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The fatty acid composition of oophagous tadpoles (Chirixalus eiffingeri) fed conspecific or chicken egg yolk
Comparative Biochemistry and Physiology Part A 135 (2003) 329–336
The fatty acid composition of oophagous tadpoles (Chirixalus eiffingeri) fed conspecific or chicken egg yolk Chen-Huei Huanga, Min-Fang Liangb, Yeong-Choy Kamb,c,* a
Department of Aquatic Biosciences, National Chiayi University, Chiayi, Taiwan, ROC Department of Biology, National Changhua University of Education, Changhua, Taiwan, ROC c Department of Biology, Tunghai University, Taichung 407, Taiwan, ROC
b
Received 6 October 2002; received in revised form 12 March 2003; accepted 12 March 2003
Abstract We compared the lipid content and fatty acid composition of (1) the egg yolk of three anuran species (Chirixalus eiffingeri, Rhacophorus moltrechti and Buergeria robustus) and chicken eggs; and (2) C. eiffingeri tadpoles fed conspecific eggs or chicken egg yolk. Anuran and chicken egg yolk contained more non-polar than polar lipids but the proportions varied among species. Chicken egg yolk contained low amounts of 22:5n-3 in the polar lipid fraction, and B. robustus eggs did not contain any n-3 or n-6 non-polar lipids. The specific variation of lipid contents and fatty acid composition may relate to the maternal diet andyor breeding biology. In C. eiffingeri tadpoles that fed chicken yolk or frog egg yolk, the dominant components of polar and non-polar lipids were 16:0, 18:0, 18:1, and 18:2n-6, or 20:4n-6 fatty acids. C. eiffingeri eggs contained more n-3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and tadpoles fed conspecific eggs contained more of these fatty acids than tadpoles fed chicken egg yolk. The compositional differences in the fatty acids between C. eiffingeri tadpoles that fed frog egg or chicken egg yolk are the reflection of the variation in the dietary sources. Our results suggest a direct incorporation of fatty acids into the body without or minimal modification, which provide an important insight into the physiological aspects of cannibalism. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Amphibian; Cannibalism; Cannibals; Nutrition; Oophagy
1. Introduction Diets of larval anurans are diverse and vary widely across taxa and environments, and the nutritional quality among diets varies greatly. The diets may contain vascular plant and animal tissues, algae, cyanobacteria, dissolved organic matter, protozoans and pollen (Kupferberg, 1997). Earlier studies have repeatedly shown that food type has significant effects on development, growth and metamorphosis of anuran larvae *Corresponding author. Tel.: q886-4-23550609; fax: q 886-4-23590296. E-mail address: [email protected] (Y.-C. Kam).
(Steinwachser and Travis, 1983; Ahlgren and Bowen, 1991; Britson and Kissell, 1996; Kupferberg, 1997). Nutritional elements such as protein affect growth and development in tadpoles, with experimental evidence that growth and development can be protein limited (Nathan and James, 1972; Steinwachser and Travis, 1983; Pandian and Marian, 1985). On the other hand, lipids are also important nutritional components of the many living organisms. In addition to being the major energy source, lipids provide a range of essential nutrients for tissue development and functions (Neuringer et al., 1988; Noble and Cocchi, 1990; Maldjian et
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 3 . 0 0 0 8 2 - 5
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al., 1995). A ‘correct’ lipid composition in egg yolk is important for the viability, growth, andyor development of larvae or embryos (Delaunay et al., 1993; Noble and McCartney, 1993; Noble et al., 1996). Despite the clear importance of egg lipid composition to the developing organisms, there has been little study of amphibian embryos and larvae in general (Barassi and Bazan, 1974; Alonso et al., 1986; Rizzo et al., 1994; Caldironi and Alonso, 1996; Rizzo et al., 1999). Furthermore, the relationship between the diet and the lipid composition of larvae is virtually unknown in tadpoles. The purpose of this study was to assess whether diet could affect the chemical composition of amphibian larvae by examining the fatty acid profiles of oophagous tadpoles (Chirixalus eiffingeri) fed with conspecific eggs or chicken eggs. Specifically, we compared (1) the fatty acid composition of the yolks of anuran eggs (C. eiffingeri, Rhacophorus moltrechti and Buergeria robustus) and chicken eggs, and (2) the fatty acid composition of C. eiffingeri tadpoles fed yolks of conspecific or chicken eggs. 2. Materials and methods 2.1. Natural history of studied animals C. eiffingeri is a small rhacophorid frog (SVL ;30–40 mm), endemic to Taiwan and two adjacent small islands, Iriomote and Ishigaki (Kuramoto, 1973; Ueda, 1986). Bamboo is the predominant plant in the study sites. C. eiffingeri breeds in arboreal water pools, and deposits fertilized eggs on the inner walls of tree holes or bamboo stumps, above the waterline (Kam et al., 1996, 1998). Upon hatching, tadpoles drop into the pool of water where they grow and develop until metamorphosis. Tadpoles are obligate oophages. They are fed by female frogs that deposit unfertilized eggs directly into the water (Ueda, 1986). Female frogs visit and feed tadpoles at intervals of approximately 8 days, and feeding occurs only at night (Kam et al., 2000). R. moltrechti breeds in lentic waters. Females deposit eggs in a foam nest over the water and the tadpoles drop into the water after hatching. In contrast, B. robustus breeds in lotic waters and where females attach individual eggs to submerged rock. No parental care is present in these two
species and herbivores.
