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Changes in lipid composition during sealed brood development of African worker honeybees
Comp. Biochera. Physiol. Vol. 68B, pp. 351 to 353
0305-0491/81/0201-0351102.00]0
© Pergamon Press Ltd 1981. Printed in Great Britain
CHANGES IN LIPID COMPOSITION DURING SEALED BROOD DEVELOPMENT OF AFRICAN WORKER HONEYBEES R. C. CANTRILLl, H. R. HEPBURN2 and S. J. WARNER2 Department of Medical Biochemistry ~ and Department of Physiology 2, University of the Witwatersrand, Johannesburg, South Africa (Received 19 May 1980) Abstract--l. The lipid composition of whole worker honeybees and of abdominal fat body were analysed over the course of sealed brood development. 2. Decreasing fat body wet weight is paralleled by decreases in extractable lipid particularly of the triglyceride and fatty acid components. 3. Triglyceride stored in the fat body constitutes a major source of energy for brood development and of precursor material for phospholipid synthesis during development of honeybee workers.
INTRODUCTION
Developmental biochemical and physiological studies on insects have long lacked precision because of the difficulty in reproducibly defining particular stages of development in morphological terms and in a way that obviates the intrusion of temporal variation. This major difficulty has recently been resolved for the African bee, Apis mellifera adansonii L., (Thompson, 1978) and has facilitated a gross chemical audit at least for honeybees. Thus, it is known that during sealed brood development (which includes larval, pupal and adult forms) metabolism is largely based on the utilization of lipid stores and, as adult emergence is approached, some protein (Hepburn et al., 1979). We now briefly extend these observations to include changes in different lipid compartments in both whole honeybee workers and in isolates of fat body during the course of sealed brood development in the African worker honeybee.
of lipid extracted from each stage was determined using colorimetric (cholesterol, free fatty acids and phospholipids) and enzymatic (triglyceride) test kits (Boehringer Mannheim, Mannheim, Germany). Assays were checked with authentic standards and were found to estimate about 50~ of the added lipid. Although reproducibility was constant in each assay, the results given for individual lipids are those obtained under assay conditions and are not corrected for this difference. Thus in all cases, the total lipid extracted (mg) considerably exceeds the sum of the component lipids.
RESULTS AND DISCUSSION
The relative changes in whole bee dry weight and extractable abdominal fat body are shown in Figs 1 and 2. Whereas the decrease in whole bee dry weight has previously been attributed to a loss of both lipid and some protein (Hepburn et al., 1979) the decrease 30
MATERIALS AND METHODS
25
Livestock 2o
Sealed worker brood were removed intact from their combs and classified as to stage of development using the criteria of Thompson (1978).
t-
Procedures Whole bees were dried to constant weight at 60°C for 2 weeks. Fat body tissue was obtained from the abdomens of bees simply by teasing the material from the dissected bee with a fine paint brush under distilled water. The unextracted portions of the deceased bees were also dried to constant weight to determine the average mass of fat body for each developmental stage. The lipids of whole bees were extracted as previously described (Hepburn et al., 1979). Lipids of the fat body-distilled water mixture were extracted using the method of Folch et al. (1957). Both whole bee and fat body lipids were identified by comparison with authentic standards (Sigma, St Louis, U.S.A.) after chromatographic separation on silica gel G thin layer plates in a solvent containing: petroleum etherdiethyl ether-acetic acid (60:40:1 respectively). The volume 351
~
15
-o
10
m S 0
days 0 stage
I
II
III
IV
V VIVII VIII
Fig. 1. Dry weight of sealed worker brood expressed in mg/bee (O) and extracted lipid in mg/bee (It) plotted in terms of morphological and temporal development. Each point is the mean value of two separate determinations from pooled material and each pool contained about 20 bees..
R. C. CANTRILL,HI R. HEPBURNand S. J. WARNER
352 20
S E 15
.E ol
"O o
..£1
O .IZl
'6
Ii
5
days 0 stage
lk
li I 6 I& lbl I 121
I
II
III
IV
14
V VlVll Vlll
Fig. 2. Extractable abdominal fat body expressed as mg/ bee (©) and extracted lipid mg/fat body (I) expressed in terms of morphological and temporal development.
