- Email: [email protected]
The fat body of bullfrog (Lithobates catesbeianus) tadpoles during metamorphosis: Changes in mass, histology, and melatonin content and effect of food deprivation
Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
The fat body of bullfrog (Lithobates catesbeianus) tadpoles during metamorphosis: Changes in mass, histology, and melatonin content and effect of food deprivation Mary L. Wright ⁎, Shaun E. Richardson, Jill M. Bigos Biology Department, College of Our Lady of the Elms, Chicopee, Massachusetts 01013, USA
a r t i c l e
i n f o
Article history: Received 25 May 2011 Received in revised form 11 August 2011 Accepted 11 August 2011 Available online 18 August 2011 Keywords: Fat bodies Adipocytes Bullfrog tadpoles Melatonin Food deprivation
a b s t r a c t The fat body of Lithobates catesbeianus (formerly Rana catesbeiana) tadpoles was studied during metamorphosis and after food deprivation in order to detect changes in its weight, adipocyte size, histology, and melatonin content. Bullfrog tadpoles have large fat bodies throughout their long larval life. Fat bodies increase in absolute weight, and weight relative to body mass, during late stages of prometamorphosis, peaking just before climax, and then decreasing, especially during the latter stages of transformation into the froglet. The climax decrease is accompanied by a reduction in size of adipocytes and a change in histology of the fat body such that interstitial tissue becomes more prominent. Food deprivation for a month during early prometamorphosis significantly decreased fat body weight and adipocyte size but did not affect the rate of development. However, food restriction just before climax retarded development, suggesting that the increased nutrient storage in the fat body before climax is necessary for metamorphic progress. Melatonin, which might be involved in the regulation of seasonal changes in fat stores, stayed approximately at the same level during most of larval life, but increased sharply in the fat body during the late stages of climax. The findings show that the rate of development of these tadpoles is not affected by starvation during larval life as long as they can utilize fat body stores for nourishment. They also suggest that the build up of fat body stores just before climax is necessary for progress during the climax period when feeding stops. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The fat bodies of anuran amphibians are large masses of adipose tissue in the form of fingerlike projections located anterior to the gonads (Duellman and Trueb, 1986). They are the primary fat depot, although the subcutaneous tissue, liver, muscle, gonads, and tail also store lipids (Jørgensen, 1992; Sheridan and Kao, 1998). Fat bodies in adult frogs cycle annually in size, correlated with temperature, food availability, and the status of the breeding cycle. Since they provide food reserves for reproduction, they are reported to be at maximum size just before hibernation and smallest just after breeding (Duellman and Trueb, 1986). Fat bodies contain lipids mainly in the form of triglycerides (Fitzpatrick, 1976). The fat body of Rana esculenta frogs shows histological and ultrastructural features similar to adipose tissue in higher vertebrates (Zancanaro et al., 1996). There have been few studies of the fat bodies of larval anurans or their changes during metamorphosis. Anuran metamorphosis consists of an early period of larval growth (premetamorphosis), a subsequent interval of continued growth and development of some adult organs (prometamorphosis) and a final period when animals cease feeding, larval organs are resorbed, and final transformation to the juvenile ⁎ Corresponding author at: Biology Department, College of Our Lady of the Elms, Chicopee, MA 01013, USA. Tel.: + 1 413 265 2298; fax: + 1 413 592 4871. E-mail address: [email protected] (M.L. Wright). 1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.08.010
frog occurs (climax). Gramapurohit et al. (1998) examined the time of appearance of fat bodies and the accumulation of fat during metamorphosis in five anuran species, including the ranids R. curtipes, R. cyanophlyctis, and R. tigrina. They found that the mass of the fat bodies varied according to species and the length of the larval period, generally being greater where the larval stage was extended. In some species, fat bodies were larger before metamorphic climax, which might indicate a stockpiling of food reserves for the non-feeding climax period. In support of this idea, whole body, liver, and tail lipids increased during prometamorphosis and declined during metamorphic climax in several anuran species (reviewed in Sheridan and Kao, 1998). Food deprivation can affect fat body size and histology in the adult frog. Weekly instead of daily feeding resulted in a decrease of fat body mass of R. tigrina. (Girish and Saidapur, 2000). After 4 months of starvation, fat body cells of R. esculenta became nearly devoid of lipid, and the fat bodies regressed to a mesenchymal-like condition (Zancanaro et al., 1996). The effect of starvation on fat body mass or histology has not been studied in tadpoles, although food deprivation may influence the rate of development depending on the stage at which it was initiated. In some species, food restriction beginning at early larval stages retarded tadpole development (D'Angelo et al., 1941; Audo et al., 1995; Denver et al., 1998), while if it was begun later in larval life, development was accelerated (D'Angelo et al., 1941; Denver et al., 1998) or not affected (Audo et al., 1995).
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
The hormone leptin is derived from adipocytes and is involved in regulation of food intake and body weight (Gündüz, 2002). Leptin was characterized in the African clawed frog, Xenopus laevis, and found to decrease food intake in tadpoles (Crespi and Denver, 2006). A leptin-like gene was expressed in various tissues, including the fat body, in the tiger salamander (Boswell et al., 2006). There is substantial evidence that melatonin, derived mainly from the pineal gland (Gern and Norris, 1979) and the eyes (Wright et al., 2006) in amphibians, is also involved in the regulation of seasonal changes in fat stores and that melatonin influences plasma leptin, although the specific effects vary. Melatonin treatment decreased body weight and suppressed visceral fat and plasma leptin in rats (Wolden-Hanson et al., 2000; Puchalski et al., 2003; Yang et al., 2005), while it increased plasma leptin in the mink (Mustonen et al., 2000). Body weight gain decreased after melatonin treatment although plasma leptin was unchanged in goldfish (De Pedro et al., 2008). Melatonin has not been identified in the fat body to our knowledge, but it is concentrated in various digestive organs in mammals (Bubenik et al., 1996), frogs (Bubenik and Pang, 1997), and tadpoles (Wright et al., 2008). The enterochromaffin cells of the digestive tract are a major source of extrapineal melatonin (Raikhlin and Kvetnoy, 1976), and because of the length of the gut in tadpoles, may represent a large reservoir of tissue melatonin. We studied changes in fat body weight in L. catesbeianus (bullfrog) tadpoles during metamorphosis, and the effect of starvation in early prometamorphosis on the fat body mass and tadpole developmental progress. We also examined the effect of fasting late in prometamorphosis on the rate of climax, reasoning that accumulation of food reserves might be necessary for progress through this period of inanition. We also compared the histology of the fat body and the size of fat cells in control and fasted prometamorphic tadpoles as well as in climax tadpoles. Finally, because melatonin might be involved in lipid regulation, changes in this hormone's level in the fat body during metamorphosis were documented. 2. Materials and methods 2.1. Animals L. catesbeianus tadpoles were obtained from Connecticut Valley Biological Supply Co. (Southampton, MA, U.S.A.) and Charles D. Sullivan Co. (Nashville, TN, U.S.A.) and kept a maximum of a few weeks in the laboratory before being used. Experiments were done in the spring or early summer. Young tadpoles were kept in plastic dishpans in 10% Holtfreter's solution, while tadpoles in late climax and froglets were housed in small tanks. Containers were cleaned and solutions changed three times weekly. All animals were in light and temperature controlled incubators at 22° on a 12L:12D cycle (light onset at 0800 h). Until feeding stopped at the beginning of climax, tadpoles were fed canned, washed spinach daily, while froglets were fed Tenebrio larvae. Staging was done according to Taylor and Kollros (1946). All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC at the College of Our Lady of the Elms. Animals were sacrificed by pithing or by immersion in 0.1% MS-222 when they reached the appropriate stage. They were blotted with kimwipes to remove excess fluid before being weighed on a Fisher Scientific Accuseries 413 top-loading balance. Following weighing, animals were dissected and the fat bodies removed, blotted on bibulous paper, and weighed on the same balance as the tadpoles and froglets. The two fat bodies of each tadpole were weighed together. 2.2. Food deprivation experiments In the first such experiment, tadpoles were at Stage XVIII (late prometamorphosis) at the start. Control animals were fed daily until
499
they ceased feeding at Stage XX, the beginning of climax, while experimental tadpoles were not fed. Animals were staged at intervals throughout the experiment to compare metamorphic progress in fed and starved groups. In the second experiment, tadpoles were at Stages X to XIII (late pre- to early prometamorphosis) at the start, with an average stage of 11.5. Control animals were fed every weekday, while experimental tadpoles were not fed at all. Animals were sacrificed after 28 days, and stage progression, body weight, and fat body weight were measured as noted above. 2.3. Histology Fat bodies from three control and three starved animals at the end of the second food deprivation experiment, and a sampling of fat bodies from climax tadpoles, were processed for morphometry and histological study. The fat bodies were initially stored in 70% alcohol. Prior to embedding, they were fixed in Carnoy's fixative for 6 h, dehydrated through a graded series of butyl alcohol, and embedded in paraffin and piccolyte. Five micron sections were mounted on slides and stained in hematoxylin and eosin or toluidine blue. Photomicrographs were taken with an AxioCam HRc camera under magnification by a Zeiss Axioimager.M2. AxioVision 4.8 software was used. 2.4. Morphometry For each sample, ten randomly-selected groups of five fat cells from different sections were projected with a microscope drawing attachment and traced onto paper, along with a 10 μm line from a stage micrometer for calibration. Four diameters of each fat cell were measured and the average diameter calculated for each sample. 2.5. Assay for fat body melatonin Tadpoles at various stages ranging from X to XXV were used. They were obtained in spring and early summer and sacrificed in the light. Thus, adipocyte melatonin represents daytime melatonin levels at these seasons of the year. Tadpoles and fat bodies were weighed and fat bodies collected according to procedures given above. Fat bodies were frozen at −20 C until processing for radioimmunoassay. Before the assay, the paired fat bodies were partially defrosted, and homogenized with a glass rod in 500 μl of 0.7% saline. Following homogenization, the mixture was centrifuged to remove all solids and the supernatant was collected and frozen at −20 °C. Melatonin in the fat bodies was assayed directly, without extraction, using a melatonin kit from IBL America (Minneapolis, MN, USA). The assay had previously been validated for use with amphibian plasma and tissue samples as described in Wright et al. (1999; 2003). The assay was further validated for use with fat body samples. The mean of the slope of the inhibition curves (−1.7670) generated from serially diluted fat body homogenate supernatant using a logit curve fit was not significantly different from the slope of the curves constructed from kit-supplied melatonin standards (−1.9987). Further characteristics (detection limit, cross reactivities, etc.) of the assay are given in Wright et al. (2003). The intra- and inter-assay coefficients of variation were 13.2 and 14.0%, respectively. Tubes were counted in a Packard RIASTAR gamma counter with a counting efficiency greater than 80%. The calculations producing the standard curve and sample hormone content were performed by computer using manufacturer-installed software. 2.6. Statistical analysis The data were evaluated using Student's t test, or ANOVA, as appropriate, with the ANOVA followed by Duncan's Multiple Range test to isolate differences among the means of the groups. Differences were considered to be significant if P b 0.05.
