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The effect of photoperiod on changes in plasma glucose, cholesterol, and free fatty acids during cold acclimation in frogs
THE EFFECT OF PHOTOPERIOD ON CHANGES IN PLASMA GLUCOSE, CHOLESTEROL. AND FREE FATTY ACIDS DURING COLD ACCLIMATION IN FROGS E. DON STIWNS Department
.4bstract
of Zoology.
University
of Guelph.
Guelph.
Ontario.
Canada
N IG ZW I
I. Many
prebious experiments on cold acclimation in frogs were carried out on animals in total darkness. I tested the effect of photopcriod on changes In plasma FFA. glucose. and cholesterol durmg cold acclimation. 2. Hematocrit levels were lowe!- whereas plasma FFA Icvcls wcrc higher in frogs kept in total darkness during cold acclimation. 3. Plasma glucose and plasma cholesterol levels wcrc not affected by photoperiod during cold acclimntion. maIntamed
IKTRODUCTION
Harri
and
his
colleagues
have
carried
out
many
studies concerning the control mechanisms and changes that occur during temperature acclimation in frogs. Many of these studies arc published in this Journal (e.g. Harri & Hendenstam. 1972; Harri PL uI., 1972: Harri. 1975; Harri & Tale. 1975a; Harri & Tale. 1975b). In their experiments when they acclimate frogs to the cold. they also acclimate them to total darkness. This protocol is appropriate in Finland where Harri and his colleagues carry out their cxpcrimcnts; because during winter days are very short (Turku. Finland is about latitude 60.5). Howcvcr. frogs also “hibernate” during winter at lower latitudes. for example to at least latitude 35 ~ in the United States. In temperate locations. at least some frog species (Ranu sp. in particular) do not burrow deep in the mud. Rather they sit on the bottom of ponds where the water temperature is about 4 C (Emcry c’t trl.. 1972). Thus they are cold acclimated but arc not in total darkness. The present experiment was carried out to see if total darkness influenced the changes that occur during cold acclimation in frogs. \I.4TERI-\LS
AND \IETHODS
Adult l’rogx. Rumr p~picr~s of both sexes were obtained from a local supphc~- (they wcrc caught m Quebec. <‘anada). Expcrimcnts were carried out in September 1979. tt-ogs were maintamcd in large stream tanks (2 m long x 0.5 m wide) with constantly running well water at 23 25 C IOm deep. They were fed mealworms daily at II00 hr. Photoperiod was LD 12: 12. .After 3 weeks. accllmatlon to the abobe conditions. a sample of 9 frogs were removed as controls. The remamlng frogs ut’rc diwdcd into two groups of 16 each and were placed :nto 100 I. aquaria filled with well water. Water tcmpcrature In these tanks was maintained at 6 i I C. was filtcrcd. and wah aerated. One tank was kept in total darkness. and in the other a LD I?:12 photopcrlod was maintained. Except for the Ilght. the tanks and other conditions were Identical. The frogs were not fed during cold acclimation. One-half of the frogs wcrc removed from each
tank for sampling after being in the cold for 9 days: the remainder w&e removed after 44 days in the cold. All samnline was done at 1400hr. Frogs in the dark tank wcw , sampled in extremely dim red light. Frogs were weighed. double-plthed. and opened to cxposc the heart. Blood samples were drawn into capillary tubes (ammonium hcparm coated) by capillary actlon. and centrifuged. Plasma samples were frozen in liquid mtrogen and stored at ~ 10 C until analysis. Glucose was estimated on a 10 111 allquot of plasma using glucose oxldase (Sigma kit No. 510). Total plasma cholesterol was cstlmated on a IO itl aliquot wing the method of Rao c’tal. (1977). Plasma FFA was estimated on a 100 ~1 ahquot using the method of Noma (Y t/l. (1973). In some cases it has nccehsary to pool wmples from two or three frogs. L
RESI. LT.5
Mean body weight was 36.3 g (range 23~ 52.5). There were no significant differences related to sex. photoperiod. or acclimation temperature.
Hematocrits were lower after 9 davs in the cold and then increased from 9 to 44 days (Fig. 1A). Frogs in the 12: 12 photoperiod had significantly higher hematocrits (33.3 & 1.33) than those held in constant darkness (2X.2 i 1.99). There were no significant differences related to sex.