,°] ~
in the wet weight of the fat body through successive developmental stages is paralleled by a decrease in the amount of extractable lipid (Fig. 2). The proportions of triglyceride, free fatty acid, phospholipid and cholesterol at each stage of sealed brood development are shown in Fig. 3. It can be seen that cholesterol levels remain more or less constant while phospholipid increases by some 42%. The sum of triglyceride and free fatty acid decreases to only 30% of that recovered from the larval bees of stage 1. Figure 4 shows the fall in total lipid extracted from the abdominal fat body as described. This decrease in lipid mass is matched by a dramatic fall in triglyceride and fatty acid content as well. The cholesterol content of the fat body lipid extract was difficult to determine since the low concentrations visualised by TLC were outside the range of sensitivity of the assay system used and thus have been recorded as trace amounts. Since the lipid preparation procedure from dried bees is open to criticism because of the elevated temperature used to dry the bees, the triglyceride and fatty acid content have been summed up (Table 1) to compensate for any hydrolytic reactions which may have occurred durin~ drvin~ of the bees. It is unlikely that these results can be explained in terms of a high level of unesterified fatty acid in the
• Total [] TG [] FA [] PL [] ChoI
3.0
1.0 E Q. .I
InlnI011. . 0.6~
II.InlH. IHmn. IIH.
.011
II
Ill
IV
V
Vl
VII
E
tO Q,.
0.2 E O 0 c)
VIII
Fig. 3. Total lipid (mg/'bee) and lipid composition (mg/bee) for each of the stages of development assessed. The results are uncorrected for low sensitivity of the assay systems. All values are the means of duplicate determinations of pooled material where each pool contained about 20 bees for each stage.
UTotaL I~TG ITI]FA I~PL I=lChol
1.0 O
2.C
"6 "O O
0.5 "~_ E 1.0 "O .h O ea
.i
-6 0
tr
~]tr II
tr III
t_r IV
~nt_r V
~]n t_r Vl
~lgt_r Vll
I Jm.' o VllI
Fig. 4. Total extractable lipid from thc abdominal fat body (rag/fat body) and lipid composition (rag/fat body) for each stage of development. Sampling as in Fig. 3.
Bee lipids in development Table 1. Triglyceride, fatty acid and phospholipid composition of lipid extracted from whole honeybee workers during sealed brood development* Stage
TG
FA
Total
PL
PL/Total
1 2 3 4 5 6 7 8
1.03 0.46 0.27 0.48 0.10 0.51 0.18 0.07
0.29 0.45 0.36 0.25 0.35 0.34 0.36 0.33
1.32 0.91 0.63 0.73 0.45 0.85 0.54 0.40
0.26 0.28 0.27 0.26 0.28 0.42 0.40 0.37
0.20 0.31 0.42 0.36 0.62 0.49 0.74 0.92
* Each value given is the mean of duplicate determinations on the lipid extracts of pooled bees where each pool contains about 20 bees per stage. Table 2. Triglyceride, fatty acid and phospholipid composition of lipid extracted from the abdominal fat body of developing worker honeybees* Stage
TG
FA
Total
PL
PL/Total
1 2 3 4 5 6 7 8
0.79 0.65 0.78 0.56 0.20 0.22 0.14 0.09
0.11 0.17 0.19 0.14 0.08 0.08 0.08 0.05
0.90 0.82 0.97 0.70 0.28 0.30 0.22 0.14
0.15 0.07 0.09 0.11 0.03 0.03 0.04 0.03
0.17 0.08 0.10 0.16 0.12 0.10 0.17 0.20
* Each value given is the mean of duplicate determinations on the lipid extracts of pooled bees where each pool contains about 20 bees per stage. developing bee since fatty acid levels in the fat body extract are low. Thus, less hydrolysis appears to have taken place in the more rapid preparation of a lipid extract from the fat body. Variations in triglyceride and fatty acid content may be explained in terms of the length of time required to remove sufficient bees of each stage from the brood frame. It is noteworthy that as the concentration of triglyceride and fatty acid decreases in the fat body, the phospholipid component changes concommittantly so that a constant P L : T G + FA ratio is maintained (Table 2). There are two obvious explanations for this observation, either
353