500
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
3. Results Each tadpole had two fat bodies located dorsally anterior to the intestine. The left fat body was larger than the one on the right side. During most of larval life, they were a bright yellow-orange, taking on a deeper orange cast at the end of climax. Both fat bodies were used to obtain fat body mass. The fat bodies in L. catesbeianus remained at approximately the same weight, with an average weight of 44.5 ± 7.4 mg during preand about one-half of prometamorphosis (Table 1). After Stage XIV, a gradual increase in the fat body weights occurred, culminating in a major rise from Stages XVIII to XIX, just before the onset of metamorphic climax, representing, at Stage XIX, an approximately sixfold increase over early larval stages. After this peak, fat body weights decreased significantly through the rest of climax, to reach the lowest weight of larval life at Stage XXV, the end of climax. Body mass increased gradually, with the difference from early larval stages becoming significant at Stage XVIII, with a peak at Stage XIX. Body weight significantly decreased thereafter through climax stages. At the end of metamorphosis body weight was about the same as that of the young tadpole. When fat body weight was calculated as a percentage of body weight (relative fat body weight), there were no significant changes until a gradual rise in the relative weight after Stage XIV culminated in a statistically significant peak at Stage XIX, just before the onset of metamorphic climax (Fig. 1). Relative fat body weight remained at a constant high during climax, not significantly different from the Stage XIX peak, except for a transient fall at Stage XX. It then significantly decreased from Stage XXIV to the end of climax at Stage XXV. The absolute and relative increases in fat body weight prior to climax suggested that fat stores were being stockpiled for metamorphic climax when tadpoles cease feeding. To determine if metamorphic progress near and into climax was affected by prior food deprivation, a group of tadpoles at Stage XVIII was starved and compared with controls, which were fed until climax began at Stage XX. Fooddeprived tadpoles began to develop more slowly than controls at the ninth day, when fed animals had reached Stage XIX (Fig. 2). The difference between stages of control and starved animals became significant on Day 20 of the experiment, as the food-deprived animals were about to enter climax. The starved animals were then fully two stages behind the controls in development. On the other hand, when food deprivation was begun earlier in larval life (late pre- to mid prometamorphosis), and continued for 28 days, developmental
Table 1 Fat body and tadpole body weights at different stages of development of Lithobates catesbeianus (means ± SEM)1. Stage III IV VII X XI XII XIV XVII XVIII XIX XX XXI XXII XXIV XXV Froglet
No. samples 3 5 5 2 2 5 5 5 11 9 9 12 16 6 6 8
Fat body weight (mg)2 a
31.3 ± 14.3 49.8 ± 12.4a 69.0 ± 22.9a 46.0 ± 35.0a 24.0 ± 1.0a 17.4 ± 5.9a 57.2 ± 23.7a 120.0 ± 31.3a,b 237.8 ± 27.0a,b 615.1 ± 109.0c 182.2 ± 50.2a,b 350.9 ± 69.3b,d 282.4 ± 53.0a,b 153.7 ± 40.7a,b 10.5 ± 2.7a 86.5 ± 28.1a,d
Body weight (g)3 4.0 ± 0.5a 4.6 ± 0.2a,b 5.3 ± 0.6a,b,c 6.3 ± 1.1a,b,c 5.9 ± 0.1a,b,c 6.9 ± 0.3a,b,c 7.7 ± 0.6a,b,c,d 9.9 ± 1.6a,b,c,d 13.9 ± 1.5d,e,f 17.5 ± 2.5e 10.9 ± 1.1b,f,h 11.6 ± 1.7c,f,g 10.9 ± 0.9b,f,h 6.3 ± 0.6a,g,h 5.4 ± 0.4a,g,h 8.5 ± 0.6a,f
1 Means with different letters within a column are significantly different with Duncan's test (P b 0.05). 2 Anova P = 8.6E-10; F = 6.55; df = 114. 3 Anova P = 5.5E-009; F = 6.01; df = 114.
progress did not change, although the absolute and relative weights of the fat bodies decreased (Fig. 3). Selected samples of fat bodies from the second food deprivation experiment and Stages XXII to XXV of climax were fixed and processed for histological observations and measurement of fat cell size. Large adipocytes made up the bulk of the tissue in fed prometamorphic tadpoles (Fig. 5a). The fat cell diameters ranged from 17 to 70 μm. Adipocyte cell size diminished after 4 weeks of food deprivation (Figs. 4, 5b) indicating that the decrease in mass of the fat bodies shown in Fig. 3 was due mainly to utilization of stored fat. Fat body cell size also decreased significantly during late climax (Figs. 4; 5c–d) and the histological appearance of the tissue changed. Capillaries and interstitial tissue around the adipocytes appeared more prominent (Fig. 5c–d) as the mean adipocyte diameter was reduced approximately 50% from 37.6± 0.3 μm in prometamorphosis to 18.2 ± 3.2 μm at the end of climax (P b 0.004; t = 8.0; df = 3). Melatonin in the fat bodies at various stages of larval life was also assessed. Fat body melatonin remained essentially the same through Stage XX, the onset of metamorphic climax, and then rose from an overall average of 13.02 ± 3.65 pg/g before climax to a high of 51.5 ± 17.6 pg/g (P b .0025; t = − 3.27; df = 34) at the last climax stages (Fig. 6). 4. Discussion In the present work, bullfrog tadpoles were deemed to be useful for fat body studies because of their large size and long larval life. They generally overwinter for one or two seasons (Pinder et al., 1992) and their progress through metamorphic climax is relatively slow. In this species, fat body weights rose in late prometamorphosis to peak at Stage XIX, just before metamorphic climax. This significant rise was also observed when fat body weights were adjusted for body weight, indicating that before climax, fat body mass increased proportionally more than body weight. The increase in fat body weight likely denotes stockpiling of food materials for climax, when feeding typically stops as mouth and digestive organs are remodeled for juvenile life. This assumption is supported by the decrease in fat body weight during climax, reaching its lowest point at Stage XXV, the end of metamorphosis. Transformation is considered to end with the complete resorption of the tail (Duellman and Trueb, 1986). We observed that disappearance of the tail stub took two to three weeks, and juveniles did not begin to feed until the tail stub was completely resorbed. The sharp decrease in relative fat body weight from Stage XXIV to XXV indicates that remaining use of fat body reserves takes place when the animals no longer get nourishment from resorbing tails, gills and other larval organs. Stockpiling of fat body reserves and their use at the end of metamorphosis appears to characterize those tadpoles that cease feeding during climax. Such might not be the case in carnivorous larvae, such as Ceratophrys ornata and Lepidobatrachus laevis, which continue to feed during metamorphic climax (Hourdry et al., 1996). Gramapurohit et al. (1998) studied changes in the fat bodies of five anuran species during metamorphosis. All of these had much shorter larval lives and much smaller body weights than L. catesbeianus in our present study. Fat body mass was proportional to body size of the species and length of larval life. The smallest species examined was Bufo melanostictus, where fat bodies were barely visible in the larva. Fat body weight of the largest species studied, R. curtipes, peaked at 64 mg just before climax. In contrast, the maximum fat body mass at the comparable stage in L. catesbeianus was 615 mg. The relative fat body mass of the two largest species examined by Gramapurohit et al. (1998) R. curtipes and R. cyanophlyctis, decreased during climax as in our study, but the difference was not statistically significant. However, climax took place much more rapidly in these species than in L. catesbeianus. Total lipid levels were proportional to body size as well in the salamanders Ambystoma opacum and
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
501
Fat Body Weight / Body Weight
0.05
0.04
d cdf
de
cdf
0.03
cdf abce abc
0.02 abc
abc
abc ab
ab
0.01
abf
ab ab
ab
b
0.00 I
II
III
IV
V
(3) (5)
VI
VII
(5)
VIII
IX
X
XI
XII
(2) (2) (5)
XIII
XIV
(5)
XV
XVI XVII XVIII XIX
XX
XXI
XXII XXIII XXIV XXV froglets
(5) (11) (9) (9)(12)(16) (6) (6) (6) (8)
Stage of Development Fig. 1. Graph of changes in the ratio of fat body weight to body weight during larval development and in the froglet. Roman numerals on the X axis indicate stages of larval development. Numbers in parentheses indicate the number of samples at each stage. Means with different letters are significantly different with Duncan's Multiple Range test (P b 0.05). Anova: P = 1.8E-10; F = 7.034; df = 114.