the
larvae
are
self-sustaining
2.2. Egg collection and storage We collected eggs from May to August 1999. Eggs of C. eiffingeri were collected from bamboo stumps in the National Taiwan University Experimental Forest at Chitou (Nantou County) and R. moltrechti eggs from the cattle tanks at the Agricultural Experiment Station at Wu-Sir (Nantou County). Amplectant B. robustus were collected from streams in the National Chung-Hsing University Experimental Forest at Hui-Sun, immediately returned to the laboratory and allowed to deposit their eggs in terraria on the same night on which they were collected. Fresh chicken eggs were bought from a grocery store each day the tadpoles were fed. We collected at least four clutches of each species and processed the ova as soon as possible (within 2 h) after oviposition to minimize lipid metabolism by the developing embryos. Only eggs whose developing embryos were Gosner stage 11 or younger were used (Gosner, 1960). All eggs were frozen at y20 8C. 2.3. Tadpole collection and storage On May 12, 2000, we collected 70 C. eiffingeri tadpoles (Gosner stage 35–41) from bamboo stumps at Chitou which were fed trophic eggs by female frogs. We fasted them for 7 days, and then placed them in air-tight containers, frozen quickly and stored at y20 8C. For the tadpoles fed chicken egg yolk, we collected fertilized eggs from the field on March 24, 2000, and transported them to the lab. We incubated the eggs on moist filter paper until they hatched, which took 10–14 days at 17 8C. We reared a total of 70 tadpoles for the experiment, and every 10 C. eiffingeri tadpoles were reared in a container (f500 ml) with dechlorinated tap water and fed chicken egg yolk every 4 days. The water was changed weekly. To feed C. eiffingeri tadpoles, they were removed from the rearing container and placed in a petri dish (10 cm in diameter) containing dechlorinated tap water. An entire chicken egg yolk was placed in the petri dish. Each tadpole swam toward the yolk, attached its mouth and began sucking up the yolk. We left the tadpoles in the dish for 20 min, but the tadpoles
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Table 1 Proportion of non-polar and polar lipids in yolks of C. eiffingeri, R. moltrechti, B. robustus and chicken eggs
C. eiffingeri R. moltrechti B. robustus Chicken
Total lipids (% wet egg wt.)
Non-polar (% lipid)
Polar (% lipid)
Sample size
2.19"0.46c 6.76"0.57b 6.07"0.22b 36.7"1.83a
65.1"0.66c 67.4"1.60bc 90.4"1.50a 70.4"0.50b
34.9"0.66a 32.6"1.60ab 9.6"1.50c 29.6"0.50b
4 4 3 9
Means"S.D. with different letters are significantly different (P-0.05).
appeared to be satiated only after 10 min. We checked for yolk in the stomach of each tadpole to confirm that it had eaten. After feeding, tadpoles were returned to their rearing container. As the tadpoles reached Gosner stage 34–41, we fasted them for 7 days, and then placed them in air-tight containers, frozen quickly and stored at y20 8C. 2.4. Fatty acid analysis We extracted lipids from the yolks of chicken and frogs (Folch et al., 1957). Because the size of the frog eggs was very small, the eggs used for fatty acid determination were composed samples from at least four egg clutches. The lipids were dissolved in hexane and passed through a Sep-pak cartridge (Waters Associates, Milford, MA) to obtain non-polar and polar lipid fractions (Bitman et al., 1984). The polar and non-polar lipid fractions were methyl-esterified (Lee et al., 1990). Fatty acid profiles were analyzed with a HewlettPackard HP 5890 II plus Gas Chromatograph equipped with a flame ionization detector and an HP-INNOWax capillary column (30 m=0.25 mm, 0.5 mm film thickness) as described by Huang et al. (1998). Nitrogen was used as the carrier gas with 0.5 mlymin flow rate and 30:1 split ratio. The initial temperature was 210 8C. The temperature was increased from 1 8Cymin to 240 8C, where it was held constant for 17 min. Injector and detector temperatures were 250 and 300 8C, respectively. A 1-ml sample of the methyl esters of fatty acids was injected into the gas chromatograph. Fatty acid methyl ester reference standards for gas chromatography analysis were obtained from Nu Chek (Elysian, MN) and Sigma (St. Louis, MO). The data were analyzed on a personal computer running chromatographic data analysis system software (SIS Corp., Taiwan).
2.5. Statistical analysis Experimental data were evaluated with a oneway ANOVA, followed by Duncan’s multiple range comparison to determine significant differences between treatment means at 5% significance level. Because the non-polar and polar lipid fraction data were expressed as percentages, prior to running the ANOVA, the data were transformed by arcsine square root to make their distribution normal (Sokal and Rohlf, 1994). 3. Results 3.1. Lipid content and fatty acid composition of frog and chicken egg yolks Eggs of anuran species and chicken contained more non-polar than polar lipids but the proportions varied among species (Table 1). C. eiffingeri eggs contained significantly fewer total lipids than the eggs of the other species. B. robustus eggs had a significantly greater proportion of non-polar lipids (and smaller proportion on polar lipids) than the eggs of C. eiffingeri, R. moltrechti and chickens. In general, the fatty acid composition of the non-polar and polar lipids of three anuran and chicken eggs was dominated by 16:0, 18:1 and 18:2n-6 with the exception of B. robustus which lacked 18:2n-6 (Table 2). In the non-polar lipids, B. robustus eggs did not contain 18:2n-6 and 18:3n-3 whereas C. eiffingeri and R. moltrechti and chicken contained high and similar levels of these fatty acids. In contrast, B. robustus eggs contained 20:1 which was lacked in other anuran and chicken eggs. In the polar lipids, all anuran eggs contained 18:3n-3 and 20:5n-3 but was absent in the chicken eggs. The 22:6n-3 was present in chicken and R. moltrechti eggs but absent in other two anuran species (Table 2).
