The predominance of polyunsaturated fatty acids in the butterfly Morpho peleides before and after metamorphosis

Abstract

We hypothesized that the polyunsaturated fatty acids of the butterfly were probably derived from the diet and that there might be a great loss of body fat during metamorphosis. To substantiate these hypotheses, we analyzed the fatty acid composition and content of the diet, the larva, and the butterfly Morpho peleides. Both the diet and the tissues of the larva and butterfly had a high concentration of polyunsaturated fatty acids. In the diet, linolenic acid accounted for 19% and linoleic acid for 8% of total fatty acids. In the larva, almost 60% of the total fatty acids were polyunsaturated: linolenic acid predominated at 42% of total fatty acids, and linoleic acid was at 17%. In the butterfly, linolenic acid represented 36% and linoleic acid represented 11% of total fatty acids. The larva had a much higher total fatty acid content than the butterfly (20.2 vs. 6.9 mg). Our data indicate that the transformation from larva to butterfly during metamorphosis drastically decreased the total fatty acid content. There was bioenhancement of polyunsaturated fatty acids from the diet to the larva and butterfly. This polyunsaturation of membranes may have functional importance in providing membrane fluidity useful in flight. We hypothesized that the polyunsaturated fatty acids of the butterfly were probably derived from the diet and that there might be a great loss of body fat during metamorphosis. To substantiate these hypotheses, we analyzed the fatty acid composition and content of the diet, the larva, and the butterfly Morpho peleides. Both the diet and the tissues of the larva and butterfly had a high concentration of polyunsaturated fatty acids. In the diet, linolenic acid accounted for 19% and linoleic acid for 8% of total fatty acids. In the larva, almost 60% of the total fatty acids were polyunsaturated: linolenic acid predominated at 42% of total fatty acids, and linoleic acid was at 17%. In the butterfly, linolenic acid represented 36% and linoleic acid represented 11% of total fatty acids. The larva had a much higher total fatty acid content than the butterfly (20.2 vs. 6.9 mg). Our data indicate that the transformation from larva to butterfly during metamorphosis drastically decreased the total fatty acid content. There was bioenhancement of polyunsaturated fatty acids from the diet to the larva and butterfly. This polyunsaturation of membranes may have functional importance in providing membrane fluidity useful in flight. Insects and vertebrates share many common metabolic pathways. Although lipid metabolism in vertebrates has been well studied, there are fewer studies in insects. Insects may be useful models that can facilitate our general understanding of biology (1Law J.H. Wells M.A. Insects as biochemical models.J. Biol. Chem. 1989; 264: 16335-16338Google Scholar). For most insects, there is a dietary requirement for polyunsaturated fatty acids (2Canavoso L.E. Jouni Z.E. Karnas K.J. Penington J.E. Wells M.A. Fat metabolism in insects.Annu. Rev. Nutr. 2001; 21: 23-46Google Scholar, 3Kerkurt G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon, Oxford, UK1985Google Scholar). Using an analysis of the fatty acid composition of seven insect orders, Thompson (4Thompson S.N. A review and comparative characterization of the fatty acid composition of seven insect orders.Comp. Biochem. Physiol. 1973; 45B: 467-482Google Scholar) identified possible phylogenetic trends. It was also observed that in the order Lepidoptera, most of the species had a high content of polyunsaturated fatty acids. The linolenic acid of the 49 species analyzed varied from 0.1% to 51.0% of total fatty acids, with a mean of 22.2%. These trends provided insights into potential relationships between insect orders. The fatty acid composition of many insect species has been analyzed in the past (5Fast P.G. Insect lipids: a review.Mem. Entomol. Soc. Can. 1964; 37: 1-50Google Scholar, 6Gilbert L.I. Lipid metabolism and function in insects.Adv. Insect Physiol. 1967; 4: 69-211Google Scholar). However, in view of the multitude of insect species that exist, most knowledge of the fatty acid compositions of insects is derived from studies of a relatively small number of insect species. The butterfly of the order Lepidoptera undergoes metamorphosis from larva to butterfly. The larva feed on the leaves of the plants and then spin a cocoon. The butterfly feeds on flower nectar, which is available later in the year. We found no information about the fatty acid composition and fat content of the butterfly before and after metamorphosis and no information about how the diet of the larva might influence its fatty acid composition. On a recent trip, we visited the Butterfly Farm at Chaa Creek, Belize, in Central America. Chaa Creek is a tourist attraction and hostel for travelers in Belize. As we have developed an interest in the evolutionary patterns of fatty acids, having previously studied snails and slugs (7Zhu N. Dai X. Lin D.S. Connor W.E. The lipids of slugs and snails: evolution, diet and biosynthesis.Lipids. 