24
*
Fed Starved
23
Fat Body Weight (g)
relatively little stored fat are those most likely to be affected by starvation. Starvation in late prometamorphosis beginning at Stage XVIII did not affect larval development until the tadpoles were about to enter climax (Stage XX), at which time metamorphosis was retarded. This finding correlates with the rise in fat body weight at Stage XIX, and suggests that this last accumulation of food reserves is necessary for normal progress through climax since body growth has stopped. Tadpole fat bodies contain leptin, an appetite-regulating hormone which decreases food intake in tadpoles and accelerates limb development
0.25
*
0.20 0.15 0.10 0.05 0.00
Fed Fat Body Wt./Body Wt.
Ambystoma talpoideum, and lipid levels at the end of metamorphosis correlated positively with adult survival (Scott et al., 2007). Studies of the energetic costs of metamorphosis (Orlofske and Hopkins, 2009) indicate that more slowly developing tadpoles such as ranids require more energy to support metamorphosis than more rapidly transforming larvae such as bufonids. When early prometamorphic tadpoles were subjected to food deprivation for 28 days, development was not affected, although absolute and relative fat body weights decreased. It should be noted that approximately one month is a very short period in a larval life span of one to two or more years. Thus, in this species, fasting earlier in larval life has no influence on development, at least not as long as fat stores can supply nourishment. Several other studies of fasting in younger bullfrog tadpoles performed in our laboratory had similar results (unpublished data). Food deprivation retarded development in Rana sylvatica and Rana pipiens in early metamorphosis and accelerated it later on (D'Angelo et al., 1941). Similarly, fasting inhibited growth and development in Scaphiopus hammondii tadpoles in early prometamorphosis, but accelerated transformation in mid- to late prometamorphosis (Denver et al., 1998). Short term food restriction in Spea hammondii tadpoles retarded development in both pre- and prometamorphosis (Crespi and Denver, 2005), while starvation in Hyla chrysoscelis retarded metamorphosis in early stages and had no effect later on (Audo et al., 1995). Thus, there seem to be species and developmental stage differences in the effect of food deprivation, and its results may also depend on the nutritional state of the tadpole and the length of larval life. It is probable that small species with
Starved
0.020
*
0.015 0.010 0.005 0.000
Fed
Starved
Fed
Starved
18.0
21
14.4
20
Stage
Stage
22
19 18 17 0
10.8 7.2 3.6
3
6
9
12
15
18
21
24
Day of Experiment Fig. 2. Effect of pre-climax food deprivation on development into climax (n = 8 fed, 12 starved). Tadpoles were starved beginning at Stage XVIII. Stages on the y axis in Arabic numerals represent the Roman numeral staging system of Taylor and Kollros (1946). There was a significant difference on Day 20 between control and starved (P b 0.01; t = 2.89; df = 18).
0.0 Fig. 3. Effect of four weeks of late pre- to early prometamorphic food deprivation on fat body weight and final developmental stage (n = 8 fed and 8 starved). Stages on the y axis in Arabic numerals in the stage graph represent the Roman numeral staging system of Taylor and Kollros (1946). Significant differences indicated by asterisks (P b .002; t = 3.84 and 4.05; df = 14).
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
50
a
40
b
Melatonin (pg/g)
Fat Cell Diameter (um)
502
b
30 20 10 0
Fed
Starved Climax
Fig. 4. Adipocyte cell size in fed and starved prometamorphic tadpoles and during late stages of climax (n = 3 fed, 3 starved, and 6 climax Stages XXIII to XXV). Means with different letters are significantly different with Duncan's Multiple Range test (P b 0.05). Anova: P = 0.027; F = 5.517; df = 11.
(Crespi and Denver, 2006), suggesting that this hormone might signal that there are enough food reserves for progress through metamorphosis. If the leptin level is not high enough because food restriction depletes the fat body, progress through the non-feeding stage of climax could be retarded or stopped. Although we did not weigh individual fat bodies, but only the pairs found in each tadpole, our observation that the left fat body was larger than the right one is in agreement with Zancanaro et al. (1996) who noted that the left fat body of adult R. esculenta weighed more than the right. The histological structure of the fat body of prometamorphic bullfrog tadpoles also appeared to be similar to adult R. esculenta as described by Zancanaro et al. (1996). Large adipocytes make up the bulk of the tissue, although the range of cell diameters observed (17–70 μm) was less than reported in adult frog (15–140 μm; Zancanaro et al., 1996). After four weeks of fasting, adipocyte cell size significantly decreased, suggesting that food reserves in the fat body were utilized to offset lack of feeding. These results confirm the loss of fat body mass observed during fasting, and show that, as in vertebrates in general and adult frogs (Zancanaro, et al., 1996) tadpole fat cells can enlarge and shrink depending upon the amount of stored lipid. Fat body cell size also decreased during the late stages of metamorphic climax, again correlating with other data above indicating that tadpoles utilize stored food in the fat body during climax. Reduction of adipocyte size by the end of climax was even more extreme than after a month of fasting at earlier stages. As Zancanaro, et al. (1996) noted in adult
a
c
80 70 60 50 40 30 20 10 0
b
a
a a
6
9
12
9
X-XII XVII-XVIII XIX-XX XXIV-XXV
Stages Fig. 6. Melatonin in the fat body at different stages of development. Numbers in the bars indicate the number of samples in each stage range. Means with different letters are significantly different with Duncan's Multiple Range test (p b 0.05). Anova: P = 0.028; F = 3.455; df = 35.
frogs after starvation, some adipocytes were virtually lipid-free and there was an increased prominence of connective tissue and capillaries in the fat body. Melatonin in the fat body remained essentially the same until late climax when it rose nearly four-fold. Bullfrog tadpole melatonin levels also rise at climax in the gills, whereas they fall in the thyroid gland and tail, and remain the same in gut, limb, and pineal glands (Wright et al., 2008). Plasma melatonin also significantly decreases at climax (Wright et al., 2003). The increased concentration of melatonin in the fat body might come from the plasma, since injected low doses of the hormone comparable to plasma concentrations cause a rise in tissue melatonin (Wright et al., 2001). The different trends in melatonin levels in various tissues, however, might indicate a functional role of melatonin in these tissues at climax. Melatonin is a potent antioxidant and apoptosis inhibitor (Karbownik and Reiter, 2000). It protects against oxidative stress and ultraviolet radiation damage in tissues such as the skin (Slominski et al., 2008). Melatonin reduced body weight gain and increased lipid mobilization in goldfish (De Pedro et al., 2008) so that melatonin in the tadpole fat body might contribute to the utilization of fat stores during climax. Alternatively, the rise in melatonin in the fat body might only be related to the depletion of fat stores in climax as a result of cessation of feeding.
b
d
Fig. 5. Fat body histology of fed prometamorphic (a), starved prometamorphic (b), climax Stage XXIV (c), and climax Stage XXV (d) tadpoles. Scale bar (100 μm) in 5a. applies to all the photomicrographs. Hematoxylin and eosin. Adipocytes decrease in size after food restriction (b) and at the end of climax (c, d).