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Table 2 Fatty acid composition1 (%) of non-polar and polar lipids in the yolks of C. eiffingeri (CE), R. moltrechti (RM), B. robustus (BR) and chicken (C) eggs Fatty acid C14:0 C16:0 C16:1 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:1 C20:4n-6 C20:5n-3 C22:6n-3
Non-polar CE (ns3) 1.9"0.3 22.9"0.8 10.1"1.1 3.7"0.1 28.5"3.4 20.6"0.8 7.4"1.4 nd 2.6"0.1 2.3"0.4 nd
Polar RM (ns2) 2
nd 26.8"0.1 8.1"0.0 4.6"0.0 31.0"0.9 20.6"0.0 8.0"0.9 nd nd nd nd
BR (ns2)
C (ns2)
CE (ns3)
RM (ns2)
BR (ns2)
C (ns2)
nd 38.0"1.7 8.2"0.3 8.3"0.1 38.2"1.2 nd nd 5.7"0.3 nd nd nd
nd 24.6"0.4 2.8"0.1 6.8"0.5 39.0"0.3 24.7"0.0 1.2"0.0 nd 0.9"0.3 nd nd
nd 26.1"1.1 4.2"0.3 8.9"0.2 17.7"0.3 23.2"1.0 3.4"0.6 nd 10.3"1.0 6.2"0.3 nd
nd 25.8"1.1 3.5"0.1 8.8"0.3 17.7"0.7 21.8"0.5 4.0"0.2 nd 9.9"0.6 4.1"0.5 4.4"1.3
nd 33.2"0.4 3.9"0.4 10.2"0.3 15.8"0.7 16.4"0.2 4.2"0.3 nd 10.4"0.4 5.9"0.5 nd
nd 25.0"0.6 nd 21.6"0.1 21.2"0.1 18.2"0.0 nd nd 9.7"0.0 nd 4.2"0.3
Means"S.D. 1 Fatty acids that were less than 0.5% of the total fatty acids are not listed. 2 nd, not detected. Table 3 Proportion of non-polar and polar lipids in the bodies of C. eiffingeri tadpoles fed yolks of conspecific or chicken eggs Egg yolk C. eiffingeri Chicken
Total lipids (% body wt.) 1.56"0.10b 3.69"0.67a
Non-polar (% lipid)
Polar (% lipid)
Sample size
47.4"3.05b 73.2"1.11a
52.6"3.05a 26.9"1.11b
3 3
Means"S.D. with different letters are significantly different (P-0.05). Table 4 Fatty acid composition (%) of non-polar and polar lipids in tadpoles of C. eiffingeri fed conspecific (CE) and chicken egg yolks Fatty acid
C16:0 C16:1 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C22:6n-3
Non-polar
Polar
CE (ns3)
Chicken (ns2)
CE (ns3)
Chicken (ns2)
26.1"0.2 8.4"1.8 4.7"1.3 25.8"0.8 29.6"0.2 5.3"0.4 nd nd nd
22.8"0.8 4.9"0.2 5.0"0.4 36.4"2.1 26.1"0.7 nd 2.4"0.1 nd 1.9"0.2
28.9"0.4 2.9"0.7 11.6"0.3 13.7"0.1 16.9"1.0 1.5"0.2 17.9"0.2 5.0"0.2 1.6"0.2
27.2"0.6 nd1 11.8"0.4 17.9"0.3 22.5"0.5 nd 17.0"0.1 nd 3.8"0.1
Means"S.D. 1 Fatty acids that were less than 0.5% of the total fatty acids are not listed.
3.2. Lipid content and fatty acid composition of tadpoles fed frog or chicken egg yolk C. eiffingeri tadpoles fed chicken egg yolk contained significantly more lipids than those fed frog eggs (P-0.05), and the proportion of nonpolar and polar lipids varied among groups. Tadpoles fed frog eggs contained fewer non-polar
lipids (47.4%) than polar lipids (52.6%), whereas tadpoles fed chicken egg yolk had threefold more non-polar lipids (73.2%) than polar lipids (26.9%). Differences between treatments were significant (P-0.05, Table 3). The non-polar lipids of tadpoles in both treatments were comprised primarily of 16:0, 18:1 and 18:2n-6 fatty acids (Table 4). Tadpoles fed frog
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Table 5 Fatty acid composition of the total lipids from the yolks of C. eiffingeri (CE) and chicken eggs, and from the bodies of C. eiffingeri tadpoles fed C. eiffingeri or chicken egg yolks Fatty acid
Egg yolk
Tadpole
CE (ns3)
Chicken (ns2)
CE (ns3)
Chicken (ns2)
C16:0 C16:1 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C20:6n-3
24.0"0.3 8.0"0.8 7.5"0.1 24.7"2.2 21.5"0.3 6.0"0.7 5.2"0.4 3.7"0.3 nd
24.7"0.5 2.0"0.0 11.2"0.4 33.7"0.2 22.8"0.0 0.8"0.0 3.5"0.1 nd 1.2"0.0
27.6"0.2 5.5"0.8 8.3"0.8 19.4"0.4 22.9"0.3 3.3"0.2 9.4"0.1 2.6"0.1 0.8"0.0
24.0"0.1 3.6"0.1 6.8"0.2 31.5"0.9 25.0"0.5 nd1 6.3"0.1 nd 2.4"0.2
Total n-3 Total n-6
9.7 26.7
2.0 26.3
6.7 32.3
2.4 31.3
Means"S.D. Fatty acids that were less than 0.5% of the total fatty acids are not listed.
1
eggs contained more n-3 fatty acids than tadpoles fed chicken egg yolk. The polar lipids of tadpoles fed frog eggs also contained more n-3 fatty acids (18:3, 20:5, 22:6) than the polar lipids of tadpoles fed chicken egg yolk. Data in Tables 3 and 4 were used to calculate the fatty acid composition of the total lipids in C. eiffingeri and chicken egg yolks, and in the bodies of C. eiffingeri tadpoles fed conspecific or chicken egg yolks (Table 5). 4. Discussion The fatty acid composition and the proportion of polar and nonpolar lipids differed among three species of anuran eggs. Maternal diet may affect the fatty acid composition of amphibian eggs significantly (Rizzo et al., 1999). The embryos of female Xenopus laevis fed commercial fish food enriched with n-3 fatty acids have different patterns of fatty acids in their phospholipids than the embryos of females fed beef liver. C. eiffingeri eat coleopterans, hymenopterans, plecopterans and arachnids, whereas B. robustus eat coleopterans, orthopterans, dermapterans, hemipterans, isopterans and arachnids (Do and Lue, 1982). Dietary data is not available for R. moltrechti. Thus, it is unclear whether dietary differences account for the observed differences in the fatty acid composition of eggs collected in the wild. It is worthwhile to mention that amphibian egg fatty acid profiles are similar to those of eggs of insectivorous lizards which also show high 20:4n-6, the presence of
20:5n-3 and generally low 22:6n-3 in polar lipids (Speake and Thompson, 2000). These similarities may be partly due to the insect-based diets of both the anurans and lizards. On the other hand, differences in the proportion of polar and non-polar lipids in the eggs of 14 species of amphibians may relate to differences in breeding biology (Komoroski et al., 1998). For example, ova of Plethodon cinereus, which deposits its eggs on land and has terrestrial neonates, contain a larger fraction of non-polar lipids than ova of species that oviposit in water. However, we found just the opposite: the ova of species that oviposit in water (B. robustus) has significantly more non-polar lipids than species that oviposit above water (C. eiffingeri and R. moltrechti). Differences in the fatty acid composition of C. eiffingeri tadpoles fed frog or chicken egg yolks resulted from differences in the composition of their food. This is the first report of the effect of dietary source on the fatty acid composition of tadpoles. Similar results have been reported for fishes, reptiles, birds, mammals and invertebrates (Cowey et al., 1976; Delaunay et al., 1993; Noble and McCartney, 1993; Xu et al., 1993; Yonekubo et al., 1993; Noble et al., 1996; Helland et al., 1998; Huang et al., 1998; Speake et al., 1999). In our experiment, certain fatty acids occur in tadpoles only if they are present in the tadpole’s diet. For example, C. eiffingeri eggs contained more n3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and tadpoles fed conspecific eggs also contained more of these fatty acids than