1994; 29: 869-875Google Scholar), we saw the potentialities of butterfly research and discussed our ideas with the scientific director, Mike Green. We decided to collaborate on fatty acid and sterol research. At the Butterfly Farm, the butterfly Morpho peleides, or blue morpho, is raised in a controlled environment. The sole food of these butterfly larva is the leaves of the rain forest tree Pterocarpus, on which the butterfly lays its eggs. Because the larva grow on this sole food source and the butterfly expends energy for metamorphosis and later flight with limited food intake, we hypothesized that diet must play an important role in the fatty acid composition of the larva, and the butterfly may have similar or even enhanced polyunsaturated fatty acid composition as the larva, but with lower body fat and body weight. To test these hypotheses, we measured the food consumption of the larva on a daily basis and analyzed the fatty acid composition and content of the diet as well as the larva. After the larva had undergone metamorphosis, we examined the fatty acid composition and content of the butterfly, which had emerged and which had a beautiful blue color. Both butterfly larva and rain forest tree leaves were collected at the Butterfly Farm at Chaa Creek. The larva were individually raised in glass jars and fed only the leaves of the Pterocarpus tree, which is the preferred diet of this species. We measured the amount of leaves consumed over a 24 h period. The larva and leaves were frozen and transported by air to the United States. Both larva and leaves were stored at −70°C. Adult blue morpho butterflies at the end of life were later collected from the Oregon Zoo in Portland. All weights were wet weight. The fatty acid composition of butterfly larva, leaves, and butterfly was analyzed by methods described previously (8Anderson G.J. Connor W.E. Corliss J.D. Lin D.S. Rapid modulation of the n-3 docosahexaenoic acid levels in the brain and retina of the newly hatched chick.J. Lipid Res. 1989; 30: 433-441Google Scholar). All samples were ground carefully, and the total lipids were extracted with chloroform-methanol (2:1; with added 3,5-di-tert-butyl-4-hydroxytoluene for antioxidation). Aliquots of the lipid extracts were saponified by incubation with 2 ml of 6% ethanolic KOH for 1 h at 37°C. After the addition of water, neutral sterols were removed by hexane extraction. The aqueous phase was acidified, and the fatty acids were recovered by hexane extraction. Methyl esters were prepared by heating the dried extract with 1 ml of 10% boron trifluoride (BF3) in methanol for 10 min at 100°C in tightly sealed tubes with Teflon-coated screw caps. All solvent evaporation was carried out under a gentle stream of nitrogen to prevent lipid peroxidation. Fatty acid methyl esters were analyzed by GC (Perkin-Elmer, Norwalk, CT) on an apparatus equipped with a 30 meter Supelco SP 2330 fused silica capillary column (column temperature of 185–190°C) attached to a HP 85 computer/3390A integrator. Results are reported as percentage total fatty acids by weight. A mixture of fatty acid standards was run daily for calibration purposes. To analyze the total fatty acid contents of the leaves, larva, and adult butterfly, a known amount of internal standard (C-17, heptadecaenoic acid) was added to each sample analyzed, so that the mass of individual fatty acids could be calculated after GC analysis. The GC system was the same as that described above. To analyze the fatty acid compositions of lipid classes, another aliquot of lipid extract was subjected to TLC using the solvent system hexane-chloroform-diethyl ether-acetic acid (80:10:10:1.5, v/v), and the lipid classes (phospholipids, free fatty acids, triglycerides, and cholesteryl esters) were separated. The TLC bands of each lipid class were scraped into the centrifuge tubes and heated (100°C) for 10 min in BF3 for phospholipids and free fatty acids and for 45 min in BF3, benzene, and methanol for triglyceride and cholesteryl esters. Fatty acid methyl esters were also analyzed with GC, as described above. To quantify the fatty acid content in