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
Bubenik et al. (1992) found that reduction of food intake increased melatonin levels in tissues such as stomach, intestine, and brain of mice. Determining the role of melatonin in the fat bodies and a possible influence of melatonin on leptin in the tadpole awaits further study. Acknowledgments The authors are grateful to Sarah Rowe, Lara Demoracski, and Christian Covert for technical help in some experiments. Dr. Jane Kaltenbach of Mt. Holyoke College and Dr. Ellen Faszewski of Wheelock College assisted with photography. This work was supported by NSF grant IBN-0236251. References Audo, M.C., Mann, T.M., Polk, T.L., Loudenslager, C.M., Diehl, W.J., Altig, R., 1995. Food deprivation during different periods of tadpole (Hyla chrysoscelis) ontogeny affects metamorphic performance differently. Oecologia 103, 518–522. Boswell, T., Dunn, I.C., Wilson, P.W., Joseph, N., Burt, D.W., Sharp, P.J., 2006. Identification of a non-mammalian leptin-like gene: characterization and expression in the tiger salamander (Ambystoma tigrinum). Gen. Comp. Endocrinol. 146, 157–166. Bubenik, G.A., Pang, S.F., 1997. Melatonin levels in the gastrointestinal tissues of a fish, amphibians, and a reptile. Gen. Comp. Endocrinol. 106, 415–419. Bubenik, G.A., Ball, R.O., Pang, S.F., 1992. The effect of food deprivation on brain and gastrointestinal tissue levels of tryptophan, serotonin, 5-hydroxyindoleacetic acid, and melatonin. J. Pineal Res. 12, 7–16. Bubenik, G.A., Pang, S.F., Hacker, R.R., Smith, P.S., 1996. Melatonin concentrations in serum and tissues of porcine gastrointestinal tract and their relationship to the intake and passage of food. J. Pineal Res. 21, 251–256. Crespi, E.J., Denver, R.J., 2005. Role of stress hormones in food intake regulation in anuran amphibians throughout the life cycle. Comp. Biochem. Physiol. A 141, 381–390. Crespi, E.J., Denver, R.J., 2006. Leptin (ob gene) of the South African clawed frog Xenopus laevis. Proc. Natl. Acad. Sci. U. S. A. 103, 10092–10097. D'Angelo, S.A., Gordon, A.S., Charipper, H.A., 1941. The role of thyroid and pituitary glands in the anomalous effect of inanition on amphibian metamorphosis. J. Exp. Zool. 87, 259–277. De Pedro, N., Martínez-Álvarez, R.M., Delgado, M.J., 2008. Melatonin reduces body weight in goldfish (Carassius auratus): effects on metabolic resources and some feeding regulators. J. Pineal Res. 45, 32–39. Denver, R.J., Mirhadi, N., Philips, M., 1998. Adaptive plasticity in amphibian metamorphosis: response of Scaphiopus hammondii tadpoles to habitat dessication. Ecology 79, 1859–1872. Duellman, W.E., Trueb, L., 1986. Biology of amphibians. The Johns Hopkins University Press, Maryland. Fitzpatrick, L.C., 1976. Life history patterns of storage and utilization of lipids for energy in amphibians. Am. Zool. 16, 725–732. Gern, W.A., Norris, D.O., 1979. Plasma melatonin in the neotenic tiger salamander (Ambystoma tigrinum): effects of photoperiod and pinealectomy. Gen. Comp. Endocrinol. 38, 393–398. Girish, S., Saidapur, S.K., 2000. Interrelationship between food availability, fat body, and ovarian cycles in the frog, Rana tigrina, with a discussion on the role of the fat body in anuran reproduction. J. Exp. Zool. 286, 487–493. Gramapurohit, N.P., Shanbhag, B.A., Saidapur, S.K., 1998. Pattern of growth and utilization of abdominal fat bodies during larval development and metamorphosis in five South Indian anurans. Curr. Sci. 75, 1188–1192.
503
Gündüz, B., 2002. Daily rhythm in serum melatonin and leptin levels in the Syrian hamster (Mesocricetus auratus). Comp. Biochem. Physiol. A 132, 393–401. Hourdry, J., Hermite, A.L., Ferrand, R., 1996. Changes in the digestive tract and feeding behavior of anuran amphibians during metamorphosis. Physiol. Zool. 69, 219–251. Jørgensen, C.B., 1992. Growth and reproduction. In: Feder, M.E., Burggren, W.W. (Eds.), Environmental Physiology of the Amphibians. The University of Chicago Press, Chicago, pp. 439–466. Karbownik, M., Reiter, R.J., 2000. Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proc. Soc. Exp. Biol. Med. 225, 9–22. Mustonen, A.M., Nieminen, P., Hyvärinen, H., Asikainen, J., 2000. Exogenous melatonin elevates the plasma leptin and thyroxine concentrations of the mink (Mustela vison). Z. Naturforsch. 55, 806–813. Orlofske, S.A., Hopkins, W.A., 2009. Energetics of metamorphic climax in the pickerel frog (Lithobates palustris). Comp. Biochem. Physiol. A 154, 191–196. Pinder, A.W., Storey, K.B., Ultsch, G.R., 1992. Estivation and hibernation. In: Feder, M.E., Burggren, W.W. (Eds.), Environmental Physiology of the Amphibians. The University of Chicago Press, Chicago, pp. 250–274. Puchalski, S.S., Green, J.N., Rasmussen, D.D., 2003. Melatonin effects on metabolism independent of gonad function. Endocrine 21, 169–173. Raikhlin, N.T., Kvetnoy, I.M., 1976. Melatonin and enterochromaffine cells. Acta Histochem. Bel. 55, 19–24. Scott, D.E., Casey, E.D., Donovan, M.F., Lynch, T.K., 2007. Amphibian lipid levels at metamorphosis correlate to post-metamorphic terrestrial survival. Oecologia 153, 521–532. Sheridan, M.A., Kao, Y.H., 1998. Regulation of metamorphosis-associated changes in the lipid metabolism of selected vertebrates. Am. Zool. 38, 350–368. Slominski, A., Tobin, D.J., Zmijewski, M.A., Wortsman, J., Paus, R., 2008. Melatonin in the skin: synthesis, metabolism and functions. Trends Endocrinol. Metabol. 19, 17–29. Taylor, A.C., Kollros, J.J., 1946. Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94, 7–23. Wolden-Hanson, T., Mitton, D.R., McCants, R.L., Yellon, S.M., Wilkinson, C.W., Matsumoto, A.M., Rasmussen, D.D., 2000. Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of food intake and total body fat. Endocrinology 141, 487–497. Wright, M.L., Proctor, K.L., Alves, C.D., 1999. Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles. Comp. Biochem. Physiol. C 124, 109–116. Wright, M., Alves, C., Francisco, L., Shea, T., Visconti, R., Bruni, N., 2001. Regulation of the plasma melatonin level in anuran tadpoles. In: Goos, H.J.Th., Rastogi, R.K., Vaudry, H., Pierantoni, R. (Eds.), Perspectives in Comparative Endocrinology. Monduzzi Editore, Bologna, pp. 669–674. Wright, M.L., Duffy, J.L., Guertin, C.J., Alves, C.D., Szatkowski, M.C., Visconti, R.F., 2003. Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 130, 120–128. Wright, M.L., Francisco, L.L., Scott, J.L., Richardson, S.R., Carr, J.A., King, A.B., Noyes, A.G., Visconti, R.F., 2006. Effects of bilateral and unilateral ophthalmectomy on plasma melatonin in Rana tadpoles and froglets under various experimental conditions. Gen. Comp. Endocrinol. 147, 158–166. Wright, M.L., Shea, T.A., Visconti, R.F., Ramah, B.L., Francisco, L.L., Scott, J.L., Wickett, A.L., 2008. Melatonin in anuran development and metamorphosis. In: Haldar, C., Singaravel, M., Pandi-Perumal, S.R., Cardinali, D.P. (Eds.), Experimental Endocrinology and Reproductive Biology. Science Publishers, Enfield, pp. 211–225. Yang, L.H., Liu, Z.M., Li, W.T., Xia, P.J., 2005. Effect of melatonin on metabolism of intraabdominal fat of rat. Pharm. Care Res. 5, 23–24. Zancanaro, C., Merigo, F., Digito, M., Pelosi, G., 1996. Fat body of the frog, Rana esculenta: an ultrastructural study. J. Morphol. 227, 321–334.