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tadpoles fed chicken eggs (Table 4). Animals, to varying degrees, have the ability to convert less unsaturated, short-chain fatty acids to more unsaturated long-chain fatty acids by elongation and desaturation at the carboxylic end of the fatty acids. For example, many fish can convert C18:3n3 and C18:2n-6 to C22:6n-3 and C20:4n-6, respectively (Wilson, 1991). Therefore, the percentage of the total fatty acids within each unsaturation series was used to assess the effect of dietary fatty acids on tadpole fatty acids. In this study, the calculated fatty acid composition of tadpole body lipids was affected by the dietary fatty acid profile. For example, the cumulative percentage of n-6 fatty acids in tadpoles fed C. eiffingeri or chicken egg yolks was 32.3 and 31.3%, respectively, and the n-6 fatty acids in C. eiffingeri and chicken egg yolks were 26.7 and 26.3%, respectively. Similar results were obtained for the n-3 fatty acids. Tadpoles fed C. eiffingeri egg yolk, which had more n-3 fatty acids than chicken egg yolk, had n-3 fatty acids in their bodies than tadpoles fed chicken egg yolk (Table 5). In conclusion, the fatty acid composition of amphibian eggs differs among species, and diet affects the fatty acid composition of C. eiffingeri tadpoles. These findings provide some insight into the physiological aspects of cannibalism. Cannibalism in amphibians occurs within and between all life stages, particularly on the larval stages (Crump, 1992). The growth and development of cannibals are enhanced compared to non-cannibals (Crump, 1990; Wildy et al., 1998). Controlling for mass (Crump, 1990) or diet energy content (Wildy et al., 1998), larvae fed conspecifics grow significantly faster than larvae fed heterospecifics or commercial food. Because the biochemical composition of cannibals and their prey are virtually identical, cannibals may directly incorporate nutrients to meet their metabolic needs, resulting in normal or faster than normal growth and development (Crump, 1992). However, the hypothesis of direct incorporation of nutrient in cannibals has never been shown experimentally. In our experiment where we fed tadpoles with conspecific egg yolk or chicken egg yolk, certain fatty acid, especially essential fatty acids, were found in the tadpoles only if it was present in their diet. For example, C. eiffingeri eggs contain more n-3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and this difference was reflected in the fatty acid composition of tadpoles. This
finding suggests a direct incorporation of fatty acids into the body without or minimal modification. In contrast, tadpoles that receive conspecific feed show no differences in the relative amino acid composition compared to those received artificial and fish-meal feeds (Nagai et al., 1971). If direct incorporation of nutritional factors (e.g. fatty acids) is metabolically more efficient, then this may explain why cannibals grow and develop faster than non-cannibals. In addition, although C. eiffingeri tadpoles fed with chicken egg yolk had significantly more lipid than those fed with frog egg yolk, our preliminary data show experimentally that C. eiffingeri tadpoles fed with conspecific eggs grow faster and reached metamorphosis earlier than those fed on chicken egg yolk (Liang et al., 2002). Thus, qualitative rather than quantitative effects of diet may be more important in determining tadpole growth and development (Kupferberg et al., 1994). Whether differences in fatty acid composition are responsible for the differential development and growth of C. eiffingeri tadpoles is pending because other nutrients have not been evaluated. Thus, further works need to be done to reveal the differences in nutritional compositions between frog and chicken egg yolk and to assess the possible causal–effect relationship between the nutritional composition and the tadpole growth and development. Acknowledgments This study was supported by National Science Council grants (NSC 89-2313-B-021-012 and 892311-B-018-004) to CHH and YCK, respectively. We thank the staff of the Experimental Forest of the National Taiwan University at Chitou for providing accommodation and permitting us to work in the experimental forest. Comments and suggestions on an earlier draft of this manuscript by M.L. Crump, B.K. Speake, M.B. Thompson and A.F. Warneke are appreciated. References Ahlgren, M.O., Bowen, S.H., 1991. Growth and survival of tadpoles (Bufo americanus) fed amorphous detritus derived from natural waters. Hydrobiologia 218, 49–51. Alonso, T.S., Bonini de Romanelli, I.C., Bazan, N.G., 1986. Changes in triacyglycerol, diacyglycerol and free fatty acids after fertilization in developing toad embryos. Biochem. Biophys. Acta 875, 465–472.
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Kupferberg, S.J., 1997. The role of larval diet in anuran metamorphosis. Am. Zool. 37, 146–159. Kupferberg, S.J., Marks, J.C., Power, M.E., 1994. Effects of variation in natural algal and detrital diets on larval anuran (Hyla regilla) life history traits. Copeia 1994, 446–457. Kuramoto, M., 1973. The amphibians of Iriomote of the Ryukyu Islands: ecological and zoogeographical notes. Bull. Fukuoka Univ. Edu. 22, 139–151. Lee, M.H., Wang, M.L., Min, B.W., 1990. Effect of methylation on determination of fatty acids. Food Sci. 17, 1–10. Liang, M.-F., Huang, C.-H., Kam, Y.-C., 2002. Effects of intermittent feeding on the growth of oophagous (Chirixalus eiffingeri) and herbivorous (Chirixalus idiootocus) tadpoles from Taiwan. J. Zool. 256, 207–213. Maldjian, A., Farkas, K., Noble, R.C., Cocchi, M., Speake, B.K., 1995. The transfer of decosahexaenoic acid from the yolk to the tissues of the chick embryo. Biochim. Biophys. Acta 1258, 81–89. Nagai, Y., Nagai, S., Tishikawa, T., 1971. The nutritional efficiency of cannibalism and an artificial feed for the growth of tadpoles of Japanese toad (Bufo vulgaris). Agri. Biol. Chem. 35, 697–703. Nathan, J.M., James, V.G., 1972. The role of protozoa in the nutrition of tadpoles. Copeia 1972, 669–679. Neuringer, M., Anderson, G.J., Connor, W.E., 1988. The essentiality of n-3 fatty acids for the development and function of the retina and brain. Annu. Rev. Nutr. 8, 517–541. Noble, R.C., Cocchi, M., 1990. Lipid metabolism and the neonatal chicken. Prog. Lipid Res. 29, 107–140. Noble, R.C., McCartney, R., 1993. Lipid and fatty acid compostional differences between eggs of wild and captivebreeding alligators (Alligator mississippiensis): an association with reduced hatchability? J. Zool. Lond. 230, 639–649. Noble, R.C., Speake, B.K., McCartney, R., Foggin, C.M., Deeming, D.C., 1996. Yolk lipids and their fatty acids in the wild and captive Ostrich (Struthio camelus). Comp. Biochem. Physiol., Part B 113, 753–756. Pandian, T.J., Marian, M.P., 1985. Predicting anuran metamorphosis and energetics. Physiol. Zool. 58, 538–552. Rizzo, A.M., Gornati, R., Galli, C., Bernardini, G., Berra, B., 1994. Cholesterol, triacyglicerols and phospholipids during Xenopus embryo development. Cell Biol. Int. 18, 1085–1090. Rizzo, A.M., Gornati, R., Rossi, F., Bernardini, G., Berra, B., 1999. Effect of maternal diet on the distribution of phospholipids and their fatty acid composition in Xenopus laevis embryos. J. Nutr. Biochem. 10, 44–48. Sokal, R.R., Rohlf, F.J., 1994. Biometry: The Principles and Practice of Statistics in Biological Research. W. H. Freeman and Company, New York. Speake, B.K., Decrock, F., Surai, P.F., Groscolas, R., 1999. Fatty acids composition of the adipose tissue and yolk lipids of a bird with a Marine-based diet, the Emperor Penguin (Aptenodytes forsteri). Lipids 34 (3), 283–290. Speake, B.K., Thompson, M.B., 2000. Lipids of eggs and neonates of oviparous and viviparous lizards. Comp. Biochem. Physiol. A 127, 453–467. Steinwachser, K., Travis, J., 1983. Influence of food quality and quantity on early growth of two anurans. Copeia 1983, 238–242.
