Insect Biochem., i972 , 2, I6 I - I66 . [Scientechnica (Publishers) Ltd.] x6I

INCORPORATION OF [I-14C]PALMITATE INTO LIPIDS

AND THE PATTERN OF HAEMOLYMPH PROTEINS IN

DIAPAUSE AND NON-DIAPAUSE LARVAE OF THE BEETLE

TROGODERMA GRANARIUM

EPHRAIM COHEN* Department of Entomology, Hebrew University of Jerusalem, Jerusalem, Israel

(Received 24 September, I97I )

ABSTRACT Trogoderma granarium lipids were separated into io classes by thin-layer

chromatography. The lipids detected were phospholipids, monoglycerides, diglycerides, sterols, free fatty acids (FFA), triglycerides, sterol esters, and hydrocarbons.

[i-14C]Palmitate is esterified rapidly into TG. After 0"5 hour a third of the total label was found in the TG fraction with a concomitant loss in the FFA. The rate of incorporation was similar in non-diapause and diapause larvae.

Electropherograms from diapause and control larvae showed differences in the protein pattern which may reflect the physiological and nutritional state of the two groups of animals.

THE Khapra beetle, Trogoderma granarium, is known as an important pest of stored cereals in hot countries. Several investigations have been carried out on the biological and ecological aspects of the insect (Voelkel, i924; Shulov, i955; Hadaway, i956; Burges, i959b ).

The characteristic of T. granarium of entering a state of weak facultative diapause or quiescence was studied thoroughly by Burges (I959a,c , i96o , i962, i963). A delay in pupation was affected by accumulation of larval faeces in the food (Stanic, Shaaya, and Shulov, t963) and further investigations demonstrated the role of faecal lipids in such a delay (Karnavar and Nair, i969b ; Ikan, Stanic, Cohen, and Shulov, i97o ). Karnavar and Nair (i969a) have shown differences in body-weight, fat, glycogen, and proteins between diapause and non-diapause larvae.

The present paper deals with the investigation of metabolic changes in the rate of palmitic acid incorporation into higher lipid molecules as well as with differences in the haemolymph proteins between non-diapause and diapause larvae of T. granarium.

M A T E R I A L S A N D METHODS EXPERIMENTAL MATERIAL

Trogoderma granarium was raised on broken wheat and kept at 380 C. and about 4o per cent r.h. Diapause and non-diapause larvae were obtained according to a procedure described by Ikan and others (i97o).

* Present address: Department of Biological Sciences, Northwestern University, Evanston, Illinois 6o2oi, U.S.A.

62 COHEN Insect Biochem.

PREPARATION OF AQUEOUS SOLUTION OF [ I - t 4 C ] P A L M I T A T E

According to the method of Masironi and Decopas (1961) the benzene solution of [ i -HC]palmit ic acid (Radiochemical Centre, Amersham) was evaporated and a sodium salt prepared by adding o 'o4 M methanolic sodium hydroxide. T h e methanol was removed and the labelled sodium palmitate conjugated to aqueous bovine serum albumin.

INJECTIONS

The injections were per formed with a syringe microburet te (Model No. SB2, Micrometr ic Ins t rument Co., Cleveland, Ohio) using hypodermic needles 3oG, { in. Diapause and non- diapause larvae were anaesthetized with carbon dioxide and o '4 lal. of labelled palmitate solution was injected into the ventral side of the abdomen of each larva. The animals were then re turned to their original diets.

EXTRACTION AND ISOLATION OF THE L I P I D S

Whole larvae were homogenized with a glass grinder in a chloroform/methanol (2 : 1) solution and the lipids were extracted according to the method of Folch, Lees, and Sloane Stanley (I957). T h e lipid extract was appl ied on silica gel G plates (2o × 20 cm., o'25 mm.) and developed three times with petrol ether/diethyl ether/acetic acid (85 : 15 : 1). T h e classes of lipids were identified by comparison with pure standards after spraying with 5o per cent sulphuric acid and subsequent charr ing at 17 °° C. For experimental purposes the extract was applied on preparative plates (o'5o mm.) and developed in the manner ment ioned above. Exposure to iodine vapour revealed yellow spots of the separated lipids which were scraped off into tubes. T w o ml. of chloroform were added and the tubes were centrifuged for IO minutes at 45oo r.p.m. T h e supernatant was decanted into scintillation vials and dried. T e n ml. of a toluene-based scintillation l iquid were poured into each vial and the radioactivity was measured in a Packard Tr icarb l iquid scintillation counter.