Contents lists available at SciVerse ScienceDirect
Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
The fat body of bullfrog (Lithobates catesbeianus) tadpoles during metamorphosis: Changes in mass, histology, and melatonin content and effect of food deprivation Mary L. Wright ⁎, Shaun E. Richardson, Jill M. Bigos Biology Department, College of Our Lady of the Elms, Chicopee, Massachusetts 01013, USA
a r t i c l e
i n f o
Article history: Received 25 May 2011 Received in revised form 11 August 2011 Accepted 11 August 2011 Available online 18 August 2011 Keywords: Fat bodies Adipocytes Bullfrog tadpoles Melatonin Food deprivation
a b s t r a c t The fat body of Lithobates catesbeianus (formerly Rana catesbeiana) tadpoles was studied during metamorphosis and after food deprivation in order to detect changes in its weight, adipocyte size, histology, and melatonin content. Bullfrog tadpoles have large fat bodies throughout their long larval life. Fat bodies increase in absolute weight, and weight relative to body mass, during late stages of prometamorphosis, peaking just before climax, and then decreasing, especially during the latter stages of transformation into the froglet. The climax decrease is accompanied by a reduction in size of adipocytes and a change in histology of the fat body such that interstitial tissue becomes more prominent. Food deprivation for a month during early prometamorphosis significantly decreased fat body weight and adipocyte size but did not affect the rate of development. However, food restriction just before climax retarded development, suggesting that the increased nutrient storage in the fat body before climax is necessary for metamorphic progress. Melatonin, which might be involved in the regulation of seasonal changes in fat stores, stayed approximately at the same level during most of larval life, but increased sharply in the fat body during the late stages of climax. The findings show that the rate of development of these tadpoles is not affected by starvation during larval life as long as they can utilize fat body stores for nourishment. They also suggest that the build up of fat body stores just before climax is necessary for progress during the climax period when feeding stops. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The fat bodies of anuran amphibians are large masses of adipose tissue in the form of fingerlike projections located anterior to the gonads (Duellman and Trueb, 1986). They are the primary fat depot, although the subcutaneous tissue, liver, muscle, gonads, and tail also store lipids (Jørgensen, 1992; Sheridan and Kao, 1998). Fat bodies in adult frogs cycle annually in size, correlated with temperature, food availability, and the status of the breeding cycle. Since they provide food reserves for reproduction, they are reported to be at maximum size just before hibernation and smallest just after breeding (Duellman and Trueb, 1986). Fat bodies contain lipids mainly in the form of triglycerides (Fitzpatrick, 1976). The fat body of Rana esculenta frogs shows histological and ultrastructural features similar to adipose tissue in higher vertebrates (Zancanaro et al., 1996). There have been few studies of the fat bodies of larval anurans or their changes during metamorphosis. Anuran metamorphosis consists of an early period of larval growth (premetamorphosis), a subsequent interval of continued growth and development of some adult organs (prometamorphosis) and a final period when animals cease feeding, larval organs are resorbed, and final transformation to the juvenile ⁎ Corresponding author at: Biology Department, College of Our Lady of the Elms, Chicopee, MA 01013, USA. Tel.: + 1 413 265 2298; fax: + 1 413 592 4871. E-mail address: [email protected] (M.L. Wright). 1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.08.010
frog occurs (climax). Gramapurohit et al. (1998) examined the time of appearance of fat bodies and the accumulation of fat during metamorphosis in five anuran species, including the ranids R. curtipes, R. cyanophlyctis, and R. tigrina. They found that the mass of the fat bodies varied according to species and the length of the larval period, generally being greater where the larval stage was extended. In some species, fat bodies were larger before metamorphic climax, which might indicate a stockpiling of food reserves for the non-feeding climax period. In support of this idea, whole body, liver, and tail lipids increased during prometamorphosis and declined during metamorphic climax in several anuran species (reviewed in Sheridan and Kao, 1998). Food deprivation can affect fat body size and histology in the adult frog. Weekly instead of daily feeding resulted in a decrease of fat body mass of R. tigrina. (Girish and Saidapur, 2000). After 4 months of starvation, fat body cells of R. esculenta became nearly devoid of lipid, and the fat bodies regressed to a mesenchymal-like condition (Zancanaro et al., 1996). The effect of starvation on fat body mass or histology has not been studied in tadpoles, although food deprivation may influence the rate of development depending on the stage at which it was initiated. In some species, food restriction beginning at early larval stages retarded tadpole development (D'Angelo et al., 1941; Audo et al., 1995; Denver et al., 1998), while if it was begun later in larval life, development was accelerated (D'Angelo et al., 1941; Denver et al., 1998) or not affected (Audo et al., 1995).
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
The hormone leptin is derived from adipocytes and is involved in regulation of food intake and body weight (Gündüz, 2002). Leptin was characterized in the African clawed frog, Xenopus laevis, and found to decrease food intake in tadpoles (Crespi and Denver, 2006). A leptin-like gene was expressed in various tissues, including the fat body, in the tiger salamander (Boswell et al., 2006). There is substantial evidence that melatonin, derived mainly from the pineal gland (Gern and Norris, 1979) and the eyes (Wright et al., 2006) in amphibians, is also involved in the regulation of seasonal changes in fat stores and that melatonin influences plasma leptin, although the specific effects vary. Melatonin treatment decreased body weight and suppressed visceral fat and plasma leptin in rats (Wolden-Hanson et al., 2000; Puchalski et al., 2003; Yang et al., 2005), while it increased plasma leptin in the mink (Mustonen et al., 2000). Body weight gain decreased after melatonin treatment although plasma leptin was unchanged in goldfish (De Pedro et al., 2008). Melatonin has not been identified in the fat body to our knowledge, but it is concentrated in various digestive organs in mammals (Bubenik et al., 1996), frogs (Bubenik and Pang, 1997), and tadpoles (Wright et al., 2008). The enterochromaffin cells of the digestive tract are a major source of extrapineal melatonin (Raikhlin and Kvetnoy, 1976), and because of the length of the gut in tadpoles, may represent a large reservoir of tissue melatonin. We studied changes in fat body weight in L. catesbeianus (bullfrog) tadpoles during metamorphosis, and the effect of starvation in early prometamorphosis on the fat body mass and tadpole developmental progress. We also examined the effect of fasting late in prometamorphosis on the rate of climax, reasoning that accumulation of food reserves might be necessary for progress through this period of inanition. We also compared the histology of the fat body and the size of fat cells in control and fasted prometamorphic tadpoles as well as in climax tadpoles. Finally, because melatonin might be involved in lipid regulation, changes in this hormone's level in the fat body during metamorphosis were documented. 2. Materials and methods 2.1. Animals L. catesbeianus tadpoles were obtained from Connecticut Valley Biological Supply Co. (Southampton, MA, U.S.A.) and Charles D. Sullivan Co. (Nashville, TN, U.S.A.) and kept a maximum of a few weeks in the laboratory before being used. Experiments were done in the spring or early summer. Young tadpoles were kept in plastic dishpans in 10% Holtfreter's solution, while tadpoles in late climax and froglets were housed in small tanks. Containers were cleaned and solutions changed three times weekly. All animals were in light and temperature controlled incubators at 22° on a 12L:12D cycle (light onset at 0800 h). Until feeding stopped at the beginning of climax, tadpoles were fed canned, washed spinach daily, while froglets were fed Tenebrio larvae. Staging was done according to Taylor and Kollros (1946). All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC at the College of Our Lady of the Elms. Animals were sacrificed by pithing or by immersion in 0.1% MS-222 when they reached the appropriate stage. They were blotted with kimwipes to remove excess fluid before being weighed on a Fisher Scientific Accuseries 413 top-loading balance. Following weighing, animals were dissected and the fat bodies removed, blotted on bibulous paper, and weighed on the same balance as the tadpoles and froglets. The two fat bodies of each tadpole were weighed together. 2.2. Food deprivation experiments In the first such experiment, tadpoles were at Stage XVIII (late prometamorphosis) at the start. Control animals were fed daily until
499
they ceased feeding at Stage XX, the beginning of climax, while experimental tadpoles were not fed. Animals were staged at intervals throughout the experiment to compare metamorphic progress in fed and starved groups. In the second experiment, tadpoles were at Stages X to XIII (late pre- to early prometamorphosis) at the start, with an average stage of 11.5. Control animals were fed every weekday, while experimental tadpoles were not fed at all. Animals were sacrificed after 28 days, and stage progression, body weight, and fat body weight were measured as noted above. 2.3. Histology Fat bodies from three control and three starved animals at the end of the second food deprivation experiment, and a sampling of fat bodies from climax tadpoles, were processed for morphometry and histological study. The fat bodies were initially stored in 70% alcohol. Prior to embedding, they were fixed in Carnoy's fixative for 6 h, dehydrated through a graded series of butyl alcohol, and embedded in paraffin and piccolyte. Five micron sections were mounted on slides and stained in hematoxylin and eosin or toluidine blue. Photomicrographs were taken with an AxioCam HRc camera under magnification by a Zeiss Axioimager.M2. AxioVision 4.8 software was used. 2.4. Morphometry For each sample, ten randomly-selected groups of five fat cells from different sections were projected with a microscope drawing attachment and traced onto paper, along with a 10 μm line from a stage micrometer for calibration. Four diameters of each fat cell were measured and the average diameter calculated for each sample. 2.5. Assay for fat body melatonin Tadpoles at various stages ranging from X to XXV were used. They were obtained in spring and early summer and sacrificed in the light. Thus, adipocyte melatonin represents daytime melatonin levels at these seasons of the year. Tadpoles and fat bodies were weighed and fat bodies collected according to procedures given above. Fat bodies were frozen at −20 C until processing for radioimmunoassay. Before the assay, the paired fat bodies were partially defrosted, and homogenized with a glass rod in 500 μl of 0.7% saline. Following homogenization, the mixture was centrifuged to remove all solids and the supernatant was collected and frozen at −20 °C. Melatonin in the fat bodies was assayed directly, without extraction, using a melatonin kit from IBL America (Minneapolis, MN, USA). The assay had previously been validated for use with amphibian plasma and tissue samples as described in Wright et al. (1999; 2003). The assay was further validated for use with fat body samples. The mean of the slope of the inhibition curves (−1.7670) generated from serially diluted fat body homogenate supernatant using a logit curve fit was not significantly different from the slope of the curves constructed from kit-supplied melatonin standards (−1.9987). Further characteristics (detection limit, cross reactivities, etc.) of the assay are given in Wright et al. (2003). The intra- and inter-assay coefficients of variation were 13.2 and 14.0%, respectively. Tubes were counted in a Packard RIASTAR gamma counter with a counting efficiency greater than 80%. The calculations producing the standard curve and sample hormone content were performed by computer using manufacturer-installed software. 2.6. Statistical analysis The data were evaluated using Student's t test, or ANOVA, as appropriate, with the ANOVA followed by Duncan's Multiple Range test to isolate differences among the means of the groups. Differences were considered to be significant if P b 0.05.