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Ueda, H., 1986. Reproduction of Chirixalus eiffingeri (Boettger). Sci. Rep. Lab. Amphibian Biol., Hiroshima Univ. 8, 109–116. Wildy, E.L., Chivers, D.P., Kiesecker, J.M., Blaustein, R.B., 1998. Cannibalism enhances growth in larval long-toed salamanders, (Ambystoma macrodactylum). J. Herpetol. 32, 286–289. Wilson, R.P., 1991. Handbook of Nutrient Requirements of Finfish. CRC Press Inc, Boca Raton.
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The fatty acid composition of oophagous tadpoles (Chirixalus eiffingeri) fed conspecific or chicken egg yolk Chen-Huei Huanga, Min-Fang Liangb, Yeong-Choy Kamb,c,* a
Department of Aquatic Biosciences, National Chiayi University, Chiayi, Taiwan, ROC Department of Biology, National Changhua University of Education, Changhua, Taiwan, ROC c Department of Biology, Tunghai University, Taichung 407, Taiwan, ROC
b
Received 6 October 2002; received in revised form 12 March 2003; accepted 12 March 2003
Abstract We compared the lipid content and fatty acid composition of (1) the egg yolk of three anuran species (Chirixalus eiffingeri, Rhacophorus moltrechti and Buergeria robustus) and chicken eggs; and (2) C. eiffingeri tadpoles fed conspecific eggs or chicken egg yolk. Anuran and chicken egg yolk contained more non-polar than polar lipids but the proportions varied among species. Chicken egg yolk contained low amounts of 22:5n-3 in the polar lipid fraction, and B. robustus eggs did not contain any n-3 or n-6 non-polar lipids. The specific variation of lipid contents and fatty acid composition may relate to the maternal diet andyor breeding biology. In C. eiffingeri tadpoles that fed chicken yolk or frog egg yolk, the dominant components of polar and non-polar lipids were 16:0, 18:0, 18:1, and 18:2n-6, or 20:4n-6 fatty acids. C. eiffingeri eggs contained more n-3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and tadpoles fed conspecific eggs contained more of these fatty acids than tadpoles fed chicken egg yolk. The compositional differences in the fatty acids between C. eiffingeri tadpoles that fed frog egg or chicken egg yolk are the reflection of the variation in the dietary sources. Our results suggest a direct incorporation of fatty acids into the body without or minimal modification, which provide an important insight into the physiological aspects of cannibalism. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Amphibian; Cannibalism; Cannibals; Nutrition; Oophagy
1. Introduction Diets of larval anurans are diverse and vary widely across taxa and environments, and the nutritional quality among diets varies greatly. The diets may contain vascular plant and animal tissues, algae, cyanobacteria, dissolved organic matter, protozoans and pollen (Kupferberg, 1997). Earlier studies have repeatedly shown that food type has significant effects on development, growth and metamorphosis of anuran larvae *Corresponding author. Tel.: q886-4-23550609; fax: q 886-4-23590296. E-mail address: [email protected] (Y.-C. Kam).
(Steinwachser and Travis, 1983; Ahlgren and Bowen, 1991; Britson and Kissell, 1996; Kupferberg, 1997). Nutritional elements such as protein affect growth and development in tadpoles, with experimental evidence that growth and development can be protein limited (Nathan and James, 1972; Steinwachser and Travis, 1983; Pandian and Marian, 1985). On the other hand, lipids are also important nutritional components of the many living organisms. In addition to being the major energy source, lipids provide a range of essential nutrients for tissue development and functions (Neuringer et al., 1988; Noble and Cocchi, 1990; Maldjian et
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 3 . 0 0 0 8 2 - 5
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al., 1995). A ‘correct’ lipid composition in egg yolk is important for the viability, growth, andyor development of larvae or embryos (Delaunay et al., 1993; Noble and McCartney, 1993; Noble et al., 1996). Despite the clear importance of egg lipid composition to the developing organisms, there has been little study of amphibian embryos and larvae in general (Barassi and Bazan, 1974; Alonso et al., 1986; Rizzo et al., 1994; Caldironi and Alonso, 1996; Rizzo et al., 1999). Furthermore, the relationship between the diet and the lipid composition of larvae is virtually unknown in tadpoles. The purpose of this study was to assess whether diet could affect the chemical composition of amphibian larvae by examining the fatty acid profiles of oophagous tadpoles (Chirixalus eiffingeri) fed with conspecific eggs or chicken eggs. Specifically, we compared (1) the fatty acid composition of the yolks of anuran eggs (C. eiffingeri, Rhacophorus moltrechti and Buergeria robustus) and chicken eggs, and (2) the fatty acid composition of C. eiffingeri tadpoles fed yolks of conspecific or chicken eggs. 2. Materials and methods 2.1. Natural history of studied animals C. eiffingeri is a small rhacophorid frog (SVL ;30–40 mm), endemic to Taiwan and two adjacent small islands, Iriomote and Ishigaki (Kuramoto, 1973; Ueda, 1986). Bamboo is the predominant plant in the study sites. C. eiffingeri breeds in arboreal water pools, and deposits fertilized eggs on the inner walls of tree holes or bamboo stumps, above the waterline (Kam et al., 1996, 1998). Upon hatching, tadpoles drop into the pool of water where they grow and develop until metamorphosis. Tadpoles are obligate oophages. They are fed by female frogs that deposit unfertilized eggs directly into the water (Ueda, 1986). Female frogs visit and feed tadpoles at intervals of approximately 8 days, and feeding occurs only at night (Kam et al., 2000). R. moltrechti breeds in lentic waters. Females deposit eggs in a foam nest over the water and the tadpoles drop into the water after hatching. In contrast, B. robustus breeds in lotic waters and where females attach individual eggs to submerged rock. No parental care is present in these two
species and herbivores.
