Digestion and absorption of lecithin in larvae of the cabbage butterfly, Pieris brassicae

OXX-9629,79

0401~0933SO2oO:O

DIGESTION AND ABSORPTION OF LECITHIN IN LARVAE OF THE CABBAGE BUTTERFLY, PZERZS BRASSICAE S. TURUNEN* and T. KASTARI Department of Zoology, Division of Physiology, University of Helsinki, 00100 Helsinki 10, Finland (Received 23 June 1978) Abstract-l. Dietary lecithin [phosphatidyl (N-methyl-14C) choline] is converted to the surface-active lysolecithin and to water-soluble metabolites in the intestinal lumen of the butterfly. Pieris hrassicae. 2. In the midgut cells radioactivity was found in lecithin, lysolecithin and sphingomyelin. 3. In the haemolymph over 907, of the radioactivity was water-soluble after 6 hr. but only 327” was water-soluble after 48 hr of feeding. Labeled lipids in the haemolymph were lecithin (ca. 90% of lipid radioactivity) and sphingomyelin. 4. Based on an assay of lipid phosphorus, dietary lecithin was found to be utilized by the larvae almost completely. The titre and specific activity of midgut lysolecithin suggest a role for this lipid in absorption.

INTRODUCTION Most research on the digestion and absorption of lipids in insects has concerned neutral lipids (Weintraub & Tietz, 1973; Turunen, 1975; Hoffman & Downer, 1976; Turunen & Chippendale, 1977), and to date no comparable studies on the more complex dietary lipids seem to have been published. Our recent results in the cabbage butterfly, Pieris brassicae, showed, however, that this phytophagous species utilizes dietary phospholipids and glycolipids in preference to neutral lipids (Kastari & Turunen, 1977). Owing to the relatively simple endogenous mechanisms available in insects for the emulsification of oils in the intestinal lumen. the utilization of dietary triglycerides may not be as efficient in insects as it is in mammals. In this study we have examined the digestion and absorption of lecithin, a phospholipid found commonly in plant and animal tissues. Substantial hydrolysis of lecithin to the surface-active agent, lysolecithin, was found. The results suggest efficient utilization of lecithin by P. brassicae larvae. MATERIALS AND METHODS Test insects

The laboratory culture of P. brassicae was maintained on an artificial diet at 18L:6D, 23°C and about 60% r.h. (Turunen, 1973a. 1978a). Under these conditions the larvae molt into the fifth instar about 10 days after hatching. Recently ecdyzed fifth instars of both sexes were placed on labeled diets before the larvae had started feeding. The tissue distribution of the radioactivity between phospholipid classes was determined after 6, 15, 48 and 85 hr. The last group were prepupae. Labeled diet

Phosphatidyl (N-methyl-‘4C) choline. specific activity 60 mCi/mmol, was obtained from the Radiochemical * Present address: Department of Entomology, University of Missouri, Columbia, MO 65211, U.S.A.

Centre, and incorporated into the diet at a concentration of 5.2 &i/100 g wet weight. The solvent (benzene-ethanol) was evaporated by thorough mixing of the diet at ca. 65”C.The procedure has been shown to give a uniform distribution of the label in the diet (Turunen, 1973b). Radiopurity of the labeled compound in the diet was determined when the insects were removed for analysis. Ca. 98.6% of the label was present in phosphatidyl choline and 1.4% in lysophosphatidyl choline. Analytical procedures

The preparation of tissues for Iipid extraction has been described (Turunen. 1975; Turunen & Chippendale, 1977). All samples were homogenized immediately in chloroformmethanol (2:1) (v/v), and the total lipid fraction was extracted and purified (Folch et al., 1957). The lipids were separated by TLC on pre-coated 0.25 mm silica gel plates. Phospholipids were routinely separated by one-dimensional chromatography with chloroform-methanol-acetic acid-water (65:25:8:4). In twodimensional separation the lipids were first developed ca. 14 cm in chloroform-methanol-25”/, ammonia-water (65:20:2:2). air dried for 30 min, and redeveloped 14cm in the same system (Luukkonen er al., 1976). The plates were then developed 14cm in the second direction in chloroform-methanol-acetic acid-acetone-water (50: 10: 10:20:5) (Rouser et al.. 1965). Lipids were detected with iodine vapor. After the iodine had sublimed the areas containing lipid were scraped into plastic scintillation vials. to which 2 ml methanol had been added to elute the lipids. The scintillation cocktail was Packard’s Insta-gel (lOml/ vial). Radioactivity was counted with a Wallac-LKB Ultrabeta scintillation counter which uses the external standard count method to determine counting efficiency. Each analysis was repeated two or three times. Inorganic phosphorus was determined from individual phospholipids after two-dimensional chromatography by the method of Bartlett (1959). and identical samples were co-developed for scintillation counting to determine the specific activity of individual lipid classes. For the detection of lipid phosphorus on thin-layer plates the chromatograms were sprayed with the molybdenum trioxide reagent of Dittmer & Lester (1964). Radioactivity of larval haemolymph lipoproteins was determined after electrophoresis on acrylamide gels as de933