500
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
3. Results Each tadpole had two fat bodies located dorsally anterior to the intestine. The left fat body was larger than the one on the right side. During most of larval life, they were a bright yellow-orange, taking on a deeper orange cast at the end of climax. Both fat bodies were used to obtain fat body mass. The fat bodies in L. catesbeianus remained at approximately the same weight, with an average weight of 44.5 ± 7.4 mg during preand about one-half of prometamorphosis (Table 1). After Stage XIV, a gradual increase in the fat body weights occurred, culminating in a major rise from Stages XVIII to XIX, just before the onset of metamorphic climax, representing, at Stage XIX, an approximately sixfold increase over early larval stages. After this peak, fat body weights decreased significantly through the rest of climax, to reach the lowest weight of larval life at Stage XXV, the end of climax. Body mass increased gradually, with the difference from early larval stages becoming significant at Stage XVIII, with a peak at Stage XIX. Body weight significantly decreased thereafter through climax stages. At the end of metamorphosis body weight was about the same as that of the young tadpole. When fat body weight was calculated as a percentage of body weight (relative fat body weight), there were no significant changes until a gradual rise in the relative weight after Stage XIV culminated in a statistically significant peak at Stage XIX, just before the onset of metamorphic climax (Fig. 1). Relative fat body weight remained at a constant high during climax, not significantly different from the Stage XIX peak, except for a transient fall at Stage XX. It then significantly decreased from Stage XXIV to the end of climax at Stage XXV. The absolute and relative increases in fat body weight prior to climax suggested that fat stores were being stockpiled for metamorphic climax when tadpoles cease feeding. To determine if metamorphic progress near and into climax was affected by prior food deprivation, a group of tadpoles at Stage XVIII was starved and compared with controls, which were fed until climax began at Stage XX. Fooddeprived tadpoles began to develop more slowly than controls at the ninth day, when fed animals had reached Stage XIX (Fig. 2). The difference between stages of control and starved animals became significant on Day 20 of the experiment, as the food-deprived animals were about to enter climax. The starved animals were then fully two stages behind the controls in development. On the other hand, when food deprivation was begun earlier in larval life (late pre- to mid prometamorphosis), and continued for 28 days, developmental
Table 1 Fat body and tadpole body weights at different stages of development of Lithobates catesbeianus (means ± SEM)1. Stage III IV VII X XI XII XIV XVII XVIII XIX XX XXI XXII XXIV XXV Froglet
No. samples 3 5 5 2 2 5 5 5 11 9 9 12 16 6 6 8
Fat body weight (mg)2 a
31.3 ± 14.3 49.8 ± 12.4a 69.0 ± 22.9a 46.0 ± 35.0a 24.0 ± 1.0a 17.4 ± 5.9a 57.2 ± 23.7a 120.0 ± 31.3a,b 237.8 ± 27.0a,b 615.1 ± 109.0c 182.2 ± 50.2a,b 350.9 ± 69.3b,d 282.4 ± 53.0a,b 153.7 ± 40.7a,b 10.5 ± 2.7a 86.5 ± 28.1a,d
Body weight (g)3 4.0 ± 0.5a 4.6 ± 0.2a,b 5.3 ± 0.6a,b,c 6.3 ± 1.1a,b,c 5.9 ± 0.1a,b,c 6.9 ± 0.3a,b,c 7.7 ± 0.6a,b,c,d 9.9 ± 1.6a,b,c,d 13.9 ± 1.5d,e,f 17.5 ± 2.5e 10.9 ± 1.1b,f,h 11.6 ± 1.7c,f,g 10.9 ± 0.9b,f,h 6.3 ± 0.6a,g,h 5.4 ± 0.4a,g,h 8.5 ± 0.6a,f
1 Means with different letters within a column are significantly different with Duncan's test (P b 0.05). 2 Anova P = 8.6E-10; F = 6.55; df = 114. 3 Anova P = 5.5E-009; F = 6.01; df = 114.
progress did not change, although the absolute and relative weights of the fat bodies decreased (Fig. 3). Selected samples of fat bodies from the second food deprivation experiment and Stages XXII to XXV of climax were fixed and processed for histological observations and measurement of fat cell size. Large adipocytes made up the bulk of the tissue in fed prometamorphic tadpoles (Fig. 5a). The fat cell diameters ranged from 17 to 70 μm. Adipocyte cell size diminished after 4 weeks of food deprivation (Figs. 4, 5b) indicating that the decrease in mass of the fat bodies shown in Fig. 3 was due mainly to utilization of stored fat. Fat body cell size also decreased significantly during late climax (Figs. 4; 5c–d) and the histological appearance of the tissue changed. Capillaries and interstitial tissue around the adipocytes appeared more prominent (Fig. 5c–d) as the mean adipocyte diameter was reduced approximately 50% from 37.6± 0.3 μm in prometamorphosis to 18.2 ± 3.2 μm at the end of climax (P b 0.004; t = 8.0; df = 3). Melatonin in the fat bodies at various stages of larval life was also assessed. Fat body melatonin remained essentially the same through Stage XX, the onset of metamorphic climax, and then rose from an overall average of 13.02 ± 3.65 pg/g before climax to a high of 51.5 ± 17.6 pg/g (P b .0025; t = − 3.27; df = 34) at the last climax stages (Fig. 6). 4. Discussion In the present work, bullfrog tadpoles were deemed to be useful for fat body studies because of their large size and long larval life. They generally overwinter for one or two seasons (Pinder et al., 1992) and their progress through metamorphic climax is relatively slow. In this species, fat body weights rose in late prometamorphosis to peak at Stage XIX, just before metamorphic climax. This significant rise was also observed when fat body weights were adjusted for body weight, indicating that before climax, fat body mass increased proportionally more than body weight. The increase in fat body weight likely denotes stockpiling of food materials for climax, when feeding typically stops as mouth and digestive organs are remodeled for juvenile life. This assumption is supported by the decrease in fat body weight during climax, reaching its lowest point at Stage XXV, the end of metamorphosis. Transformation is considered to end with the complete resorption of the tail (Duellman and Trueb, 1986). We observed that disappearance of the tail stub took two to three weeks, and juveniles did not begin to feed until the tail stub was completely resorbed. The sharp decrease in relative fat body weight from Stage XXIV to XXV indicates that remaining use of fat body reserves takes place when the animals no longer get nourishment from resorbing tails, gills and other larval organs. Stockpiling of fat body reserves and their use at the end of metamorphosis appears to characterize those tadpoles that cease feeding during climax. Such might not be the case in carnivorous larvae, such as Ceratophrys ornata and Lepidobatrachus laevis, which continue to feed during metamorphic climax (Hourdry et al., 1996). Gramapurohit et al. (1998) studied changes in the fat bodies of five anuran species during metamorphosis. All of these had much shorter larval lives and much smaller body weights than L. catesbeianus in our present study. Fat body mass was proportional to body size of the species and length of larval life. The smallest species examined was Bufo melanostictus, where fat bodies were barely visible in the larva. Fat body weight of the largest species studied, R. curtipes, peaked at 64 mg just before climax. In contrast, the maximum fat body mass at the comparable stage in L. catesbeianus was 615 mg. The relative fat body mass of the two largest species examined by Gramapurohit et al. (1998) R. curtipes and R. cyanophlyctis, decreased during climax as in our study, but the difference was not statistically significant. However, climax took place much more rapidly in these species than in L. catesbeianus. Total lipid levels were proportional to body size as well in the salamanders Ambystoma opacum and
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
501
Fat Body Weight / Body Weight
0.05
0.04
d cdf
de
cdf
0.03
cdf abce abc
0.02 abc
abc
abc ab
ab
0.01
abf
ab ab
ab
b
0.00 I
II
III
IV
V
(3) (5)
VI
VII
(5)
VIII
IX
X
XI
XII
(2) (2) (5)
XIII
XIV
(5)
XV
XVI XVII XVIII XIX
XX
XXI
XXII XXIII XXIV XXV froglets
(5) (11) (9) (9)(12)(16) (6) (6) (6) (8)
Stage of Development Fig. 1. Graph of changes in the ratio of fat body weight to body weight during larval development and in the froglet. Roman numerals on the X axis indicate stages of larval development. Numbers in parentheses indicate the number of samples at each stage. Means with different letters are significantly different with Duncan's Multiple Range test (P b 0.05). Anova: P = 1.8E-10; F = 7.034; df = 114.