the
larvae
are
self-sustaining
2.2. Egg collection and storage We collected eggs from May to August 1999. Eggs of C. eiffingeri were collected from bamboo stumps in the National Taiwan University Experimental Forest at Chitou (Nantou County) and R. moltrechti eggs from the cattle tanks at the Agricultural Experiment Station at Wu-Sir (Nantou County). Amplectant B. robustus were collected from streams in the National Chung-Hsing University Experimental Forest at Hui-Sun, immediately returned to the laboratory and allowed to deposit their eggs in terraria on the same night on which they were collected. Fresh chicken eggs were bought from a grocery store each day the tadpoles were fed. We collected at least four clutches of each species and processed the ova as soon as possible (within 2 h) after oviposition to minimize lipid metabolism by the developing embryos. Only eggs whose developing embryos were Gosner stage 11 or younger were used (Gosner, 1960). All eggs were frozen at y20 8C. 2.3. Tadpole collection and storage On May 12, 2000, we collected 70 C. eiffingeri tadpoles (Gosner stage 35–41) from bamboo stumps at Chitou which were fed trophic eggs by female frogs. We fasted them for 7 days, and then placed them in air-tight containers, frozen quickly and stored at y20 8C. For the tadpoles fed chicken egg yolk, we collected fertilized eggs from the field on March 24, 2000, and transported them to the lab. We incubated the eggs on moist filter paper until they hatched, which took 10–14 days at 17 8C. We reared a total of 70 tadpoles for the experiment, and every 10 C. eiffingeri tadpoles were reared in a container (f500 ml) with dechlorinated tap water and fed chicken egg yolk every 4 days. The water was changed weekly. To feed C. eiffingeri tadpoles, they were removed from the rearing container and placed in a petri dish (10 cm in diameter) containing dechlorinated tap water. An entire chicken egg yolk was placed in the petri dish. Each tadpole swam toward the yolk, attached its mouth and began sucking up the yolk. We left the tadpoles in the dish for 20 min, but the tadpoles
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Table 1 Proportion of non-polar and polar lipids in yolks of C. eiffingeri, R. moltrechti, B. robustus and chicken eggs
C. eiffingeri R. moltrechti B. robustus Chicken
Total lipids (% wet egg wt.)
Non-polar (% lipid)
Polar (% lipid)
Sample size
2.19"0.46c 6.76"0.57b 6.07"0.22b 36.7"1.83a
65.1"0.66c 67.4"1.60bc 90.4"1.50a 70.4"0.50b
34.9"0.66a 32.6"1.60ab 9.6"1.50c 29.6"0.50b
4 4 3 9
Means"S.D. with different letters are significantly different (P-0.05).
appeared to be satiated only after 10 min. We checked for yolk in the stomach of each tadpole to confirm that it had eaten. After feeding, tadpoles were returned to their rearing container. As the tadpoles reached Gosner stage 34–41, we fasted them for 7 days, and then placed them in air-tight containers, frozen quickly and stored at y20 8C. 2.4. Fatty acid analysis We extracted lipids from the yolks of chicken and frogs (Folch et al., 1957). Because the size of the frog eggs was very small, the eggs used for fatty acid determination were composed samples from at least four egg clutches. The lipids were dissolved in hexane and passed through a Sep-pak cartridge (Waters Associates, Milford, MA) to obtain non-polar and polar lipid fractions (Bitman et al., 1984). The polar and non-polar lipid fractions were methyl-esterified (Lee et al., 1990). Fatty acid profiles were analyzed with a HewlettPackard HP 5890 II plus Gas Chromatograph equipped with a flame ionization detector and an HP-INNOWax capillary column (30 m=0.25 mm, 0.5 mm film thickness) as described by Huang et al. (1998). Nitrogen was used as the carrier gas with 0.5 mlymin flow rate and 30:1 split ratio. The initial temperature was 210 8C. The temperature was increased from 1 8Cymin to 240 8C, where it was held constant for 17 min. Injector and detector temperatures were 250 and 300 8C, respectively. A 1-ml sample of the methyl esters of fatty acids was injected into the gas chromatograph. Fatty acid methyl ester reference standards for gas chromatography analysis were obtained from Nu Chek (Elysian, MN) and Sigma (St. Louis, MO). The data were analyzed on a personal computer running chromatographic data analysis system software (SIS Corp., Taiwan).
2.5. Statistical analysis Experimental data were evaluated with a oneway ANOVA, followed by Duncan’s multiple range comparison to determine significant differences between treatment means at 5% significance level. Because the non-polar and polar lipid fraction data were expressed as percentages, prior to running the ANOVA, the data were transformed by arcsine square root to make their distribution normal (Sokal and Rohlf, 1994). 3. Results 3.1. Lipid content and fatty acid composition of frog and chicken egg yolks Eggs of anuran species and chicken contained more non-polar than polar lipids but the proportions varied among species (Table 1). C. eiffingeri eggs contained significantly fewer total lipids than the eggs of the other species. B. robustus eggs had a significantly greater proportion of non-polar lipids (and smaller proportion on polar lipids) than the eggs of C. eiffingeri, R. moltrechti and chickens. In general, the fatty acid composition of the non-polar and polar lipids of three anuran and chicken eggs was dominated by 16:0, 18:1 and 18:2n-6 with the exception of B. robustus which lacked 18:2n-6 (Table 2). In the non-polar lipids, B. robustus eggs did not contain 18:2n-6 and 18:3n-3 whereas C. eiffingeri and R. moltrechti and chicken contained high and similar levels of these fatty acids. In contrast, B. robustus eggs contained 20:1 which was lacked in other anuran and chicken eggs. In the polar lipids, all anuran eggs contained 18:3n-3 and 20:5n-3 but was absent in the chicken eggs. The 22:6n-3 was present in chicken and R. moltrechti eggs but absent in other two anuran species (Table 2).
