DIgestion and absorption of glycerophospholipid in Pieris brassicae

Camp. Biochem.

Physiol.

Vol. 89A,

No.

I, pp. 19-24,

0300-9629/88

1988

$3.00 + 0.00

0 1988Pergamon Journals Ltd

Printed in Great Britain

DIGESTION AND ABSORPTION OF GLYCEROPHOSPHOLIPID IN PIERIS BRASSICAE SEPPO TURUNEN Department of Zoology, University of Helsinki, 00100 Helsinki, Finland (Receiued 13 April

1987)

Abstract-l. In larvae of Pieris brussicae ingestion of dietary di(l-‘4C)oleoyl phosphatidylcholine was followed within 4 hr by release of free fatty acids (ca 50% of radioactivity) in the midgut lumen. 2. The remaining radioactivity was present chiefly in new polar lipids. 3. Within 24 hr, polar lipids were the major radiolabeled luminal lipids. 4. Synthesis of triacylglycerol (TG) was the predominant early absorption-related event in midgut cells, followed and then exceeded by synthesis of phosphatidylcholine (PC). 5. Lipid was absorbed into the anterior midgut to a greater extent than into the posterior midgut, and released into the hemolymph at a greater rate from the anterior, than from the posterior, midgut. 6. Half-life of lipid radioactivity was about 18 hr in the anterior, and about 26 hr in the posterior

midgut. 7. Enterocyte lipids with the shortest half-life (ca 15 hr) in fifth instar larvae were TG and PC. 8. The data imply a role for TG and PC in the release of diacylglycerol into the hemolymph.

present study more conclusive evidence was obtained on differences in the rate of lipid absorption along the midgut of larval P. brussicae.

INTRODUCTION

The digestion and absorption of various lipids in the insect intestine has been studied in recent years in locusts, cockroaches, flies and lepidopteran larvae, and the subject was reviewed recently (Turunen, 1985). Diacylglycerol is the major lipid released from the midgut into the hemolymph in Locusta migratoriu (Weintraub and Tietz, 1973, 1978), Schistocerca gregaria (Thomas, 1984), Peripfuneta americana (Chino and Downer, 1979), Pieris brussicae (Turunen, 1975) and Diatraea grandiosella (Turunen and Chippendale, 1977). Absorption of unesterified fatty acids or acylglycerols is followed by mucosal synthesis of triacylglycerol (TG) and phospholipids (PL) in these insects. The significance of this absorption-related synthesis is unknown, however. The present study provides new information on the possible role of TG and PL synthesis in midgut cells. In P. brussicae glycerol trioleate is hydrolyzed slowly and incompletely, and in analysis of midgut lumen of feeding fifth instar larvae almost a third of labeled triolein remains unhydrolyzed (Turunen, 1975). Under comparable conditions, hydrolysis and subsequent utilization of phosphatidylcholine (PC) is very efficient (Turunen and Kastari, 1979). Our data have indicated that most of dietary PC is hydrolyzed to release the base moiety. A smaller proportion is hydrolyzed by phospholipase A-like activity to lysophosphatidylcholine and free fatty acid. No information is available on the fate of the choline-free moiety of PC, which according to these data is the major product of PC hydrolysis in P. brassicae. This was examined in the present study. Previous indirect data have suggested that lipid translocation into the hemolymph is more rapid across the anterior than across the posterior region of the midgut in larvae of the pyralid moth, D. grandiosella (Turunen and Chippendale, 1977). In the