24
*
Fed Starved
23
Fat Body Weight (g)
relatively little stored fat are those most likely to be affected by starvation. Starvation in late prometamorphosis beginning at Stage XVIII did not affect larval development until the tadpoles were about to enter climax (Stage XX), at which time metamorphosis was retarded. This finding correlates with the rise in fat body weight at Stage XIX, and suggests that this last accumulation of food reserves is necessary for normal progress through climax since body growth has stopped. Tadpole fat bodies contain leptin, an appetite-regulating hormone which decreases food intake in tadpoles and accelerates limb development
0.25
*
0.20 0.15 0.10 0.05 0.00
Fed Fat Body Wt./Body Wt.
Ambystoma talpoideum, and lipid levels at the end of metamorphosis correlated positively with adult survival (Scott et al., 2007). Studies of the energetic costs of metamorphosis (Orlofske and Hopkins, 2009) indicate that more slowly developing tadpoles such as ranids require more energy to support metamorphosis than more rapidly transforming larvae such as bufonids. When early prometamorphic tadpoles were subjected to food deprivation for 28 days, development was not affected, although absolute and relative fat body weights decreased. It should be noted that approximately one month is a very short period in a larval life span of one to two or more years. Thus, in this species, fasting earlier in larval life has no influence on development, at least not as long as fat stores can supply nourishment. Several other studies of fasting in younger bullfrog tadpoles performed in our laboratory had similar results (unpublished data). Food deprivation retarded development in Rana sylvatica and Rana pipiens in early metamorphosis and accelerated it later on (D'Angelo et al., 1941). Similarly, fasting inhibited growth and development in Scaphiopus hammondii tadpoles in early prometamorphosis, but accelerated transformation in mid- to late prometamorphosis (Denver et al., 1998). Short term food restriction in Spea hammondii tadpoles retarded development in both pre- and prometamorphosis (Crespi and Denver, 2005), while starvation in Hyla chrysoscelis retarded metamorphosis in early stages and had no effect later on (Audo et al., 1995). Thus, there seem to be species and developmental stage differences in the effect of food deprivation, and its results may also depend on the nutritional state of the tadpole and the length of larval life. It is probable that small species with
Starved
0.020
*
0.015 0.010 0.005 0.000
Fed
Starved
Fed
Starved
18.0
21
14.4
20
Stage
Stage
22
19 18 17 0
10.8 7.2 3.6
3
6
9
12
15
18
21
24
Day of Experiment Fig. 2. Effect of pre-climax food deprivation on development into climax (n = 8 fed, 12 starved). Tadpoles were starved beginning at Stage XVIII. Stages on the y axis in Arabic numerals represent the Roman numeral staging system of Taylor and Kollros (1946). There was a significant difference on Day 20 between control and starved (P b 0.01; t = 2.89; df = 18).
0.0 Fig. 3. Effect of four weeks of late pre- to early prometamorphic food deprivation on fat body weight and final developmental stage (n = 8 fed and 8 starved). Stages on the y axis in Arabic numerals in the stage graph represent the Roman numeral staging system of Taylor and Kollros (1946). Significant differences indicated by asterisks (P b .002; t = 3.84 and 4.05; df = 14).
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
50
a
40
b
Melatonin (pg/g)
Fat Cell Diameter (um)
502
b
30 20 10 0
Fed
Starved Climax
Fig. 4. Adipocyte cell size in fed and starved prometamorphic tadpoles and during late stages of climax (n = 3 fed, 3 starved, and 6 climax Stages XXIII to XXV). Means with different letters are significantly different with Duncan's Multiple Range test (P b 0.05). Anova: P = 0.027; F = 5.517; df = 11.
(Crespi and Denver, 2006), suggesting that this hormone might signal that there are enough food reserves for progress through metamorphosis. If the leptin level is not high enough because food restriction depletes the fat body, progress through the non-feeding stage of climax could be retarded or stopped. Although we did not weigh individual fat bodies, but only the pairs found in each tadpole, our observation that the left fat body was larger than the right one is in agreement with Zancanaro et al. (1996) who noted that the left fat body of adult R. esculenta weighed more than the right. The histological structure of the fat body of prometamorphic bullfrog tadpoles also appeared to be similar to adult R. esculenta as described by Zancanaro et al. (1996). Large adipocytes make up the bulk of the tissue, although the range of cell diameters observed (17–70 μm) was less than reported in adult frog (15–140 μm; Zancanaro et al., 1996). After four weeks of fasting, adipocyte cell size significantly decreased, suggesting that food reserves in the fat body were utilized to offset lack of feeding. These results confirm the loss of fat body mass observed during fasting, and show that, as in vertebrates in general and adult frogs (Zancanaro, et al., 1996) tadpole fat cells can enlarge and shrink depending upon the amount of stored lipid. Fat body cell size also decreased during the late stages of metamorphic climax, again correlating with other data above indicating that tadpoles utilize stored food in the fat body during climax. Reduction of adipocyte size by the end of climax was even more extreme than after a month of fasting at earlier stages. As Zancanaro, et al. (1996) noted in adult
a
c
80 70 60 50 40 30 20 10 0
b
a
a a
6
9
12
9
X-XII XVII-XVIII XIX-XX XXIV-XXV
Stages Fig. 6. Melatonin in the fat body at different stages of development. Numbers in the bars indicate the number of samples in each stage range. Means with different letters are significantly different with Duncan's Multiple Range test (p b 0.05). Anova: P = 0.028; F = 3.455; df = 35.
frogs after starvation, some adipocytes were virtually lipid-free and there was an increased prominence of connective tissue and capillaries in the fat body. Melatonin in the fat body remained essentially the same until late climax when it rose nearly four-fold. Bullfrog tadpole melatonin levels also rise at climax in the gills, whereas they fall in the thyroid gland and tail, and remain the same in gut, limb, and pineal glands (Wright et al., 2008). Plasma melatonin also significantly decreases at climax (Wright et al., 2003). The increased concentration of melatonin in the fat body might come from the plasma, since injected low doses of the hormone comparable to plasma concentrations cause a rise in tissue melatonin (Wright et al., 2001). The different trends in melatonin levels in various tissues, however, might indicate a functional role of melatonin in these tissues at climax. Melatonin is a potent antioxidant and apoptosis inhibitor (Karbownik and Reiter, 2000). It protects against oxidative stress and ultraviolet radiation damage in tissues such as the skin (Slominski et al., 2008). Melatonin reduced body weight gain and increased lipid mobilization in goldfish (De Pedro et al., 2008) so that melatonin in the tadpole fat body might contribute to the utilization of fat stores during climax. Alternatively, the rise in melatonin in the fat body might only be related to the depletion of fat stores in climax as a result of cessation of feeding.
b
d
Fig. 5. Fat body histology of fed prometamorphic (a), starved prometamorphic (b), climax Stage XXIV (c), and climax Stage XXV (d) tadpoles. Scale bar (100 μm) in 5a. applies to all the photomicrographs. Hematoxylin and eosin. Adipocytes decrease in size after food restriction (b) and at the end of climax (c, d).