S. TURC‘NEKand

Yi4

scribed previously (Turunen. 1978b). Slowly migrating hpoproteins (Rm 0.09 and 0.12) and rapidly mrgrating lipoproteins (Rtn 0.40 and 0.45) of 48 hr post-ecdysis fifth instar larvae were GUI from the gels. incubated in 2 ml distilled water in scintillation vials for 24 hr. after which IO ml of scintillation cocktail was added to each vial.

RESCLTS

Hydrolysis

in the larval

intestine

TLC of dietary polar lipids showed the presence of at least 15 lipid classes, including phosphatidyl choline, phosphatidyl ethanolamine and phosphatidic acid as major components. Lysophosphatidyl choline, phosphatidyl inositol and sphingomyelin were present in smaller amounts. In addition, several polar lipids not containing phosphorus were detected. The radioactivity present in the diet was recovered almost totally from the lipid-soluble fraction (Table Il. Analysis of food in the midgut contents showed, however, that the radioactivity was distributed between water-soluble and lipid-soluble fractions, indicating hydrolysis of phosphatidyl choline. Thus after 48 hr about 75% of the radioactivity in the intestinal lumen was recovered from the water-soluble fraction. The results show that the lipid-soluble radioactivity was incorporated into larval tissues: in the faeces about 90% of the radioactivity was present in the watersoluble fraction (Table I). TLC of dietary phospholipids indicated that 98.6”; of the radioactivity was present in phosphatidyl choline (Table 2). Analysis of phospholipids in the intestinal lumen showed that phosphatidyl choline was hydrolyzed, to yield lysophosphatidyl choline. After 48 hr 90.4”; of phospholipid radioactivity was present in lysophosphatidyl choline in the intestinal lumen. These data initially suggested that hydrolysis of phosphatidyl choline could precede absorption and that

T. KASTARI

the utilization be efficient. .4hsorptiorl

of dietary

phusphatidyl

choline

may

oj phospholipid

Chromatography of dietary, intestinal and faecal phospholipids indicated that dietary lysophosphatidyl choline. phosphatidic acid. phosphatidyl choline and phosphatidyl ethanolamine were aimost completely utilized by the larvae. For an examination of absorption the anterior and posterior regions of the midgut were analyzed separately for radioactivity. The results show that in the midgut cells radioactivity is present mainly in the lipid-soluble fraction (Table 1). The anterior and posterior regions were similar in the labeling pattern of their phospholipids, although data suggest that water-soluble metabolites (probably free choline) were present in higher amount in the posterior than in the anterior region of the midgut. The specific activity of lipids in the anterior midgut increased from 409 counts/min per mg at 6 hr to 6536 counts/min per mg at 48 hr. but decreased again to 3106 counts/min per mg in prepupae (Table I ). These data indicate that phospholipids are not stationary. or merely structural. lipids in the midgut but actively involved in lipid translocation across the midgut. When midgut tissue phospholipids were separated by TLC, both lysophosphatidyl choline and phosphatidy1 choline were found to contain radioactivity (Table 2). In actively feeding (48 hr) larvae 10.54, of the radioactivity was present in lysophosphatidyl choline, 2.0”” in sphingomyelin, and 87.69, in phosphatidyl choline in the anterior midgut. When larvae were feeding less actively (I 5 hr) the proportion of labeled lysophosphatidyl choline was smaller within midgut cells. and decreased further after feeding had ceased (85 hr). These data suggest that lysophosphatidyl choline was absorbed from the midgut lumen and subse-

Table 1. Specific activity of water-soluble and lipid-soluble metabolites in larvae of Pieris hrassicae fed a diet containing phosphatidy1 (N-methyl-14C) choline

*.