MATERIALS AND METHODS

Insects The laboratory culture of P. brassicae originated from insects obtained in 1984 from the Glasshouse Crops Research Institute, Littlehampton, England. Larvae were maintained on a semi-synthetic diet at 18L:6D, 23°C and about 60% r.h. (Kastari and Turunen, 1977). Under these conditions larvae molt into the fifth instar about 10 days after hatching. Newly-ecdyzed fifth instars were placed on the diet containing radioactive lipid and analyzed for radioactivity after 24, 48 and 72 hr. In another series of experiments, 24-hr-old fifth instars were fasted for 24 hr and then placed on the radioactive diet for 4 hr, to study the initial products of hydrolysis and initial stages of absorption. For information on the rate and site of lipid translocation across the midgut into the hemolymph, larvae first fed for 48 hr on the radioactive diet, in which time a relatively stable level of midgut tissue labeling was reached. Preliminary experiments had shown that at this stage the specific activity of hemolymph had reached a plateau and the specific activity increase of midgut tissue had started to level off. The larvae were then placed on a fresh diet of identical composition, but without the labeled lipid, and feeding continued on the unlabeled diet without delay. Radioactivity was measured in the anterior and posterior halves of the midgut tissue before transfer (48-hr-old larvae), and the decline of radioactivity was then followed in the anterior and posterior halves during the next 48 hr (sampled at 24 and 48 hr). Larvae in the 48-hr sample (about 96 hr from the beginning of the fifth instar) were in the walking stage but not yet in the post-absorptive stage. Dietary radioactivity from the midgut lumen was available for these larvae for only about 2-4 hr after transfer to an unlabeled diet. Decline in the radioactivity of individual midgut tissue lipids provided information on the rate of utilization of these lipids during absorption. 19

SEPPOTURUNEN

20

Table 1. Incorporation of radioactivity from dietary di(l-“C) oleoyl phosphatidylcholine into fifth instar larvae of Pieris brassicae Approximate digestibility of radioactivity (A.D.) during the 5th instar (2472 hr) Specific activity of diet Specific activity of feces Specific activity of hemolymph 24 hr 48 hr 72 hr

84.2% 63070 dpm/g (tw.) (N = 4) 22857 dpm/g (tw.) (N = 2) 5.1 dpm/pl (32 larvae, N = 3) 8.2 dpm/+l (20 larvae, N = 2) 8.4 dpm/pl (36 larvae, N = 3)

Average radioactivity recovered from one larva at 48 hr Fat body

20201dpm* (20 larvae, N = 2) 7867dpm (22 larvae, N = 3) 5350dpm* (20 larvae, N = 2) 820dum (20 larvae. N = 2)

Midgut tissue Midgut contents Hemolvmuh f 100~1) *Not quantitative.

Labeled diet

RESULTS

Di(l-‘4C)oleoyl-L-3-phosphatidylcholine, specific activity 112 mCi/mmol (Amersham) was incorporated into the diet at a concentration of 63,07Odpm/g fresh weight. Radiopurity of the compound, determined from lipids extracted from the diet and separated by thin layer chromatography (TLC) on Silica gel 60 in a solvent system of chloroform:methanol:acetic acid: water (25: 15:4:2), was 97.0%. Some radioactivity was recovered from lysophosphatidylcholine (1.5%) and unesterified fatty acids (1.5%). Analytical procedures

Hemolymph was drawn into graduated capillary pipettes from a wound made at the base of a leg and was promptly extracted for lipids. Larvae were dissected under a cold saline solution, rinsed clear of hemolymph, the midgut was carefully opened and the contents were removed with the peritrophic membrane intact (Turunen, 1975). The isolated midgut tissue and fat body were thoroughly rinsed in the saline, all samples were extracted with chloroform: methanol (2: 1, v: v) in a glass homogenizer, and total lipids were purified (Folch et al., 1957). TLC of lipids and standards was carried out on precoated Silica gel 60 plates (Merck) in two steps. Phospholipids were first separated by developing the plate in chloroform: methanol:acetic acid:water (25: 15:4:2) to 12 cm. The plates were air-dried for 20 min and the neutral lipids then separated by developing in hexane:diethyl ether (4: 1) to 19 cm. Lipids were visualized in iodine vapor, and the spots were scraped directly into scintillation vials. Radioactivities were counted in IOml Lipoluma, using a Wallac Minibeta scintillation counter. Efficiency of counting was monitored by external standards. TLC lipid standards were from Sigma, except phosphatidylinositol (Koch Light Laboratories).