Table /.--INcoRPORATION OF [I-'4C]PALMITATE INTO LIPIDS OF DIAPAUSE AND NON-DIAPAUSE T. granarium LARVAE

TiME (hours)

o'5

22

LARVAE

Control Diapause

Control Diapause

Control Diapause

Control Diapause

Control Diapause

PERCENTAGE LABEL RECOVERED IN LIPID CLASSES

PL

o" 4 0" 3

I ' I

1"2

2"4 I 'O

I ' O

1"2

0"3 I ' I

M G

0. 3 O ' I

0" I

0 " I

0' 3

O ' I

0 '2

O ' I

0"0

0"2

D G

3"8 3"1

6"8 9"6

4"2 4"o

3"6 4"7

1"8

1" 3

F F A

87" 5 87" 9

62"3 65"6

58-0 62"7

59"6 64-i

60"9 66-i

T G

8" I

8"4

28" 9 22"8

34"0 3I'5

34"6 28"9

35"0 29"7

SE

0 " 0

0 " 0

o'8 o'8

I ' l

o" 7

I ' I

I 'O

1"4 I-6

T h e values are the averages of 3 experiments each of which contained groups of IOO non- diapause and ioo diapause larvae injected with [ '4C]palmitate solution (about 4ooo c.p.m, to each larva). T h e animals were transferred to their original diet and after an appropriate period 2o individuals of either group were sacrificed and handled as described in Materials and Methods.

I972, 2 L I P I D S A N D P R O T E I N S I N LARVAE IN DIAPAUSE i63

COLLECTION OF HAEMOLYMPH Both diapause and non-diapause larvae were punctured with a fine needle in the region of the

head, and drops of clear blood were collected with a fine glass capillary. The blood was immediately transferred to a tube placed in a mixture of crushed ice and sodium chloride to prevent darkening.

DIsc ELECTROPHORESIS The procedure described by Davis (1964) was followed. Samples of haemolymph were mixed

with an equal volume of 4 ° per cent sucrose solution containing o'o5 per cent bromophenol blue as the tracking dye. The mixture was applied directly on the separating gel (9"5-cm. columns) and the gels were subjected to a current of 2"5 mA. per gel for a period of about 3"5 hours.

RESULTS

Fig. I reveals that IO bands of lipids can be separated corresponding to 8 lipid fractions, i.e., phospholipids (PL), monoglycerides (MG), diglycerides (DG), sterols (S), free fatty

HYDROCARBONS ~ SOLVENT FRONT

STEROLESTERS O

TRIGLYCERIDES •

TRIGLYCERIDES O

FATTY ACIDS •

STEROLS O

DIGLYCERIDES (:~

DIGLYCERIDES (:D

MONOGLYCERIDES 0 PHOSPHOLIPI DS o

FIG. i.--Separation of lipids from T. granarium larvae. Spots were detected after spraying silicage gel G o'25-mm, plates with 5 ° per cent w/w sulpburic acid and subsequent charring at 17 °0 C.

acids (FFA), triglycerides (TG) , sterol esters (SE), hydrocarbons (HC). The plates were developed three times in order to obtain improved separation between the fractions. Preliminary experiments indicated that bands of sterols and hydrocarbons had negligible amounts of radioactivity, and they were therefore not collected. Th e 2 bands of D G and T G were combined.