major lipid pools, lipid extracts were subjected to TLC to separate into different lipid classes as described above. A known quantity of heptadecaenoic acid was added to each sample before methylation and GC analysis. The total fatty acid content of each lipid pool was thus calculated (phospholipid, triglyceride, and cholesteryl ester). Means and standard deviations were calculated to characterize the fatty acid composition (as a percentage of total fatty acids) in larva and butterflies and Pterocarpus leaves. Differences between the fatty acid composition means were ascertained by use of the appropriate t-test. We used Bonferroni inequality to control the overall α-level. Data were analyzed using SPSS for Windows (release 11.0.1, November 15, 2001; SPSS, Inc., Chicago, IL). The fatty acid composition of the leaf of the Pterocarpus tree (the sole diet of the larva) is presented in Table 1 as percentages of total fatty acids. There was a high content of polyunsaturated fatty acids (32.8%): 18.9% of total fatty acids as linolenic acid (18:3 n-3) and 7.5% as linoleic acid (18:2 n-6). The two major saturated fatty acids were stearic acid (18:0) at 26.7% and palmitic acid (16:0) at 25% of total fatty acids. Oleic acid (18:1 n-9) contributed only 4.5% of the total fatty acids. Among the four lipid classes, phospholipids had the highest polyunsaturated fatty acid content, with a similar fatty acid composition as for total fatty acids. Phospholipid fatty acids constitute the chief membrane fatty acids.TABLE 1.Fatty acid composition of the butterfly diet, the leaves of the Pterocarpus treeFatty AcidsTotal Fatty AcidsLipid ClassesPhospholipidsFree Fatty AcidsTriglyceridesSterol EstersSaturated14:01.82 ± 0.101.73 ± 0.471.95 ± 1.133.33 ± 0.733.44 ± 0.6016:0 palmitic25.00 ± 0.8024.31 ± 3.3830.63 ± 3.6518.48 ± 1.2812.72 ± 3.4118:0 stearic26.7 ± 0.8317.91 ± 3.4219.66 ± 1.7810.40 ± 2.5914.08 ± 3.80Monounsaturated16:1 (n-7)1.42 ± 0.541.75 ± 0.551.68 ± 0.523.89 ± 1.376.34 ± 5.8018:1 (n-9)/(n-7) oleic4.48 ± 0.384.86 ± 0.3211.69 ± 2.0720.27 ± 4.538.31 ± 2.9118:1 (n-9) trans1.62 ± 0.630.14 ± 0.211.36 ± 0.213.04 ± 3.461.62 ± 1.37Polyunsaturated16:2 (n-4)3.30 ± 0.581.60 ± 0.400.26 ± 0.201.05 ± 1.391.47 ± 1.5118:2 (n-6) linoleic7.52 ± 0.179.61 ± 2.4712.86 ± 1.344.14 ± 3.323.10 ± 1.7018:3 (n-3) α-linolenic18.89 ± 0.4421.23 ± 4.1711.09 ± 1.411.95 ± 1.821.35 ± 1.00Total saturated56.54 ± 1.7247.82 ± 2.1256.32 ± 2.0441.50 ± 4.4139.99 ± 6.59Total monounsaturated8.24 ± 0.917.52 ± 0.4815.87 ± 2.0729.46 ± 6.6019.04 ± 5.48Total polyunsaturated32.83 ± 0.2934.99 ± 2.1026.22 ± 2.6015.62 ± 4.0413.32 ± 3.80Total (n-6)9.84 ± 0.1811.49 ± 2.3713.31 ± 1.508.84 ± 2.606.12 ± 2.31Total (n-3)19.56 ± 0.5221.41 ± 4.1811.77 ± 1.604.10 ± 1.733.52 ± 1.68(n-6)/(n-3)0.50 ± 0.020.57 ± 0.231.15 ± 0.202.41 ± 1.041.93 ± 0.82Values shown are percentages of total fatty acids (means ± SD, n = 6). Open table in a new shown are percentages of total fatty acids (means ± SD, n = 6). The fatty acid composition of the larva was different from that of the diet than of the fatty acids were acid accounted for of the total fatty acids, and linoleic acid contributed of the The two saturated fatty acids, stearic and palmitic acids, were much at and A of the fatty acid composition of the leaves and larva a great in polyunsaturated fatty acids and linoleic acids) and decreased saturated fatty acids and palmitic acids) in larva with the dietary fatty acids of the leaves acid composition of the butterfly AcidsTotal Fatty AcidsLipid ClassesPhospholipidsFree Fatty AcidsTriglyceridesSterol ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (n-9)/(n-7) ± ± ± ± ± (n-9) ± ± ± ± ± ± ± ± ± ± (n-6) ± ± ± ± ± (n-3) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± small at (n-6) and (n-3) as discussed in the ± ± ± ± ± small at (n-6) and (n-3) as discussed in the ± ± ± ± ± ± ± ± ± ± shown are percentages of total fatty acids (means ± SD, n = small at (n-6) and (n-3) as discussed in the Open table in a new shown are percentages of total fatty acids (means ± SD, n = 6). In the triglyceride and free fatty acid there were the same high of linolenic acid as in the total fatty acids fatty acids the of fatty acids. The linolenic acid content in phospholipids was lower The linoleic acid levels were of acid and acid in the The sterol esters had a lower polyunsaturated fatty acid content and a higher saturated fatty acid content. The fatty acid composition of adult butterflies is in Table the larva, had a high content of polyunsaturated fatty acids of total fatty acid to be at of total fatty acids. acid content was lower vs. and acid was higher vs. than in larva. The fatty