Hydrolysis of phosphatidylcholine

The overall efficiency of utilization (approximate digestibility, AD) of dietary oleic acid esterified in phosphatidylcholine (PC) was found to be about 84% in actively-feeding fifth instar larvae (24-72 hr). AD was defined as (I - F)Z-i, where Z is the amount of labeled compound ingested between 24 and 72 hr, and F is the amount excreted in feces during this period. The overall efficiency of conversion of ingested food during this period was ca 11%. These data show a high degree of utilization of PC oleate in P. brassicae (Table 1). Data on the incorporation of radioactivity into larval tissues suggest that a plateau is reached in hemolymph radioactivity by 48 hr. At this time, most of the tissue radioactivity was recovered from fat body (Table 1). Dietary PC is hydrolyzed in the midgut lumen of P. brassicue to yield several metabolites, including choline, lysophosphatidylcholine (LPC) and free fatty acids (FFA). The distribution of radioactivity in different lipid fractions in the midgut lumen, midgut tissue, feces, hemolymph and fat body shows that PC is hydrolyzed as soon as it enters the lumen (Table 2). After 4 hr, radioactivity was found in FFA (SO.S%), LPC (7.3%), and several other polar lipids: three new labeled lipid fractions were observed (A, B, C). Very little intact PC was observed (2.2%). In larvae fed 48 hr on the radioactive diet, fractions A and B were

Table 2. Percent distribution of radioactivity (dpm) from dietary di(l-14C) oleoyl phosphatidylcholine larvae of P. brassicae

Midgut contents Lipid*

Diet7

4 hrf

LPC PC PI Ar

1.5 97.0

$ PE DG FFA TG SE

z 1.5 -

1

Midgut tissue

Feces

Hemolymph

in the lipids of fifth instar

Fat body

48 hr$

4 hrf

48 hr§

48 hr§

24 hrll

72 hrsl

24 hrlj

48 hr§

7.3 2.2

2.3 4.1

tr 29.9 2.4

2.2 3.0

6.7

9.3 -

6.0

5.2

30.7

tr 52.1 8.4 -

1.7 -

14.6 17.2 I.2 50.8 1.6 -

33.5 13.0

lLPC = lysophosphatidylcholine,

tr 7.8 5.9

5.5 5.8 1.7 54.2

18.9 3.4 tr 14.9 tr

PC = phosphatidylcholine,

29.8 II.8 4.9 1.4 45. I tr

10.6 14.2 tr 3.8 1.6

PI = phosphatidylinositol,

15.5 64.7 tr 6.0 1.7

3.9 1.8 2.4 85.3 -

1.4 1.1 1.3 93.9 tr

PE = phosphatidylethanolamine,

DG = diacylglycerol, FFA = free fatty acid, TG = triacylglycerol, SE = sterol ester. tMean of 2 determinations. fMid-fifth instar larvae (20, N = 2) fasted 24 hr before being placed on radioactive diet for 4 hr. §Larvae fed on the radioactive diet 48 hr from the beginning of the fifth instar (20, N = 2). 1132larvae, N = 2. ll36 larvae, N = 3. tr = less than I%.

Pieris digestion/absorption

of glycerophospholipid 32 PC

32

A

/ B

Midgut

contents 3 /

A/

24

h

24

h

Fig. 1. Radioactivity recovered from the midgut lumen (A) and midgut cells (B) of P. brassicae larvae receiving dietary di(l-‘4C)oleoyl phosphatidylcholine. Larvae at 4 hr were mid-fifth instars, which had been fasted for 24 hr before being placed on the labeled diet for 4 hr. Larvae at 24 hr have been feeding on the labeled diet from the beginning of the fifth instar and are ca 1 day younger than larvae at 4 hr. A, B, C = unidentified polar lipid fractions, FFA = free fatty acids, PC = phosphatidylcholine, LPC = lysophosphatidylcholine. Number of insects and range are shown (N = 2).

the major labeled lipids in the lumen. The origin and significance of these luminal lipids is unknown, but their synthesis may be preceded by release of diacylglycerol (DG), as a result of phospholipase activity hydrolyzing the base from the labeled PC. The presence of a small amount of labeled triacylglycerol (TG) in 48-hr-old fifth instar larvae (6% of luminal radioactivity) is noteworthy. In midgut cells, in contrast, TG and PC were the predominant labeled lipids after short term (4 hr) exposure to the diet (Table 2). Prolonged feeding led to a relatively smaller proportion of labeled TG but to an increase in labeled PC and PE in midgut cells. Unabsorbed radioactivity was excreted mainly as FFA, but also as fraction A, which was quantitatively a major fecal lipid fraction. The data leave unanswered the fate of the substantial amount of labeled fraction B present in the lumen. The results do not show which lipids are released from the midgut into the hemolymph as a result of absorption. After 24 hr of feeding, 74% of hemolymph radioactivity was present in DG, about 11% in PE, and 7% in PC. It is probable that hemolymph radioactivity at this stage reflects release of lipids mainly from the midgut but also from fat body. Most of fat body radioactivity after 24 and 48 hr was present in TG (Table 2). The rate of PC hydrolysis is rapid, because after 4 hr of feeding only a little intact PC was recovered in the midgut lumen. Analyses of lipid radioactivities (dpm recovered per insect) in the lumen during the first 24 hr of digestion suggest that initially there is