I Time of exposure to labeled diet. * Per cent distribution of radioactivity recovered 3 All tissues except the intestine and haemolymph was repeated 2 to 3 times.

from sample. Each analysis

Digestion Table

and absorption

2. Digestion yl-14C) choline

of lecithin

in larvae of the cabbage butterfly

935

and absorption of phosphatidyl (N-methin fifth instar larvae of Pieris brassicae 1

Distribution of (I CJ between Insects (No.1

19 15

:z Fat body nther Other

tissues tissues

lipid class&

Tim

(hr-) LysoPC Sph

iii 4e ie "5

t.: 10.' 11.5 :.a trace 3.2 -

(4)

PC

Other

trace 2.3 trax 5.8

4;.c Si.6 33.4 9O.b

-

3.5 :.1 9.2

96.9 c;., 90.3

-

15

tz P5

18

43

-

5.1

C!q.,

-

15 18

> ;::

-

4.1 5.7

%.‘j 94.;

-

Compare the legend to Table 1. Lyso PC-lysophosphatidyl choline. Sph-sphingomyelin. PC-phosphatidyl chohne.

quently converted to phosphatidyl choline in midgut cells. The larvae also incorporated absorbed choline or its derivatives into sphingomyelin in the midgut ceils. No radioactivity was found in any of the other phosphatides. Haemolymph transport The results show that water-soluble derivatives are rapidly found in larval haemolymph (Table 1). After 6 hr 91.5% of haemolymph radioactivity was recovered from the water-soluble fraction, but the proportion of water-soluble radioactivity had decreased to 32.2% after 48 hr. Analysis of individual haemolymph phospholipids showed that in early fifth instar larvae (6 hr, 15 hr) lysophosphatidyl choline contained little or no radioactivity, and most of the radioactivity was in phosphatidyl choline. In actively feeding 48 hr larvae the radioactivity of haemolymph phospholipids was distributed between lysophosphatidyl choline (3.2x), sphingomyelin (9.5%). and phosphatidyl choline (87.3%) (Table 2). No radioactivity was found in any of the other haemolymph phospholipids. To examine the possible role of haemolymph proteins in phospholipid transport, the radioactivity Table 3. Specific activity of phospholipids larvae fed a diet containing phosphatidyl choline

sample

in fifth instar (N-methyl-“‘C)

Tim

Counts/rGn/n(-

Z ph&pholipi%J

(hr)

phospholipid

in total lipid

6 :;

640 5571 9062

792 79 77

f? Pk

3424 1405P 11214

41 34 l4U

4P

21%@

6 15 148 31

572 3265 7747 12598

Compare the legend to Table I. ’ Anterior and posterior halves combined. ’ Turunen (1975). 3 Turunen (1973b).

f>f 46 37 28 16

present in larval haemolymph lipoproteins was measured in actively feeding 48 hr fifth instar larvae. Slowly migrating (Rm 0.09 and 0.12) and more rapidly migrating (Rm 0.40 and 0.45) lipoproteins were pooled separately from several gels. Both the slowly migrating and the rapidly migrating classes of lipoproteins were found to contain radioactivity: between the two classes the slowly migrating bands contained 450,; and the rapidly migrating bands 55% of the radioactivity. Specijic activity and phosphorus content of phospholipids In the fat body of actively feeding 48 hr larvae radioactivity was found mainly in the lipid-soluble fraction (98.2%) (Table 1). Most of this activity was present in phosphatidyl choline (94.9x), but some was found in sphingomyelin (5.1%) (Table 2). Absorbed lecithin (lysolecithin) thus appears to be stored mainly as tissue lecithin. The incorporation of the label in tissue phospholipids increased in the midgut and haemolymph during feeding. and after 48 hr reached 9062 counts/min per mg phospholipid in the midgut, 14058 counts/ mim per mg phospholipid in the haemolymph. and 23360counts/min per mg phospholipid in the fat body (Table 3), showing that the absorbed phosphatide is stored specifically in the fat body. In 85 hr prepupae the label had again decreased in the midgut and haemolymph. but had increased in other larvai tissues (including fat body and integument, see Table 3), suggesting retention of the absorbed phospholipid in storage tissues. Among the tissues studied the fat body had greatest specific activity of phospholipids. Our data on inorganic phosphorus coritent of dietary and faecal lipids suggest that dietary phosphatidyl choline is absorbed almost completely by the larvae (Table 4). Results further suggest that lysophosphatidyl choline is present in a high titre in the midgut cells, if compared to the diet or to other larval tissues. After cessation of absorption (85 hr prepupae) the titre of lysophosphatidyl choline decreases notably in the midgut, suggesting a role for lysophosphatidyl choline in absorption (Table 4). Defermination of the specific activities of lysophosphatidyl choline and phosphatidyl choline, based on