acid composition of free fatty acids, and was similar to the composition of the total fatty acids. sterol esters had the polyunsaturated fatty acids and higher saturated fatty acids. The fatty acid composition of larva and butterflies in general was similar that the larva had higher linoleic acid and lower acid than adult butterflies the larva, the phospholipids of the adult butterflies acid and acid acid composition of the adult AcidsTotal Fatty AcidsLipid ClassesPhospholipidsFree Fatty AcidsTriglyceridesSterol ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (n-9)/(n-7) ± ± ± ± ± (n-9) ± ± ± ± ± ± ± ± ± ± (n-6) ± ± ± ± ± (n-3) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± small at (n-6) and (n-3) as discussed in the ± ± ± ± ± small at (n-6) and (n-3) as discussed in the ± ± ± ± ± ± ± ± ± ± shown are percentages of total fatty acids (means ± SD, n = small at (n-6) and (n-3) as discussed in the Open table in a new shown are percentages of total fatty acids (means ± SD, n = 6). the amount of leaves consumed by the larva, we calculated the total dietary of fatty acids by the larva The larva consumed of fatty acids and consumed of linoleic acid and of linolenic acid consumption of fatty acids by the ± ± ± ± ± ± ± ± ± ± ± ± ± ± Open table in a new The fatty acid content in body and body is presented in Table The transformation from larva to butterfly a in fatty acids in of total body fatty acid and fatty acid of body weight. In total body fatty acid content, there was a Both saturated and polyunsaturated fatty acids decreased fatty acids decreased In of fatty acid of body there was a in total fatty acid of body weight. and polyunsaturated fatty acids decreased and and fatty acids acid content of the larva and = = = = body ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± ± ± butterfly ± ± ± ± ± ± butterfly ± ± ± ± ± ± ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± ± ± ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly ± ± butterfly fatty ± ± butterfly ± ± butterfly butterfly butterfly butterfly butterfly larva. Open table in a new There were in body and fatty acid content in the triglyceride pool during metamorphosis 6). decreased The triglyceride pool decreased The and cholesteryl of body and fatty acid content in major lipid classes during metamorphosis of the ± ± acid ± ± ± ± ± ± in are of Open table in a new in are of In this we a butterfly species that a amount of the polyunsaturated fatty acids linoleic and linolenic acids. of these polyunsaturated fatty acids are also found in the leaf of the Pterocarpus tree, the sole food of the larva. Our that the diet of the larva to the high content of polyunsaturated fatty acids in the butterfly. on the fatty acid composition of tissues has been shown in insect species Lin Fatty acids in the lipids of of and dietary fatty Scholar, N. Fatty acid composition of insect of Biochem. Physiol. Scholar, G.J. of fatty acid composition in major lipid classes of the of the Biochem. Physiol. Scholar, of diet on the composition of body fat in Scholar). In Lipid and fatty acid composition of plants in to Lipid Scholar) observed that the fatty acid composition of the diet its For most insects, there is a dietary requirement for polyunsaturated fatty acids (2Canavoso L.E. Jouni Z.E. Karnas K.J. Penington J.E. Wells M.A. Fat metabolism in insects.Annu. Rev. Nutr. 2001; 21: 23-46Google Scholar, 3Kerkurt G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon, Oxford, UK1985Google Scholar). However, in the by G.J. of linoleic acid in a and Biochem. Scholar), insect as and of linoleic acid from We found no on the of n-3 in insects. the fatty acids of the diet and the larva in our there was a in the two fatty acids, and and a in two saturated fatty acids and stearic The that the larva can saturated or fatty acids in the diet to the two fatty acids and the butterfly larva are of fatty acids from the diet, these could be by of the fatty acids at the of saturated fatty acids. we the of linoleic and linolenic acids by the for linoleic acid and for linolenic acid The pool of linoleic acid was and the pool of linolenic acid was the that the butterfly larva are of fatty acids, we from these data that the for linoleic and linolenic acids were and A of this butterfly is its high content of linolenic which was of the total fatty acids. Thompson (4Thompson S.N. A review and comparative characterization of the fatty acid composition of seven insect orders.Comp. Biochem. Physiol. 