release of FFA from PC, but within a few hours synthesis of fractions A, B and C increase, until well before 24 hr all three lipids incorporate more radioactivity than FFA (Fig. 1A). The titre of labeled luminal FFA thereafter (up to at least 48 hr) appears to remain below the value reached at 4 hr. Luminal LPC also remains at a relatively constant level during 2 days of digestion and absorption. In the enterocytes, labeled TG accumulates rapidly, followed and soon exceeded by the accumulation of PC (Fig. 1B). TG accumulation appears to reach a peak within a few hours and to decrease thereafter, parallel to the changes observed in the titre of FFA in the midgut lumen. PC is the predominant radioactive lipid in the enterocytes within 1 day of feeding. The titres of enterocyte DG and FFA remained at a relatively stable level during the period of absorption examined. It should be noted that the larvae sampled at 4 hr and those sampled at 24 hr differ physiologically (see Materials and Methods). The former have fasted for 24 hr before the experiment, and they incorporate radioactivity into the midgut tissue at a somewhat faster rate than larvae in the 24 hr sample. Larval weights were comparable and their ages differed only by about 24 hr. Thus it would seem that the 4 and 24 hr samples give an idea of the time course of digestion and absorption of lipid. lipids from the midgut Lipid radioactivity increases nearly linearly in the midgut tissue during the first two days of feeding, Release of radioactive

22

SEPPOTURUNEN

whereafter midgut tissue radioactivity increases at a much smaller rate. Some of the incorporation of radioactivity is related to tissue growth. To learn which lipids are involved in the translocation of lipid into the hemolymph, 48-hr-old radioactive larvae were transferred to an unlabeled diet of identical composition, and the decline of midgut lipid radioactivity was followed during the next 48 hr (Fig. 2, points B-C). The anterior half of the midgut contained about 73% of midgut tissue radioactivity at the start of the experiment (B) (whole midgut radioactivity ca 7867 dpm/insect). After 48 hr the radioactivity had declined in the anterior and posterior half, now containing 58 and 420/b, respectively, of total midgut radioactivity (ca 3513 dpm/insect). In pre-pupae, which have not fed for several hours, both halves contained about equal amounts of radioactivity. These data show that quantitatively the anterior midgut released over 3 times more radioactivity than the posterior midgut during the 2-day experiment (Fig. 2). It may be estimated from data in Fig. 2 that the half-life of lipid radioactivity, after transfer of larvae to an unlabeled diet, was about 18 hr in the anterior midgut, and about 26 hr in the posterior midgut. These data indicate that lipid transport is more rapid across the anterior than across the posterior half of the midgut of P. brassicae. dPrn_

w3/

midgut

22

l

I

1 3

Wholsmidgut

1I’ 4

24

40 +

B

72

96

h

c’ Fig. 2. Radioactivity recovered from midgut tissue of insects receiving dietary di(l-r4C)oleoyl phosphatidylcholine. Larvae were placed on the labeled diet at the beginning of the fifth instar (point A). At 48 hr, larvae were transferred to an unlabeled diet (point B), and the decline of radioactivity was examined in the anterior and posterior halves of the midgut. Point C denotes the attachment of larvae prior to pupation (pre-pupae). Larvae at 4 hr are not strictly comparable with the pattern of radioactivity incorporation (A-B), since they are older and have been fasted before the experiment (see Fig. 1). Number of insects, range, and number of samples are shown. f;

0

24

46

h

Fig. 3. Radioactivity recovered from midgut tissue lipids of P. brassicae larvae transferred from a labeled diet to an unlabeled diet (corresponding to EC in Fig. 2). Larvae at 0 hr have been feeding on a diet containing di(l-W)oleoyl phosphatidylcholine for 48 hr from the beginning of the fifth instar. (A) Decline of radioactivity in the anterior half of the midgut. (B) Decline of radioactivity in the posterior half of the midgut. PC = phosphatidylcholine, PE = phosphatidylethanolamine, TG = triacylglycerol, PI = phosphatidylinositol, DG = diacylglycerol. Number of insects, range, and number of samples, as in Fig. 2.