or both of which may be correct: (1) the abdominal fat body is a major store for both triglyceride and phospholipid for metamorphosis and/or (2) as triglyceride is metabolized by the fat body there is cell loss and a corresponding decrease in phospholipid. The change in the P L : T G + FA ratio seen in the whole bees may also be explained in several ways. It is possible that total phospholipid content remains constant while the levels of triglyceride and fatty acid fall. Alternatively, there may be a net synthesis of phospholipid for new cell membranes. The 4 2 ~ increase in extracted phospholipid during development may indicate net synthesis; however, the pathway for such synthesis may be more complex than initially suggested by these results. Diglycerides are an important form in which triglycerides are mobilized in insects (Gilbert & Chino, 1974) so that the route of phospholipid synthesis from triglyceride stores may be either via acylation of ct-glycerophosphate and CDP-diglyceride formation or via CDP-base incorporation directly into diglyceride. The existence of the acylation pathway is shown in the following communication (Warner et al., 1981). In any event, it is apparent that the triglyceride stores of the abdominal fat body of developing worker honeybees are both a source of energy for sealed brood development as well as of precursors for phospholipid synthesis. REFERENCES CHEFURKAW. (1965) Intermediary metabolism of nitrogenous and lipid compounds in insects. In Physiology of the Insects, Vol. 2 (Edited by ROCKSTEIN M.) pp. 669 768. Academic Press, New York. FOLCH J., LEES N. & SLOANE-STANLEYC. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. GILBERT L. |. 8¢, CHINO H. (1974) Transport of lipids in insects. J. Lipid Res. 15, 439-456. HEPBURN H. R., CANTRILL R. C., THOMPSON P. R. KENEDI E. (1979) Metabolism of carbohydrate, lipid and protein during development of sealed worker brood of the African honeybee. J. apicult. Res. 18, 30-35. THOMPSONP. R. (1978) Histological development of cuticle in the worker honeybee, Apis mellifera adansonii L. J. apicult. Res. 17, 32-40. WARNER S. J., CANTRILLR. C. t~¢ HEPBURN n. R. (1981) Metabolism of [l-14C]palmitic acid in sealed brood of the African worker honeybee. Comp. Biochem. Physiol. 6gB, 355 356.
0305-0491/81/0201-0351102.00]0
© Pergamon Press Ltd 1981. Printed in Great Britain
CHANGES IN LIPID COMPOSITION DURING SEALED BROOD DEVELOPMENT OF AFRICAN WORKER HONEYBEES R. C. CANTRILLl, H. R. HEPBURN2 and S. J. WARNER2 Department of Medical Biochemistry ~ and Department of Physiology 2, University of the Witwatersrand, Johannesburg, South Africa (Received 19 May 1980) Abstract--l. The lipid composition of whole worker honeybees and of abdominal fat body were analysed over the course of sealed brood development. 2. Decreasing fat body wet weight is paralleled by decreases in extractable lipid particularly of the triglyceride and fatty acid components. 3. Triglyceride stored in the fat body constitutes a major source of energy for brood development and of precursor material for phospholipid synthesis during development of honeybee workers.
INTRODUCTION
Developmental biochemical and physiological studies on insects have long lacked precision because of the difficulty in reproducibly defining particular stages of development in morphological terms and in a way that obviates the intrusion of temporal variation. This major difficulty has recently been resolved for the African bee, Apis mellifera adansonii L., (Thompson, 1978) and has facilitated a gross chemical audit at least for honeybees. Thus, it is known that during sealed brood development (which includes larval, pupal and adult forms) metabolism is largely based on the utilization of lipid stores and, as adult emergence is approached, some protein (Hepburn et al., 1979). We now briefly extend these observations to include changes in different lipid compartments in both whole honeybee workers and in isolates of fat body during the course of sealed brood development in the African worker honeybee.
of lipid extracted from each stage was determined using colorimetric (cholesterol, free fatty acids and phospholipids) and enzymatic (triglyceride) test kits (Boehringer Mannheim, Mannheim, Germany). Assays were checked with authentic standards and were found to estimate about 50~ of the added lipid. Although reproducibility was constant in each assay, the results given for individual lipids are those obtained under assay conditions and are not corrected for this difference. Thus in all cases, the total lipid extracted (mg) considerably exceeds the sum of the component lipids.
