L release in Schistocerca gregaria (as shown by Mayer and Candy, 1969) were confirmed using the methodology employed for studying lipid movement in the tobacco hornworm; (5) metabolic inhibitors were shown partially to inhibit FFA release from the fat-body, indicating that the process is an 'active' one (i.e., requiring an expenditure of energy); and (6) there is a definite similarity to lipid release patterns found in mammals.

A scheme of lipid release and transport in the tobacco hornworm may be constructed from the results obtained in this study: Towards the end of the larval stage there is an

Table XVIII.--DISTRIBUTION OF RADIOACTIVITY IN THE LIPID FRACTIONS AFTER INCUBATING PRE-LABELLED PUPAL FAT-BODY OF Manduca sexta IN BUFFER--SALINE CONTAINING CASEIN AND

VARIOUS CONCENTRATIONS OF BOVINE SERUM ALBUMIN (FRACTION V)


Medium Approximately I per cent BSA (11"4 mg. protein per ml.)

BSA (1.14 mg. protein per ml.) BSA (o'114 rag. protein per ml.) Casein (8-0 rag. protein per ml.) Phosphate/saline buffer

Tissue Fat-body (1I'4 mg. BSA per ml.) Fat-body (1"14 mg. BSA per ml.) Fat-body (o"114 rag. BSA per ml.) Fat-body (8 rag. casein per ml.)


FFA MGL DGL TGL

131,454 3 ,303 2,394 1,ooo lO8,424 9,I5I 2,151 970 38,030 3 ,939 1,576 364 39,030 3 ,606 1,667 1,182 38,454 4 , 2 1 2 I,OOO 969

6,364 8 ,182 21,273 208,727 14,545 11,454 39,273 274,14o 9,454 9,182 34,454 222,720 7,545 4 ,545 22,367 222,909

Pre-labelled fat-body: The conditions for labelling were the same as those outlined in TableXVI. BSA and casein were dissolved in phosphate/saline buffer and a protein analysis (Goa, 1953) carried out on the mixtures.

enormous increase in food intake accompanied by a conversion and storage of much of this nutrient as lipid in the fat-body. At this stage of development, there is little or no lipid release into the haemolymph, and the general level of lipid in the haemolymph is low. ~ During the pupal stage, intense metabolic activity and reorganization of material into adult tissue (histolysis and histogenesis) occurs. At this time, the fat-body becomes ' capable' of releasing lipid into the haemolymph, chiefly as FFA, for energy and other pur- poses. Release is stimulated by a factor or factors present in the haemolymph. Lipases present in the homworm fat-body and haemolymph (to be reported elsewhere at a later date) catalyse interconversions between the glycerides and FFA and glycerol. This mode of release, first exhibited in large measure in the pupal stage, is carried over into the adult stage, where the liberated fatty acids are used as a major source of energy by the flight muscles.

ACKNOWLEDGEMENT

This work was supported by U.S.P.H.S. Training Grant ITI GM-IO76 to F. C. and N.I.H. Grant number AI-o6345 to S. F.

80 CHANG AND FRIEDMAN

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Key Word Index: Physiology, insects, tobacco hornworm (Manduca sexta), locust (Schistocerca gregaria), cockroach (Leucophaea maderae), silkmoth (Hyalophora cecropia), fat-body, lipid content, haemolymph, lipid release, development, lipid distribution, radiochromatography.

a developmental analysis of the uptake and release of lipids by the fat-body of the tobacco...

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