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Table 2 Fatty acid composition1 (%) of non-polar and polar lipids in the yolks of C. eiffingeri (CE), R. moltrechti (RM), B. robustus (BR) and chicken (C) eggs Fatty acid C14:0 C16:0 C16:1 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:1 C20:4n-6 C20:5n-3 C22:6n-3
Non-polar CE (ns3) 1.9"0.3 22.9"0.8 10.1"1.1 3.7"0.1 28.5"3.4 20.6"0.8 7.4"1.4 nd 2.6"0.1 2.3"0.4 nd
Polar RM (ns2) 2
nd 26.8"0.1 8.1"0.0 4.6"0.0 31.0"0.9 20.6"0.0 8.0"0.9 nd nd nd nd
BR (ns2)
C (ns2)
CE (ns3)
RM (ns2)
BR (ns2)
C (ns2)
nd 38.0"1.7 8.2"0.3 8.3"0.1 38.2"1.2 nd nd 5.7"0.3 nd nd nd
nd 24.6"0.4 2.8"0.1 6.8"0.5 39.0"0.3 24.7"0.0 1.2"0.0 nd 0.9"0.3 nd nd
nd 26.1"1.1 4.2"0.3 8.9"0.2 17.7"0.3 23.2"1.0 3.4"0.6 nd 10.3"1.0 6.2"0.3 nd
nd 25.8"1.1 3.5"0.1 8.8"0.3 17.7"0.7 21.8"0.5 4.0"0.2 nd 9.9"0.6 4.1"0.5 4.4"1.3
nd 33.2"0.4 3.9"0.4 10.2"0.3 15.8"0.7 16.4"0.2 4.2"0.3 nd 10.4"0.4 5.9"0.5 nd
nd 25.0"0.6 nd 21.6"0.1 21.2"0.1 18.2"0.0 nd nd 9.7"0.0 nd 4.2"0.3
Means"S.D. 1 Fatty acids that were less than 0.5% of the total fatty acids are not listed. 2 nd, not detected. Table 3 Proportion of non-polar and polar lipids in the bodies of C. eiffingeri tadpoles fed yolks of conspecific or chicken eggs Egg yolk C. eiffingeri Chicken
Total lipids (% body wt.) 1.56"0.10b 3.69"0.67a
Non-polar (% lipid)
Polar (% lipid)
Sample size
47.4"3.05b 73.2"1.11a
52.6"3.05a 26.9"1.11b
3 3
Means"S.D. with different letters are significantly different (P-0.05). Table 4 Fatty acid composition (%) of non-polar and polar lipids in tadpoles of C. eiffingeri fed conspecific (CE) and chicken egg yolks Fatty acid
C16:0 C16:1 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C22:6n-3
Non-polar
Polar
CE (ns3)
Chicken (ns2)
CE (ns3)
Chicken (ns2)
26.1"0.2 8.4"1.8 4.7"1.3 25.8"0.8 29.6"0.2 5.3"0.4 nd nd nd
22.8"0.8 4.9"0.2 5.0"0.4 36.4"2.1 26.1"0.7 nd 2.4"0.1 nd 1.9"0.2
28.9"0.4 2.9"0.7 11.6"0.3 13.7"0.1 16.9"1.0 1.5"0.2 17.9"0.2 5.0"0.2 1.6"0.2
27.2"0.6 nd1 11.8"0.4 17.9"0.3 22.5"0.5 nd 17.0"0.1 nd 3.8"0.1
Means"S.D. 1 Fatty acids that were less than 0.5% of the total fatty acids are not listed.
3.2. Lipid content and fatty acid composition of tadpoles fed frog or chicken egg yolk C. eiffingeri tadpoles fed chicken egg yolk contained significantly more lipids than those fed frog eggs (P-0.05), and the proportion of nonpolar and polar lipids varied among groups. Tadpoles fed frog eggs contained fewer non-polar
lipids (47.4%) than polar lipids (52.6%), whereas tadpoles fed chicken egg yolk had threefold more non-polar lipids (73.2%) than polar lipids (26.9%). Differences between treatments were significant (P-0.05, Table 3). The non-polar lipids of tadpoles in both treatments were comprised primarily of 16:0, 18:1 and 18:2n-6 fatty acids (Table 4). Tadpoles fed frog
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Table 5 Fatty acid composition of the total lipids from the yolks of C. eiffingeri (CE) and chicken eggs, and from the bodies of C. eiffingeri tadpoles fed C. eiffingeri or chicken egg yolks Fatty acid
Egg yolk
Tadpole
CE (ns3)
Chicken (ns2)
CE (ns3)
Chicken (ns2)
C16:0 C16:1 C18:0 C18:1n-9 C18:2n-6 C18:3n-3 C20:4n-6 C20:5n-3 C20:6n-3
24.0"0.3 8.0"0.8 7.5"0.1 24.7"2.2 21.5"0.3 6.0"0.7 5.2"0.4 3.7"0.3 nd
24.7"0.5 2.0"0.0 11.2"0.4 33.7"0.2 22.8"0.0 0.8"0.0 3.5"0.1 nd 1.2"0.0
27.6"0.2 5.5"0.8 8.3"0.8 19.4"0.4 22.9"0.3 3.3"0.2 9.4"0.1 2.6"0.1 0.8"0.0
24.0"0.1 3.6"0.1 6.8"0.2 31.5"0.9 25.0"0.5 nd1 6.3"0.1 nd 2.4"0.2
Total n-3 Total n-6
9.7 26.7
2.0 26.3
6.7 32.3
2.4 31.3
Means"S.D. Fatty acids that were less than 0.5% of the total fatty acids are not listed.