RESULTS AND DISCUSSION
The relative changes in whole bee dry weight and extractable abdominal fat body are shown in Figs 1 and 2. Whereas the decrease in whole bee dry weight has previously been attributed to a loss of both lipid and some protein (Hepburn et al., 1979) the decrease 30
MATERIALS AND METHODS
25
Livestock 2o
Sealed worker brood were removed intact from their combs and classified as to stage of development using the criteria of Thompson (1978).
t-
Procedures Whole bees were dried to constant weight at 60°C for 2 weeks. Fat body tissue was obtained from the abdomens of bees simply by teasing the material from the dissected bee with a fine paint brush under distilled water. The unextracted portions of the deceased bees were also dried to constant weight to determine the average mass of fat body for each developmental stage. The lipids of whole bees were extracted as previously described (Hepburn et al., 1979). Lipids of the fat body-distilled water mixture were extracted using the method of Folch et al. (1957). Both whole bee and fat body lipids were identified by comparison with authentic standards (Sigma, St Louis, U.S.A.) after chromatographic separation on silica gel G thin layer plates in a solvent containing: petroleum etherdiethyl ether-acetic acid (60:40:1 respectively). The volume 351
~
15
-o
10
m S 0
days 0 stage
I
II
III
IV
V VIVII VIII
Fig. 1. Dry weight of sealed worker brood expressed in mg/bee (O) and extracted lipid in mg/bee (It) plotted in terms of morphological and temporal development. Each point is the mean value of two separate determinations from pooled material and each pool contained about 20 bees..
R. C. CANTRILL,HI R. HEPBURNand S. J. WARNER
352 20
S E 15
.E ol
"O o
..£1
O .IZl
'6
Ii
5
days 0 stage
lk
li I 6 I& lbl I 121
I
II
III
IV
14
V VlVll Vlll
Fig. 2. Extractable abdominal fat body expressed as mg/ bee (©) and extracted lipid mg/fat body (I) expressed in terms of morphological and temporal development.
,°] ~
in the wet weight of the fat body through successive developmental stages is paralleled by a decrease in the amount of extractable lipid (Fig. 2). The proportions of triglyceride, free fatty acid, phospholipid and cholesterol at each stage of sealed brood development are shown in Fig. 3. It can be seen that cholesterol levels remain more or less constant while phospholipid increases by some 42%. The sum of triglyceride and free fatty acid decreases to only 30% of that recovered from the larval bees of stage 1. Figure 4 shows the fall in total lipid extracted from the abdominal fat body as described. This decrease in lipid mass is matched by a dramatic fall in triglyceride and fatty acid content as well. The cholesterol content of the fat body lipid extract was difficult to determine since the low concentrations visualised by TLC were outside the range of sensitivity of the assay system used and thus have been recorded as trace amounts. Since the lipid preparation procedure from dried bees is open to criticism because of the elevated temperature used to dry the bees, the triglyceride and fatty acid content have been summed up (Table 1) to compensate for any hydrolytic reactions which may have occurred durin~ drvin~ of the bees. It is unlikely that these results can be explained in terms of a high level of unesterified fatty acid in the
• Total [] TG [] FA [] PL [] ChoI
3.0
1.0 E Q. .I
InlnI011. . 0.6~
II.InlH. IHmn. IIH.
.011
II
Ill
IV
V
Vl
VII
E
tO Q,.
0.2 E O 0 c)
VIII
Fig. 3. Total lipid (mg/'bee) and lipid composition (mg/bee) for each of the stages of development assessed. The results are uncorrected for low sensitivity of the assay systems. All values are the means of duplicate determinations of pooled material where each pool contained about 20 bees for each stage.
UTotaL I~TG ITI]FA I~PL I=lChol
1.0 O
2.C
"6 "O O
0.5 "~_ E 1.0 "O .h O ea
.i
-6 0
tr
~]tr II
tr III
t_r IV
~nt_r V
~]n t_r Vl
~lgt_r Vll
I Jm.' o VllI
Fig. 4. Total extractable lipid from thc abdominal fat body (rag/fat body) and lipid composition (rag/fat body) for each stage of development. Sampling as in Fig. 3.