M.L. Wright et al. / Comparative Biochemistry and Physiology, Part A 160 (2011) 498–503
Bubenik et al. (1992) found that reduction of food intake increased melatonin levels in tissues such as stomach, intestine, and brain of mice. Determining the role of melatonin in the fat bodies and a possible influence of melatonin on leptin in the tadpole awaits further study. Acknowledgments The authors are grateful to Sarah Rowe, Lara Demoracski, and Christian Covert for technical help in some experiments. Dr. Jane Kaltenbach of Mt. Holyoke College and Dr. Ellen Faszewski of Wheelock College assisted with photography. This work was supported by NSF grant IBN-0236251. References Audo, M.C., Mann, T.M., Polk, T.L., Loudenslager, C.M., Diehl, W.J., Altig, R., 1995. Food deprivation during different periods of tadpole (Hyla chrysoscelis) ontogeny affects metamorphic performance differently. Oecologia 103, 518–522. Boswell, T., Dunn, I.C., Wilson, P.W., Joseph, N., Burt, D.W., Sharp, P.J., 2006. Identification of a non-mammalian leptin-like gene: characterization and expression in the tiger salamander (Ambystoma tigrinum). Gen. Comp. Endocrinol. 146, 157–166. Bubenik, G.A., Pang, S.F., 1997. Melatonin levels in the gastrointestinal tissues of a fish, amphibians, and a reptile. Gen. Comp. Endocrinol. 106, 415–419. Bubenik, G.A., Ball, R.O., Pang, S.F., 1992. The effect of food deprivation on brain and gastrointestinal tissue levels of tryptophan, serotonin, 5-hydroxyindoleacetic acid, and melatonin. J. Pineal Res. 12, 7–16. Bubenik, G.A., Pang, S.F., Hacker, R.R., Smith, P.S., 1996. Melatonin concentrations in serum and tissues of porcine gastrointestinal tract and their relationship to the intake and passage of food. J. Pineal Res. 21, 251–256. Crespi, E.J., Denver, R.J., 2005. Role of stress hormones in food intake regulation in anuran amphibians throughout the life cycle. Comp. Biochem. Physiol. A 141, 381–390. Crespi, E.J., Denver, R.J., 2006. Leptin (ob gene) of the South African clawed frog Xenopus laevis. Proc. Natl. Acad. Sci. U. S. A. 103, 10092–10097. D'Angelo, S.A., Gordon, A.S., Charipper, H.A., 1941. The role of thyroid and pituitary glands in the anomalous effect of inanition on amphibian metamorphosis. J. Exp. Zool. 87, 259–277. De Pedro, N., Martínez-Álvarez, R.M., Delgado, M.J., 2008. Melatonin reduces body weight in goldfish (Carassius auratus): effects on metabolic resources and some feeding regulators. J. Pineal Res. 45, 32–39. Denver, R.J., Mirhadi, N., Philips, M., 1998. Adaptive plasticity in amphibian metamorphosis: response of Scaphiopus hammondii tadpoles to habitat dessication. Ecology 79, 1859–1872. Duellman, W.E., Trueb, L., 1986. Biology of amphibians. The Johns Hopkins University Press, Maryland. Fitzpatrick, L.C., 1976. Life history patterns of storage and utilization of lipids for energy in amphibians. Am. Zool. 16, 725–732. Gern, W.A., Norris, D.O., 1979. Plasma melatonin in the neotenic tiger salamander (Ambystoma tigrinum): effects of photoperiod and pinealectomy. Gen. Comp. Endocrinol. 38, 393–398. Girish, S., Saidapur, S.K., 2000. Interrelationship between food availability, fat body, and ovarian cycles in the frog, Rana tigrina, with a discussion on the role of the fat body in anuran reproduction. J. Exp. Zool. 286, 487–493. Gramapurohit, N.P., Shanbhag, B.A., Saidapur, S.K., 1998. Pattern of growth and utilization of abdominal fat bodies during larval development and metamorphosis in five South Indian anurans. Curr. Sci. 75, 1188–1192.
503
Gündüz, B., 2002. Daily rhythm in serum melatonin and leptin levels in the Syrian hamster (Mesocricetus auratus). Comp. Biochem. Physiol. A 132, 393–401. Hourdry, J., Hermite, A.L., Ferrand, R., 1996. Changes in the digestive tract and feeding behavior of anuran amphibians during metamorphosis. Physiol. Zool. 69, 219–251. Jørgensen, C.B., 1992. Growth and reproduction. In: Feder, M.E., Burggren, W.W. (Eds.), Environmental Physiology of the Amphibians. The University of Chicago Press, Chicago, pp. 439–466. Karbownik, M., Reiter, R.J., 2000. Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proc. Soc. Exp. Biol. Med. 225, 9–22. Mustonen, A.M., Nieminen, P., Hyvärinen, H., Asikainen, J., 2000. Exogenous melatonin elevates the plasma leptin and thyroxine concentrations of the mink (Mustela vison). Z. Naturforsch. 55, 806–813. Orlofske, S.A., Hopkins, W.A., 2009. Energetics of metamorphic climax in the pickerel frog (Lithobates palustris). Comp. Biochem. Physiol. A 154, 191–196. Pinder, A.W., Storey, K.B., Ultsch, G.R., 1992. Estivation and hibernation. In: Feder, M.E., Burggren, W.W. (Eds.), Environmental Physiology of the Amphibians. The University of Chicago Press, Chicago, pp. 250–274. Puchalski, S.S., Green, J.N., Rasmussen, D.D., 2003. Melatonin effects on metabolism independent of gonad function. Endocrine 21, 169–173. Raikhlin, N.T., Kvetnoy, I.M., 1976. Melatonin and enterochromaffine cells. Acta Histochem. Bel. 55, 19–24. Scott, D.E., Casey, E.D., Donovan, M.F., Lynch, T.K., 2007. Amphibian lipid levels at metamorphosis correlate to post-metamorphic terrestrial survival. Oecologia 153, 521–532. Sheridan, M.A., Kao, Y.H., 1998. Regulation of metamorphosis-associated changes in the lipid metabolism of selected vertebrates. Am. Zool. 38, 350–368. Slominski, A., Tobin, D.J., Zmijewski, M.A., Wortsman, J., Paus, R., 2008. Melatonin in the skin: synthesis, metabolism and functions. Trends Endocrinol. Metabol. 19, 17–29. Taylor, A.C., Kollros, J.J., 1946. Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94, 7–23. Wolden-Hanson, T., Mitton, D.R., McCants, R.L., Yellon, S.M., Wilkinson, C.W., Matsumoto, A.M., Rasmussen, D.D., 2000. Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of food intake and total body fat. Endocrinology 141, 487–497. Wright, M.L., Proctor, K.L., Alves, C.D., 1999. Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles. Comp. Biochem. Physiol. C 124, 109–116. Wright, M., Alves, C., Francisco, L., Shea, T., Visconti, R., Bruni, N., 2001. Regulation of the plasma melatonin level in anuran tadpoles. In: Goos, H.J.Th., Rastogi, R.K., Vaudry, H., Pierantoni, R. (Eds.), Perspectives in Comparative Endocrinology. Monduzzi Editore, Bologna, pp. 669–674. Wright, M.L., Duffy, J.L., Guertin, C.J., Alves, C.D., Szatkowski, M.C., Visconti, R.F., 2003. Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol. 130, 120–128. Wright, M.L., Francisco, L.L., Scott, J.L., Richardson, S.R., Carr, J.A., King, A.B., Noyes, A.G., Visconti, R.F., 2006. Effects of bilateral and unilateral ophthalmectomy on plasma melatonin in Rana tadpoles and froglets under various experimental conditions. Gen. Comp. Endocrinol. 147, 158–166. Wright, M.L., Shea, T.A., Visconti, R.F., Ramah, B.L., Francisco, L.L., Scott, J.L., Wickett, A.L., 2008. Melatonin in anuran development and metamorphosis. In: Haldar, C., Singaravel, M., Pandi-Perumal, S.R., Cardinali, D.P. (Eds.), Experimental Endocrinology and Reproductive Biology. Science Publishers, Enfield, pp. 211–225. Yang, L.H., Liu, Z.M., Li, W.T., Xia, P.J., 2005. Effect of melatonin on metabolism of intraabdominal fat of rat. Pharm. Care Res. 5, 23–24. Zancanaro, C., Merigo, F., Digito, M., Pelosi, G., 1996. Fat body of the frog, Rana esculenta: an ultrastructural study. J. Morphol. 227, 321–334.