Table I and Fig. 2 summarize the rate of incorporation of labelled palmitate into other compounds in diapause and non-diapause larvae. Normal larvae do not differ from the diapause animals and incorporate palmitic acid into T G only slightly faster. Generally it can be noticed that one-third of the label at zero time is detected in the T G , reaching a plateau after half an hour. Some radioactivity is found in the D G bands which decreases with time from about 6-8 per cent to 1.8 per cent in the non-diapause animals and from 9.6 per cent to 1. 3 per cent in diapause ones. Radioactivity increases after 22 hours in the sterol esters fraction.

In both larval types there is a rapid increase in incorporation of labelled palmitic acid into T G accompanied by a parallel decrease in the label of FFA, which reach a plateau

16 4 COHEN Insect Biochem.

after z hours. Fig. 3 demonstrates a different pattern of haemolymph proteins between normal and diapause larvae. The fast-migrating proteins (1-8) are qualitatively and quantitatively similar as can be judged from the intensity of staining. Band 9 in the diapause larvae is divided into 2 bands in the normal counterpart.

C P M

5 0 0 0

4000

3000

2000

1300

D

~5 74 13

I;

/ " rG . o

I [ i 1 i i I

2 3 4 5 6

T I M E (hrs)

FIG. z.--The course of [i-14C]palmitate incorporation into triglycerides of diapause larvae of T. granarium.

r o

~9

8

!

T D

N

~4 13

H

rO

9A o

G

4 ~ZI~Y

2 v D ~ m m m ~

i J Fic. 3.--Disc electropherograms of haemo-

lymph proteins of T. granarium larvae. D = Diapause larvae; N - non-diapause larvae; TD - tracking dye.

In the slow-migrating proteins z major bands appear (IO and I2) in both larval types, which are presumably lipoproteins because of their yellow colour in unstained gels. Part of the remaining proteins vary in their electrophoretic migration, and diapause larvae contain in addition one more protein band.

SUMMARY AND DISCUSSION Ikan and others (I97o) have shown an overall similarity in fatty acid pattern in diapause

and non-diapause T. granarium larvae, although the diapause type does contain slightly more unsaturated acids. The present study reveals that [14C]palmitate is incorporated into the lipid fractions of both larval groups at about the same rate. After e hours approximately a third of the label is detected in TG with a concomitant decrease in the FFA fraction. Minor amounts of radioactivity are found in DG, reaching a maximum after o. 5 hour and declining after 22 hours, while SE and PL increase gradually. Yurkiewicz and Mathur (1969) have shown that the same process occurred in blowflies, with [14C]palmitate transferred to the TG fraction, accompanied by a decrease in FFA and some radioactivity also incorporated into other lipid classes. Bhakthan and Gilbert

1972, 2 LIPIDS AND PROTEINS IN LARVAE IN DIAPAUSE 165

(i97o) noted in their in vitro experiments that palmitic acid is rapidly esterified to T G with only a small incorporation into DG. No label was found by them in the MG. Th e above authors have studied the site of esterification by light and electron microscope autoradiography and have shown it to be located in the endoplasmic reticulum, from where the product is transferred to lipid globules. The fact that diapause T. granarium larvae feed and are mobile like normal larvae may explain the similarity in [14C]palmitate incorporation between the two groups.

The small amount of blood required for protein separation by disc electrophoresis has facilitated the investigation of haemolymph proteins (Clark and Ball, 1956 ; Mitlin, Lusk, and Wiygul, 1967; Wang and Patton, 1968). This technique was applied to the separa- tion of blood proteins from T. granarium larvae. The differences in the pattern of haemo- lymph proteins in diapause and non-diapause larvae may relate to divergent physiological conditions of both larval types: their different nutritional status, the accumulating quantities of faeces in diapause populations, and their continued growth in size without pupation. On the other hand, non-diapause larvae feed on fresh food and are able to pupate.

ACKNOWLEDGEMENT

This work was supported by grant No. FG-Is-25o, United States Department of Agriculture.

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166 COHEN

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Key Word Index: Trogoderma granarium, diapause, fatty acids, lipids, [i-14C]palmitate, disc electrophoresis, haemolymph proteins.