S. TLJRLWENand

936 Table

4. Distribution of inorganic phosphorus faecal and tissue phospholipids

’ Total to Tables

lipid aliquot I and 2.

used for analysis.

an assay of inorganic phosphorus. showed that both lipid classes are more radioactive in the anterior than in the posterior midgut. A typical analysis is shown in Table 5. Data also indicate that in actively feeding 48 hr larvae the specific activity of midgut lysophosphatidyl choline reaches close to that of midgut phosphatidyl choline. Faecal phosphatidyl choline and lysophosphatidyl choline contained little or no radioactivity.

DISCUSSION

Although the dilution of the label by unlabeled lipids can hamper the interpretation of results obtained with labeled lipids incorporated into semidefined diets, our results suggest efficient utilization of dietary lecithin by fifth instar larvae of P. brassicae. Substantial hydrolysis to lysolecithin seemed to occur in the lumen of the larval intestine. The surface-active lysolecithin was subsequently found in larval midgut cells, but was possibly converted to lecithin within the cells. In comparison to previous data on the utilization of dietary neutral lipids by P. brassicue larvae (Turunen, 1973b, 1975) the present data suggest that this phytophagous larva is well adpated to the utilization of dietary phospholipids. Interest in the nutritional significance of dietary

Table 5. Specific activity of tissue lysophosphatidyl and phosphatidyl Insects

Sample

(No.)

Time

choline

choline

Counts/min/up:

(hr)

Lyso

Diet

PC

phosphorus PC

631

Anterior midgut Posterior midgut Anterior midgut Posterior midgut Whole midgut

15 15 IS 18 15

H%3lOlymph Haemolymph Haemolymph

:: 15

15 118 SCI

z

Fat

18

lie

67

body

15 4185 &S 85

No radioactivity was detected little in faecal lyso PC. Compare

T. KASTARI

211 18 74 17

49 31 104 66 53 7f

in faecal PC and to Table 4.

only

Compare

among

dietar?

the legends

phospholipids for plant-feeding insects is based on the common occurrence of polar lipids (phospholipids. galactosyl diglycerides) in photosynthetic tissues. The leaf lipids of several higher plants have been found to consist mainly of mono- and digalactosyl diglycerides and phospholipids (Rouchan & Batt, 1969), suggesting that phytophagous insects normally ingest quantitatively more of these lipids than triglycerides. Our recent data on the efficiency of utilization of Brassica oleracea var. acephala leaf lipids by larvae of the cabbage butterfly showed that the utilization of neutral, phospho, and glycolipids increased in that order (Kastari & Turunen, 1977). It is possible that P. brassicue normally obtains essential fatty acids (EFA) mainly as components of leaf polar lipids. It is also possible that, as consitituents of artificial diets, polar lipids would be preferable to triglycerides as EFA sources for P. hrussicue. The efficiency of utilization of complex lipids by insects has yet to be studied systematically before the significance of these lipids in the nutrition of phytophagous insects is understood. However, a recent study of three lepidopterous larvae. P. brassicae. Sporlopteru e?tiguen.s. and Trichoplusia ni, has shown phospholipase A activity in the digestive juice. capable of hydrolyzing phosphatidyl glycerol and phosphatidyl ethanolamine (Somerville & Pockett. 1976). It has also been recognized much earlier that Gderiu mellonella can utilize beeswax without the aid of intestinal symbionts (Niemierko. 1959). Although in the present study we used phosphatidyl choline labeled in the choline moiety and were unable to follow the fate of the lipid after removal of choline, the data with the labeled lipid coupled with quantitative phosphorus determination suggest an almost complete absorption of dietary lecithin, possibly in the form of lysolecithin. Because of its surface-active properties lysolecithin might easily dissolve in the membrane lipids of midgut cells. It is possible that lysolecithin also contributes to lipid emulsification in the intestinal lumen, thus facilitating the hydrolysis and absorption of other dietary lipids, but much further study is needed to obtain convincing evidence for this. Acknowledgemertrs-This study was supported by a grant from the Finnish Cultural Foundation to T. Kastari.

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