1
eggs contained more n-3 fatty acids than tadpoles fed chicken egg yolk. The polar lipids of tadpoles fed frog eggs also contained more n-3 fatty acids (18:3, 20:5, 22:6) than the polar lipids of tadpoles fed chicken egg yolk. Data in Tables 3 and 4 were used to calculate the fatty acid composition of the total lipids in C. eiffingeri and chicken egg yolks, and in the bodies of C. eiffingeri tadpoles fed conspecific or chicken egg yolks (Table 5). 4. Discussion The fatty acid composition and the proportion of polar and nonpolar lipids differed among three species of anuran eggs. Maternal diet may affect the fatty acid composition of amphibian eggs significantly (Rizzo et al., 1999). The embryos of female Xenopus laevis fed commercial fish food enriched with n-3 fatty acids have different patterns of fatty acids in their phospholipids than the embryos of females fed beef liver. C. eiffingeri eat coleopterans, hymenopterans, plecopterans and arachnids, whereas B. robustus eat coleopterans, orthopterans, dermapterans, hemipterans, isopterans and arachnids (Do and Lue, 1982). Dietary data is not available for R. moltrechti. Thus, it is unclear whether dietary differences account for the observed differences in the fatty acid composition of eggs collected in the wild. It is worthwhile to mention that amphibian egg fatty acid profiles are similar to those of eggs of insectivorous lizards which also show high 20:4n-6, the presence of
20:5n-3 and generally low 22:6n-3 in polar lipids (Speake and Thompson, 2000). These similarities may be partly due to the insect-based diets of both the anurans and lizards. On the other hand, differences in the proportion of polar and non-polar lipids in the eggs of 14 species of amphibians may relate to differences in breeding biology (Komoroski et al., 1998). For example, ova of Plethodon cinereus, which deposits its eggs on land and has terrestrial neonates, contain a larger fraction of non-polar lipids than ova of species that oviposit in water. However, we found just the opposite: the ova of species that oviposit in water (B. robustus) has significantly more non-polar lipids than species that oviposit above water (C. eiffingeri and R. moltrechti). Differences in the fatty acid composition of C. eiffingeri tadpoles fed frog or chicken egg yolks resulted from differences in the composition of their food. This is the first report of the effect of dietary source on the fatty acid composition of tadpoles. Similar results have been reported for fishes, reptiles, birds, mammals and invertebrates (Cowey et al., 1976; Delaunay et al., 1993; Noble and McCartney, 1993; Xu et al., 1993; Yonekubo et al., 1993; Noble et al., 1996; Helland et al., 1998; Huang et al., 1998; Speake et al., 1999). In our experiment, certain fatty acids occur in tadpoles only if they are present in the tadpole’s diet. For example, C. eiffingeri eggs contained more n3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and tadpoles fed conspecific eggs also contained more of these fatty acids than
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tadpoles fed chicken eggs (Table 4). Animals, to varying degrees, have the ability to convert less unsaturated, short-chain fatty acids to more unsaturated long-chain fatty acids by elongation and desaturation at the carboxylic end of the fatty acids. For example, many fish can convert C18:3n3 and C18:2n-6 to C22:6n-3 and C20:4n-6, respectively (Wilson, 1991). Therefore, the percentage of the total fatty acids within each unsaturation series was used to assess the effect of dietary fatty acids on tadpole fatty acids. In this study, the calculated fatty acid composition of tadpole body lipids was affected by the dietary fatty acid profile. For example, the cumulative percentage of n-6 fatty acids in tadpoles fed C. eiffingeri or chicken egg yolks was 32.3 and 31.3%, respectively, and the n-6 fatty acids in C. eiffingeri and chicken egg yolks were 26.7 and 26.3%, respectively. Similar results were obtained for the n-3 fatty acids. Tadpoles fed C. eiffingeri egg yolk, which had more n-3 fatty acids than chicken egg yolk, had n-3 fatty acids in their bodies than tadpoles fed chicken egg yolk (Table 5). In conclusion, the fatty acid composition of amphibian eggs differs among species, and diet affects the fatty acid composition of C. eiffingeri tadpoles. These findings provide some insight into the physiological aspects of cannibalism. Cannibalism in amphibians occurs within and between all life stages, particularly on the larval stages (Crump, 1992). The growth and development of cannibals are enhanced compared to non-cannibals (Crump, 1990; Wildy et al., 1998). Controlling for mass (Crump, 1990) or diet energy content (Wildy et al., 1998), larvae fed conspecifics grow significantly faster than larvae fed heterospecifics or commercial food. Because the biochemical composition of cannibals and their prey are virtually identical, cannibals may directly incorporate nutrients to meet their metabolic needs, resulting in normal or faster than normal growth and development (Crump, 1992). However, the hypothesis of direct incorporation of nutrient in cannibals has never been shown experimentally. In our experiment where we fed tadpoles with conspecific egg yolk or chicken egg yolk, certain fatty acid, especially essential fatty acids, were found in the tadpoles only if it was present in their diet. For example, C. eiffingeri eggs contain more n-3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and this difference was reflected in the fatty acid composition of tadpoles. This
finding suggests a direct incorporation of fatty acids into the body without or minimal modification. In contrast, tadpoles that receive conspecific feed show no differences in the relative amino acid composition compared to those received artificial and fish-meal feeds (Nagai et al., 1971). If direct incorporation of nutritional factors (e.g. fatty acids) is metabolically more efficient, then this may explain why cannibals grow and develop faster than non-cannibals. In addition, although C. eiffingeri tadpoles fed with chicken egg yolk had significantly more lipid than those fed with frog egg yolk, our preliminary data show experimentally that C. eiffingeri tadpoles fed with conspecific eggs grow faster and reached metamorphosis earlier than those fed on chicken egg yolk (Liang et al., 2002). Thus, qualitative rather than quantitative effects of diet may be more important in determining tadpole growth and development (Kupferberg et al., 1994). Whether differences in fatty acid composition are responsible for the differential development and growth of C. eiffingeri tadpoles is pending because other nutrients have not been evaluated. Thus, further works need to be done to reveal the differences in nutritional compositions between frog and chicken egg yolk and to assess the possible causal–effect relationship between the nutritional composition and the tadpole growth and development. Acknowledgments This study was supported by National Science Council grants (NSC 89-2313-B-021-012 and 892311-B-018-004) to CHH and YCK, respectively. We thank the staff of the Experimental Forest of the National Taiwan University at Chitou for providing accommodation and permitting us to work in the experimental forest. Comments and suggestions on an earlier draft of this manuscript by M.L. Crump, B.K. Speake, M.B. Thompson and A.F. Warneke are appreciated. References Ahlgren, M.O., Bowen, S.H., 1991. Growth and survival of tadpoles (Bufo americanus) fed amorphous detritus derived from natural waters. Hydrobiologia 218, 49–51. Alonso, T.S., Bonini de Romanelli, I.C., Bazan, N.G., 1986. Changes in triacyglycerol, diacyglycerol and free fatty acids after fertilization in developing toad embryos. Biochem. Biophys. Acta 875, 465–472.
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