The decline in midgut tissue radioactivity is primarily a result of a decrease in the labeling of PC and TG (Fig. 3). At the start of the experiment, PC was the predominant radioactive lipid in the anterior midgut. The half-life of PC and TG was estimated to be about 15 hr in the anterior midgut, but distinctly more in the posterior midgut (for these estimates the decline is drawn as a curve rather than a straight line). Both PC and TG are present in the anterior midgut at a much greater concentration than in the posterior midgut: at the start of the experiment, PC contained about 3200 dpm/insect in the anterior and 930dpm/insect in the posterior midgut. The titre of labeled DG is also about 3 times greater in the anterior than in the posterior half of the midgut. In contrast, there is less difference in PE and PI radioactivity between the two halves. The decline of radioactivity from midgut tissue PE was relatively slow if compared to that of PC. In the anterior midgut the half-life of PE was about 40 hr, and in the posterior midgut about 33 hr (Fig. 3). The metabolism of midgut tissue PE thus appears to differ from that of midgut tissue PC in relation to absorption. The present data suggest a role for PC and TG in the absorption-related release of lipid into the hemolymph.

Pieris digestion/absorption of glycerophospholipid DISCUSSION

The present data complement previous findings on the absorption of phosphatidyl (N-methyl-‘4C)choline in P. brassicae (Turunen and Kastari, 1979), and provide new information on lipid transport and metabolism in the midgut. In the midgut lumen PC is hydrolyzed by at least two types of enzymes, one releasing FFA (lipase, phospholipase A) and another releasing the base. Data obtained to date suggest that in P. brassicae the activity of the enzyme releasing the base is greater than that of the enzyme releasing fatty acid. Depending on which phosphodiester link is hydrolyzed in the lumen (unclear at present), the choline-free moiety is diacylglycerol or phosphatidic acid. The enzyme releasing phosphatidic acid (phospholipase D) has been characterized in plants (Gurr and James, 1975). The base was shown previously to be rapidly absorbed into the hemolymph (Turunen and Kastari, 1979). The fate of the diacylglycerol moiety has remained unexplored and was examined in the present study. The presence of several new labeled polar lipids, as well as TG, in the midgut lumen of P. brassicue, following ingestion and digestion of di( l-‘4C)oleoyl phosphatidylcholine, could be a result of synthesis within enterocytes, the cells then being sloughed off into the lumen, but two observations make such an explanation unlikely. First, the synthesis of these lipids is rapid, for within 4 hr about 50% of luminal radioactivity is present in these new polar lipids. Second, B is the major labeled lipid in the midgut lumen, but midgut cells contain no labeled B. It also seems unlikely that fraction A is present in midgut ceils. These lipids thus seem to be synthesized in the lumen. The identity of the new luminal fractions remains unclear. Preliminary data suggest that they could be glycolipids. Comparable synthesis of luminal polar lipids was also observed in P. brussicue fed glycerol tri(l-‘4C)oleate (Turunen, 1975). In this case, also, it was noteworthy that no labeled PC was observed in the midgut lumen. In fact, polar lipid synthesis was remarkably similar in both cases. The results suggest that part of the luminal DG, released by lipolytic activity on dietary TG or PC, is used in the synthesis of other lipids in the midgut lumen. Choline seems not to be incorporated into these lipids, possibly because it is rapidly absorbed. P. brussicue has been found to require choline for optimal growth (Bridges, 1987). The function of the lipids synthesized is unknown, although they could have a role in lipid solubilization in the lumen. The extent of this synthesis is so marked that its significance should be studied. Absorbed radioactive lipids were incorporated first into midgut TG, parallel to the hydrolytic release of FFA in the midgut lumen. Synthesis of TG was followed and soon exceeded by the accumulation of PC in midgut cells. Previous results suggest that absorbed LPC is resynthesized into PC within midgut cells (Turunen and Kastari, 1979). Synthesis of TG and PC has been found to follow absorption also when the larvae were fed FFA or TG (Turunen, 1975) and similar observations have been made in L. migratoriu (Weintraub and Tietz, 1973, 1978), D.