There were no significant effects related to sex, and no significant differences related to photoperiod. Plasma glucose was significantly lower after 9 days in the cold, and decreased even more after 44 days in the cold (Fig. 1B).
In e\ery case the concentration of plasma cholestcrol was greater in females than in males (in all samples the ranges did not even overlap) and thus
E. DON STFVENS
Fig. I. The effect of photoperiod on changes in hematocrit level (A), plasma glucose level (B). plasma cholesterol level (C). and plasma FFA level (D) during 44 days acclimation in the cold (6 C) in frogs. LD = 12: I2 indicates I2 hr light. 12 hr dark (solid lines) and LD = 0:24 indxates continuous darkness (dashed lines).
must be considered separately. There was no significant difference related to photoperiod at either 9 or 44 days in the cold. Plasma cholesterol decrease significantly in both males and females after 9 days in the cold (Fig. IC). It increased significantly between 9 and 44 days in males, but did not return to levels seen in warm-acclimated frogs. The small increase between 9 and 44 days in females was not significant.
There were no differences in plasma FFA levels related to sex. Levels decreased markedly after 9 days in the cold. but there were no differences related to photoperiod. After 44 days in the cold, levels of plasma FFA in frogs on 12: 12 photoperiod stayed low, whereas levels in frogs in darkness returned to and exceeded levels in warm adapted controls.
DlSCUSSION
Clearly, the important result of the present study is that some variables were altered (hematocrit and plasma FFA) by photoperiod during cold acclimation whereas others were not (plasma glucose and plasma cholesterol). The erects of season and temperature on
a variety of variables are well documented in amphibians (Mizell, 1965; Hong ef ul., 1968; Hermansen & Jorgensen, 1969; Harri, 1975). However the effects of photoperiod are less well understood. The interactive effects of photoperiod and tcmperature acclimation at the level of the whole organism have been shown in other groups of animals (e.g. goldfish. Hoar & Robertson. 1959; salamanders. Hutchison. 1961) as well as frogs. Vinegar & Hutchison (1965) reported that photoperiod affected whole animal oxygen uptake in frogs at some acclimation temperatures, but that this effect was short-lived and not seen after one week. Because photoperiod is one of the most reliable environmental cues. it is often used to trigger adaptive responses in anticipation of changing temperature. The changes in metabolite levels are large relative to the changes in hematocrit. and thus the changes in metabolite levels are not due to shifts in fluid between compartments. The decrease in glucose in cold is similar to that reported by Harri & Lindgren (1972). Cholesterol is a precursor of steroids in frogs and levels arc high in females (Boyd. 1938). The observation that total darkness decreases glucose levels but increases FFA and cholesterol levels suggests that the hormone levels
The effect of photoperiod
that participate in the control of substrate utilization are affected by photoperiod and that energetic pathways are altered by light. In this regard Hanke (1973) reports that long photoperiod or continuous darkness change secretory activity of hypothalamus in Runa pipirns, and that the activity of the adenohypophysis is regulated by the hypothalamus. Thus some of the effects reported to be due to cold acclimation in amphibia may in fact be due to the combined interactive effects of photoperiod and temperature and this fact should be taken into account in future experiments. 4~ ~rlo~~/~,dU~‘mrnts~~This study is supported by an NSERC operating grant. I thank J. M. Renaud and N. N. Rao for technical assistance. The manuscript was expertly typed by Marilyn Botter.
REFERENCES
HARRI M. N. E., HEDENSTAM R.. LINIXREN E. & PUc%hA M. (1972) Calorigenic effect of 5-hydroxytryptamine In the frog, Runu temporuria. Camp. Biochem. PI~wid 43A, 545-552. HARRI M. N. E. & LINDGREN E. (1972) Adrenerglc contt-ol of carbohydrate metabolism in the frog. Rtrnrr tcmpcw uriu. Comp. yen. Pharmucol,
3, 226234.