Bee lipids in development Table 1. Triglyceride, fatty acid and phospholipid composition of lipid extracted from whole honeybee workers during sealed brood development* Stage
TG
FA
Total
PL
PL/Total
1 2 3 4 5 6 7 8
1.03 0.46 0.27 0.48 0.10 0.51 0.18 0.07
0.29 0.45 0.36 0.25 0.35 0.34 0.36 0.33
1.32 0.91 0.63 0.73 0.45 0.85 0.54 0.40
0.26 0.28 0.27 0.26 0.28 0.42 0.40 0.37
0.20 0.31 0.42 0.36 0.62 0.49 0.74 0.92
* Each value given is the mean of duplicate determinations on the lipid extracts of pooled bees where each pool contains about 20 bees per stage. Table 2. Triglyceride, fatty acid and phospholipid composition of lipid extracted from the abdominal fat body of developing worker honeybees* Stage
TG
FA
Total
PL
PL/Total
1 2 3 4 5 6 7 8
0.79 0.65 0.78 0.56 0.20 0.22 0.14 0.09
0.11 0.17 0.19 0.14 0.08 0.08 0.08 0.05
0.90 0.82 0.97 0.70 0.28 0.30 0.22 0.14
0.15 0.07 0.09 0.11 0.03 0.03 0.04 0.03
0.17 0.08 0.10 0.16 0.12 0.10 0.17 0.20
* Each value given is the mean of duplicate determinations on the lipid extracts of pooled bees where each pool contains about 20 bees per stage. developing bee since fatty acid levels in the fat body extract are low. Thus, less hydrolysis appears to have taken place in the more rapid preparation of a lipid extract from the fat body. Variations in triglyceride and fatty acid content may be explained in terms of the length of time required to remove sufficient bees of each stage from the brood frame. It is noteworthy that as the concentration of triglyceride and fatty acid decreases in the fat body, the phospholipid component changes concommittantly so that a constant P L : T G + FA ratio is maintained (Table 2). There are two obvious explanations for this observation, either
353
or both of which may be correct: (1) the abdominal fat body is a major store for both triglyceride and phospholipid for metamorphosis and/or (2) as triglyceride is metabolized by the fat body there is cell loss and a corresponding decrease in phospholipid. The change in the P L : T G + FA ratio seen in the whole bees may also be explained in several ways. It is possible that total phospholipid content remains constant while the levels of triglyceride and fatty acid fall. Alternatively, there may be a net synthesis of phospholipid for new cell membranes. The 4 2 ~ increase in extracted phospholipid during development may indicate net synthesis; however, the pathway for such synthesis may be more complex than initially suggested by these results. Diglycerides are an important form in which triglycerides are mobilized in insects (Gilbert & Chino, 1974) so that the route of phospholipid synthesis from triglyceride stores may be either via acylation of ct-glycerophosphate and CDP-diglyceride formation or via CDP-base incorporation directly into diglyceride. The existence of the acylation pathway is shown in the following communication (Warner et al., 1981). In any event, it is apparent that the triglyceride stores of the abdominal fat body of developing worker honeybees are both a source of energy for sealed brood development as well as of precursors for phospholipid synthesis. REFERENCES CHEFURKAW. (1965) Intermediary metabolism of nitrogenous and lipid compounds in insects. In Physiology of the Insects, Vol. 2 (Edited by ROCKSTEIN M.) pp. 669 768. Academic Press, New York. FOLCH J., LEES N. & SLOANE-STANLEYC. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. GILBERT L. |. 8¢, CHINO H. (1974) Transport of lipids in insects. J. Lipid Res. 15, 439-456. HEPBURN H. R., CANTRILL R. C., THOMPSON P. R. KENEDI E. (1979) Metabolism of carbohydrate, lipid and protein during development of sealed worker brood of the African honeybee. J. apicult. Res. 18, 30-35. THOMPSONP. R. (1978) Histological development of cuticle in the worker honeybee, Apis mellifera adansonii L. J. apicult. Res. 17, 32-40. WARNER S. J., CANTRILLR. C. t~¢ HEPBURN n. R. (1981) Metabolism of [l-14C]palmitic acid in sealed brood of the African worker honeybee. Comp. Biochem. Physiol. 6gB, 355 356.