1973; 45B: 467-482Google Scholar) reported the fatty acid composition of seven insect orders. In the order Lepidoptera, found a high content of linolenic acid in most of the species In fatty acids in the membrane membrane fluidity fatty acids and the of membranes in Scholar). The high polyunsaturated fatty acid content of the membranes of the butterfly may possible its In our we found that docosahexaenoic acid n-3) was most in and we that polyunsaturation was for W.E. Lin D.S. of and docosahexaenoic acid in the and of Lipid Res. 30: Scholar). We only small of and by the larva or butterfly from and n-3 In was reported that of to Phospholipid and fatty acid composition of major life of Biochem. Physiol. Biochem. Biol. Scholar). the in the fatty acid composition of during and with a diet, and in the fatty acid composition of during and Physiol. Scholar) examined the fatty acid composition in different for the of our there were no fatty acids found at of This an to fatty acids. In a we found acid and acid n-3) in slugs and but no docosahexaenoic acid n-3) (7Zhu N. Dai X. Lin D.S. Connor W.E. The lipids of slugs and snails: evolution, diet and biosynthesis.Lipids. 1994; 29: 869-875Google Scholar). However, n-3 was that to the in slugs and and sterols have a role in the membrane of the The of polyunsaturated to saturated fatty acids in phospholipids has a on the of membranes in composition and composition of fatty acids of from Biochem. Scholar). In the leaf and the butterfly, the fatty acid composition of phospholipids was similar to that of the total fatty acids. In larva the linolenic acid content was lower than that of the leaf vs. the of metabolic energy in insects. The major for in insects is the fat body G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon, Oxford, UK1985Google Scholar). The triglyceride of the leaves had a much lower polyunsaturated fatty acid content than the total fatty acids. In the polyunsaturated fatty acids in triglyceride of larva and adult butterflies were similar to the composition of the total fatty acids. This in to dietary lipid is triglyceride, the butterfly probably derived its polyunsaturated fatty acids from phospholipids in the diet, which have much higher levels of polyunsaturated fatty acids than In a separate but we found a mixture of different sterols in the leaves, larva, and butterfly and The fatty acid composition of the sterol esters represented a of The polyunsaturated fatty acids of sterol esters were much higher in larva and butterfly than in the This is sterol esters are from free fatty acids or from phospholipids Both and free fatty acid classes had higher polyunsaturated fatty acid levels in larva and butterfly than in the In our the butterfly larva had a higher linoleic acid content and a lower acid content than the butterfly. This may be to the fatty acid metabolism in the butterfly. metamorphosis in a and M.A. in the lipid content during the metamorphosis of the Insect Scholar) found that the to and the to data that the of varied during metamorphosis. This was by the data of (2Canavoso L.E. Jouni Z.E. Karnas K.J. Penington J.E. Wells M.A. Fat metabolism in insects.Annu. Rev. Nutr. 2001; 21: 23-46Google Scholar), which the of to during the in insects. In our the transformation from larva to butterfly drastically decreased the total fatty acid content. Although the butterfly had a lower body than larva this the loss of fatty There was also a in fatty acid weight. Because fatty acid is the most source of energy and of metabolism in insect Biochem. Physiol. Scholar), the larva must have fat during metamorphosis and the butterfly during flight as a of energy The of triglyceride observed in our is with this total It is that saturated and polyunsaturated fatty acids decreased to the same fatty acid was only The fatty acids a than n-3 fatty acid vs. The loss of fatty acid the that the adult butterfly can acid from In a we were to the fatty acid and content of two life and adult of the butterfly species peleides. This butterfly high levels of polyunsaturated fatty acids and linoleic 60% of fatty acids in larva and of fatty acids in the butterfly were The polyunsaturated fatty acids in the diet were also high of total fatty The daily of polyunsaturated fatty acids of the larva was to 8% of the total polyunsaturated fatty acids. was a to the polyunsaturation of fatty acids of the butterfly. In metamorphosis from larva to butterfly, there was the loss of a amount of fatty acids, probably as a of energy The fatty acid content of the butterfly was only of that of the larva vs. mg). to a dietary which provided a high polyunsaturated content, the butterfly membranes that were These membranes may facilitate flight. The the of Mike of Butterfly identified the at the Oregon and of the Butterfly at the Oregon