HARKI M. N. E. & TALO A. (1975) Effect of season and temperature acclimation on the heart rate+ temperature relationship in the frog. Rtrnu temporurw. (‘wN[I. Hiw &cm. Physiol. SOA, 469-472. HARKI M. N. E. & TALO A. (1975) Effect of season and temperature acclimation on the heart rate temperature relationship in the isolated frog’s heart (Rumr temperuriu). Camp. Biochrm. Physiol. 52A. 409 -412. HERMANSEN B. & JORGENSEN C. B. (1969) Blood glucose m male toads (&f/o hufo): annual variation and hormonal regulation. Gun. Camp. Endow. 12, 313 321. HOAR W. S. & ROHERTSONG. B. (1959) Temperature reristante of goldfish maintained under controlled photoperiods. Corn. J. Zoo/. 37, 419 428. HONG S. K.. PARK C. S., PARK Y. S. & KIM .I. K. (106X1 Seasonal changes of antidiuretic hormone action on sodium transport across frog skm. Atn. .I. Plnxioi 215. 329-443.
Bov~) E. M. (1938) Lipoid substances of the ovary during ova productlon in Runu pipiens. J. P/zJ,.sio/. 91, 394 397. EMI-KY A. R.. BEKST A. H. & KOD~RIA K. (1972) Under-ice ohscrvations of wintering sites of leopard frogs. Copriu 1972. 123m 136. HAVKI W. (1973) Endocrinology of amphibia. In CheMicul ZOO/O
HUT~HINS~N V. H. (1961) manders. Physiol. Zoo/. MIZ~.LL S. (1965) Seasonal the common frog. Runu
Critical 34, 92
changes pipkns.
thermal
maxima
in \al3-
125.
in the energy reserves tn J. cc//. camp. P/I~~\Io/ 66,
251-258.
NOMA A.. H~ROAKI 0. & MASAMI K. (1973) A new colorsmetric microdetermination of free fatty acids in serum Clinica
chin]. Actu 43, 3 17- 320.
RAO R. B.. SA~HANUR R. S.. PAUMAKAR S. P. & <;I KAI II S B. (1977) A simphfied calorimetric mlcromethod fol- deof sermum cholesterol. Lipid\. 12. termination 1078~1080. VINEGAR A. & HUT~HIXIN V. H. (1965) Pulmonary and cutaneous gas exchange in the green frog, Rtrnu c,/umifuns. Zoologic~u 50, 47-53.
.4bstract
of Zoology.
University
of Guelph.
Guelph.
Ontario.
Canada
N IG ZW I
I. Many
prebious experiments on cold acclimation in frogs were carried out on animals in total darkness. I tested the effect of photopcriod on changes In plasma FFA. glucose. and cholesterol durmg cold acclimation. 2. Hematocrit levels were lowe!- whereas plasma FFA Icvcls wcrc higher in frogs kept in total darkness during cold acclimation. 3. Plasma glucose and plasma cholesterol levels wcrc not affected by photoperiod during cold acclimntion. maIntamed
IKTRODUCTION
Harri
and
his
colleagues
have
carried
out
many
studies concerning the control mechanisms and changes that occur during temperature acclimation in frogs. Many of these studies arc published in this Journal (e.g. Harri & Hendenstam. 1972; Harri PL uI., 1972: Harri. 1975; Harri & Tale. 1975a; Harri & Tale. 1975b). In their experiments when they acclimate frogs to the cold. they also acclimate them to total darkness. This protocol is appropriate in Finland where Harri and his colleagues carry out their cxpcrimcnts; because during winter days are very short (Turku. Finland is about latitude 60.5). Howcvcr. frogs also “hibernate” during winter at lower latitudes. for example to at least latitude 35 ~ in the United States. In temperate locations. at least some frog species (Ranu sp. in particular) do not burrow deep in the mud. Rather they sit on the bottom of ponds where the water temperature is about 4 C (Emcry c’t trl.. 1972). Thus they are cold acclimated but arc not in total darkness. The present experiment was carried out to see if total darkness influenced the changes that occur during cold acclimation in frogs. \I.4TERI-\LS
AND \IETHODS