23

grundiosellu (Turunen and Chippendale, 1977) P. umericunu (Chino and Downer, 1979), S. greguriu (Thomas, 1984) and Aeshnu cyuneu (Komnick et al.,

1984). The synthesized phospholipids have been suggested to be a source of hemolymph diacylglycerol in L. migratoriu (Weintraub and Tietz, 1973) and a similar function was suggested for TG in S. greguriu (Thomas, 1984). The present data support a role for both TG and PC in lipid transport and release in the midgut of P. brussicue. The half-life of both lipid types is relatively short in the midgut tissue. Although the hemolymph contained labeled PC, and some release of PC from the midgut probably occurs, its titre in hemolymph remained low, less than that of PE, for example. The rapid and quantitatively large decline of midgut PC radioactivity observed suggests that enterocyte PC is an important source of hemolymph DG. During the two-day experiment when the decline of midgut radioactivity was followed, the titre of enterocyte DG remained relatively constant, suggesting that DG originating from TG and PC is quickly released into the hemolymph and is not accumulated within cells prior to release. The rapid synthesis and turnover of midgut tissue PC and TG may be necessary for maintaining a suitable diffusion gradient for the products of luminal hydrolysis. The present data show that hydrolysis of PC is rapid and that phospholipid oleate is absorbed predominantly across the anterior half of the midgut. Comparable differentiation of midgut in glyceride and sterol absorption was implicated also by data from D. grundiosellu, in which the anterior third of the midgut contained the highest titre of labeled DG, FFA and PL following digestion of glycerol tri(l-‘4C)oleate (Turunen and Chippendale, 1977). These data were interpreted as suggesting a more rapid rate of lipid absorption across the anterior part of the midgut. It is possible that hydrolysis is a more rapid process than lipid absorption and the hydrolyzed lipids diffuse into the enterocytes as soon as food reaches the anterior region of the midgut. Alternatively, hydrolyzed lipids may be regurgitated back from the more posterior regions for absorption in the anterior midgut. These data suggest that diffusion is the rate-limiting step, because some FFA is excreted regardless of which lipid (TG, PC) the larvae ingest. Acknowledgement-This study was supported in part by funds from the Academy of Finland.

REFERENCES

Bridges R. G. (1987) Choline metabolism in larvae of Pieris brassicae. Insect Biochem. 17, 61-69. Chino H. and Downer R. G. H. (1979) The role of diacylglycerol in absorption of dietary glyceride in the American cockroach. Insect Biochem. 9, 379-382. Folch J., Lees M. and Stanley G. H. S. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Gurr M. I. and James A. T. (1975) Lioid Biochemistrv: , Introduction. Chapman and Hall, London. Kastari T. and Turunen S. (1977) Lipid utilization in Pieris brassicae reared on meridic and natural diets: implications for dietary improvement. Enf. exp. appl. 22, 71-80.

24

SEPPOTURUNEN

Komnick H., Kukulies J., Bongers J. and Fischer W. (1984) Absorption of dietary triacylglycerol by lipolysis and lipid resynthesis in the mesenteron of larval Aeshna cyanea (Insecta, Odonata). Protoplasma 123, 5749. Thomas K. K. (1984) Studies on the absorption of lipid from the gut of desert locust, Schistocerca gregariu. Comp. Biochem. Physiol. 77A, 707-712.

Turunen S. (1975) Absorption and transport of dietary lipid in Pieris brassicae. J. Insect Physiol. 21, 1521-1529. Turunen S. (1985) Absorption. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 4, pp. 241-277. Pergamon Press, Oxford.

Turunen S. and Chippendale G. M. (1977) Lipid absorptton and transport: sectional analysis of the larval midgut of the corn borer, Diatraea grandiosella. Insect Biochem. 7, 203-208.

Turunen S. and Kastari T. (1979) Digestion and absorption of lecithin in larvae of the cabbage butterfly, Pieris brassicae. Comp. Biochem. Physiol. 62A, 933-937.

Weintraub H. and Tietz A. (1973) Triglyceride digestion and absorption in the locust, Locusta migratoria. Biochim. biophys. Acta 306, 3141.

Weintraub H. and Tietz A. (1978) Lipid absorption by isolated intestinal preparations. Insecr Biochem. 8, 267-274.