Adult l’rogx. Rumr p~picr~s of both sexes were obtained from a local supphc~- (they wcrc caught m Quebec. <‘anada). Expcrimcnts were carried out in September 1979. tt-ogs were maintamcd in large stream tanks (2 m long x 0.5 m wide) with constantly running well water at 23 25 C IOm deep. They were fed mealworms daily at II00 hr. Photoperiod was LD 12: 12. .After 3 weeks. accllmatlon to the abobe conditions. a sample of 9 frogs were removed as controls. The remamlng frogs ut’rc diwdcd into two groups of 16 each and were placed :nto 100 I. aquaria filled with well water. Water tcmpcrature In these tanks was maintained at 6 i I C. was filtcrcd. and wah aerated. One tank was kept in total darkness. and in the other a LD I?:12 photopcrlod was maintained. Except for the Ilght. the tanks and other conditions were Identical. The frogs were not fed during cold acclimation. One-half of the frogs wcrc removed from each
tank for sampling after being in the cold for 9 days: the remainder w&e removed after 44 days in the cold. All samnline was done at 1400hr. Frogs in the dark tank wcw , sampled in extremely dim red light. Frogs were weighed. double-plthed. and opened to cxposc the heart. Blood samples were drawn into capillary tubes (ammonium hcparm coated) by capillary actlon. and centrifuged. Plasma samples were frozen in liquid mtrogen and stored at ~ 10 C until analysis. Glucose was estimated on a 10 111 allquot of plasma using glucose oxldase (Sigma kit No. 510). Total plasma cholesterol was cstlmated on a IO itl aliquot wing the method of Rao c’tal. (1977). Plasma FFA was estimated on a 100 ~1 ahquot using the method of Noma (Y t/l. (1973). In some cases it has nccehsary to pool wmples from two or three frogs. L
RESI. LT.5
Mean body weight was 36.3 g (range 23~ 52.5). There were no significant differences related to sex. photoperiod. or acclimation temperature.
Hematocrits were lower after 9 davs in the cold and then increased from 9 to 44 days (Fig. 1A). Frogs in the 12: 12 photoperiod had significantly higher hematocrits (33.3 & 1.33) than those held in constant darkness (2X.2 i 1.99). There were no significant differences related to sex.
There were no significant effects related to sex, and no significant differences related to photoperiod. Plasma glucose was significantly lower after 9 days in the cold, and decreased even more after 44 days in the cold (Fig. 1B).
In e\ery case the concentration of plasma cholestcrol was greater in females than in males (in all samples the ranges did not even overlap) and thus
E. DON STFVENS
Fig. I. The effect of photoperiod on changes in hematocrit level (A), plasma glucose level (B). plasma cholesterol level (C). and plasma FFA level (D) during 44 days acclimation in the cold (6 C) in frogs. LD = 12: I2 indicates I2 hr light. 12 hr dark (solid lines) and LD = 0:24 indxates continuous darkness (dashed lines).
must be considered separately. There was no significant difference related to photoperiod at either 9 or 44 days in the cold. Plasma cholesterol decrease significantly in both males and females after 9 days in the cold (Fig. IC). It increased significantly between 9 and 44 days in males, but did not return to levels seen in warm-acclimated frogs. The small increase between 9 and 44 days in females was not significant.
There were no differences in plasma FFA levels related to sex. Levels decreased markedly after 9 days in the cold. but there were no differences related to photoperiod. After 44 days in the cold, levels of plasma FFA in frogs on 12: 12 photoperiod stayed low, whereas levels in frogs in darkness returned to and exceeded levels in warm adapted controls.
DlSCUSSION
Clearly, the important result of the present study is that some variables were altered (hematocrit and plasma FFA) by photoperiod during cold acclimation whereas others were not (plasma glucose and plasma cholesterol). The erects of season and temperature on
a variety of variables are well documented in amphibians (Mizell, 1965; Hong ef ul., 1968; Hermansen & Jorgensen, 1969; Harri, 1975). However the effects of photoperiod are less well understood. The interactive effects of photoperiod and tcmperature acclimation at the level of the whole organism have been shown in other groups of animals (e.g. goldfish. Hoar & Robertson. 1959; salamanders. Hutchison. 1961) as well as frogs. Vinegar & Hutchison (1965) reported that photoperiod affected whole animal oxygen uptake in frogs at some acclimation temperatures, but that this effect was short-lived and not seen after one week. Because photoperiod is one of the most reliable environmental cues. it is often used to trigger adaptive responses in anticipation of changing temperature. The changes in metabolite levels are large relative to the changes in hematocrit. and thus the changes in metabolite levels are not due to shifts in fluid between compartments. The decrease in glucose in cold is similar to that reported by Harri & Lindgren (1972). Cholesterol is a precursor of steroids in frogs and levels arc high in females (Boyd. 1938). The observation that total darkness decreases glucose levels but increases FFA and cholesterol levels suggests that the hormone levels
The effect of photoperiod
that participate in the control of substrate utilization are affected by photoperiod and that energetic pathways are altered by light. In this regard Hanke (1973) reports that long photoperiod or continuous darkness change secretory activity of hypothalamus in Runa pipirns, and that the activity of the adenohypophysis is regulated by the hypothalamus. Thus some of the effects reported to be due to cold acclimation in amphibia may in fact be due to the combined interactive effects of photoperiod and temperature and this fact should be taken into account in future experiments. 4~ ~rlo~~/~,dU~‘mrnts~~This study is supported by an NSERC operating grant. I thank J. M. Renaud and N. N. Rao for technical assistance. The manuscript was expertly typed by Marilyn Botter.
REFERENCES
HARRI M. N. E., HEDENSTAM R.. LINIXREN E. & PUc%hA M. (1972) Calorigenic effect of 5-hydroxytryptamine In the frog, Runu temporuria. Camp. Biochem. PI~wid 43A, 545-552. HARRI M. N. E. & LINDGREN E. (1972) Adrenerglc contt-ol of carbohydrate metabolism in the frog. Rtrnrr tcmpcw uriu. Comp. yen. Pharmucol,
3, 226234.
HARKI M. N. E. & TALO A. (1975) Effect of season and temperature acclimation on the heart rate+ temperature relationship in the frog. Rtrnu temporurw. (‘wN[I. Hiw &cm. Physiol. SOA, 469-472. HARKI M. N. E. & TALO A. (1975) Effect of season and temperature acclimation on the heart rate temperature relationship in the isolated frog’s heart (Rumr temperuriu). Camp. Biochrm. Physiol. 52A. 409 -412. HERMANSEN B. & JORGENSEN C. B. (1969) Blood glucose m male toads (&f/o hufo): annual variation and hormonal regulation. Gun. Camp. Endow. 12, 313 321. HOAR W. S. & ROHERTSONG. B. (1959) Temperature reristante of goldfish maintained under controlled photoperiods. Corn. J. Zoo/. 37, 419 428. HONG S. K.. PARK C. S., PARK Y. S. & KIM .I. K. (106X1 Seasonal changes of antidiuretic hormone action on sodium transport across frog skm. Atn. .I. Plnxioi 215. 329-443.
Bov~) E. M. (1938) Lipoid substances of the ovary during ova productlon in Runu pipiens. J. P/zJ,.sio/. 91, 394 397. EMI-KY A. R.. BEKST A. H. & KOD~RIA K. (1972) Under-ice ohscrvations of wintering sites of leopard frogs. Copriu 1972. 123m 136. HAVKI W. (1973) Endocrinology of amphibia. In CheMicul ZOO/O
HUT~HINS~N V. H. (1961) manders. Physiol. Zoo/. MIZ~.LL S. (1965) Seasonal the common frog. Runu
Critical 34, 92
changes pipkns.
thermal
maxima
in \al3-
125.
in the energy reserves tn J. cc//. camp. P/I~~\Io/ 66,
251-258.
NOMA A.. H~ROAKI 0. & MASAMI K. (1973) A new colorsmetric microdetermination of free fatty acids in serum Clinica
chin]. Actu 43, 3 17- 320.
RAO R. B.. SA~HANUR R. S.. PAUMAKAR S. P. & <;I KAI II S B. (1977) A simphfied calorimetric mlcromethod fol- deof sermum cholesterol. Lipid\. 12. termination 1078~1080. VINEGAR A. & HUT~HIXIN V. H. (1965) Pulmonary and cutaneous gas exchange in the green frog, Rtrnu c,/umifuns. Zoologic~u 50, 47-53.