Retardation of thyroxine-induced metamorphosis by Amphenone B in toad tadpoles

The effect of Amphenone B, an inhibitor of corticoid synthesis, on thyroxine (T4)-induced metamorphosis was studied in toad tadpoles kept in thiourea. Amphenone injections retarded T4-induced tail resorption markedly. The effect of Amphenone was nullified by aldosterone and corticosterone added to the water in which tadpoles were kept. Steroidogenic cells of adrenals in Amphenone-injected animals were enlarged markedly as compared with those in the saline-injected tadpoles or the Amphenone-injected tadpoles which were supplemented with corticoids. The results strongly suggest that endogenous corticoids act together with thyroid hormone to accelerate metamorphosis.     

Effect of temperature of imbibition on phospholipid metabolism in pea embryonic axes

Pea seeds were imbibed in radioactive choline and then germinated. Treatments were either at 5° or 25° and the seeds were imbibed for 5 hr at one temperature and then transferred to the other. [$\text{Me}$-¹⁴C]Choline incorporation into phosphatidyl choline in the ER and the plasma membrane obtained from the embryonic axes after germination was measured. Seeds kept constantly at 25° had a very rapid initial incorporation of choline followed by a loss of label. Seeds kept at 5° had a very much lower rate of incorporation. However, seeds transferred from 5 to 25° behaved for at least 48 hr as if continuously kept at 5°, while in seeds transferred from 25 to 5° incorporation stopped after 15 hr. The seeds apparently respond to transient exposure to temperature by a changed metabolism of phospholipid. Data are also given for the choline content of the seeds under the different treatments and for the changes in total phospholipid.     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.     

THE EFFECTS OF ACTINOMYCIN D ON MUSCLE CELLS OF ASCIDIAN EMBRYO*

The effect of actinomycin D on muscle cells development of the ascidian, Herdmania momus was studied ultrastructurally. No myofilament was formed when the drug was given at any stage before early tail-bud stage (stage 3). Some aggregates of myofilaments in various size were formed when the treatment was started at stage 4 (4.5 hr after fertilization at about 28°C). Above 60% of myofibrils of fully differentiated muscle cells were formed when the treatment was initiated at stage 5 (5 hr after fertilization). Muscle cells of the tadpoles treated from stage 7 (6 hr after fertilization), at which myofilaments were first detectable in normal development, differentiated almost normally. It is therefore suggested that most mRNAs for muscle proteins are synthesized preceding the onset of myofilament formation and are relatively stable. It is also suggested that mRNAs for myosin, actin and Z band materials are almost simultaneously synthesized. One of the characteristic features of the muscle cell development of the ascidian embryo is almost synchronous differentiation of relatively small numbers of cells (about 54–60 cells). The significance of these results is discussed in relation to mosaic natures of the muscle development.     

Hymenolepis diminuta (Cestoda): Effects of parasite density and temperature on the water balance of Tenebrio molitor and subsequent infectivity of the cysticercoids

The effects of the density of Hymenolepis diminuta and the effects of thermal acclimation on the water balance of Tenebrio molitor were examined. Also, the subsequent infectivity of the cysticercoids for rats were investigated. T. molitor beetles were fed known numbers of H. diminuta eggs and then were kept at 15° or 25°C for 14 days. After 14 days, beetles were desiccated and water loss was determined. Parasite density did not significantly affect transpiratory water loss in T. molitor kept at 15° or 25°C following 24 or 48 hr of desiccation. However, after 72 hr of desiccation, beetles maintained at 15°C evidently could not regulate water efficiently since there was a significant increase in the transpiratory water loss as parasite density increased. Beetles acclimated at 15°C produced fewer cysticercoids than did beetles maintained at 25°C. Also, fewer adult worms were recovered from rats intubated with cysticercoids from heavily infected, 15°C-acclimated beetles. Apparently, heavily infected beetles acclimated to 15°C do not produce viable cysticercoids.     

Effect of a light pulse during the dark on photoperiodic regulation of the rate of thyroxine-induced, spontaneous, and prolactin-inhibited metamorphosis in Rana pipiens tadpoles

Since Rana pipiens tadpoles injected with thyroxine (T4) early in the dark develop more slowly than those injected in the light, we studied the effect of giving a light pulse of 1 hr early in the dark. Tadpoles injected under a 7.5-W red light bulb in a darkened room with 0.2 microgram T4 daily at 2200 hr went through metamorphosis faster on a 12L:3D:1L:8D cycle with a light pulse after injection than on a 12L:12D cycle without a light pulse, and even faster on a 12L:1.5D:1L:9.5D cycle with a light pulse before the injection. Thus a 1-hr light pulse counteracted the metamorphic delay resulting from administration of T4 in the dark, and set in motion the conditions that resulted in a more rapid response to an injection of T4. However, a 1-hr light pulse in the early dark had no effect on growth and development of older or younger untreated tadpoles or those constantly immersed in 30 micrograms/liter T4. Larvae on 21L:3D with T4 injection in the dark and on 12L:3D:1L:8D with T4 injection at 0700 hr just before the start of the main light phase progressed faster than 12L:3D:1L:8D with injection at 2200 hr in the dark before only a 1-hr light pulse. Thus the length of the light phase immediately after T4 injection was significant. There was no difference on 12L:12D and 12L:3D:1L:8D cycles in the effectiveness of daily injections of 10 micrograms prolactin (PRL) in the early dark at 2200 hr in promoting tail growth or antagonizing tail resorption induced by T4 immersion. Under these conditions, PRL utilization did not appear to be inhibited by the light pulse.     

Temperature-limited Activity in the Garter Snake Thamnophis melanogaster (Colubridae)

Many garter snakes, Thamnophis melanogaster, at a desert pond first started foraging for tadpoles when mean water surface temperature was about 20 °C (at 0945–1015 h), and the number of snakes tripled when water temperature reached about 24 °C (at 1100–1130 h). In two years, snakes foraged in April and May, but not in March when water never reached 23 °C and only exceeded 20 °C for a few hours after the usual foraging hours. Snakes in the laboratory dedicated increasing amounts of time to underwater foraging as air and water temperatures increased from 9 °C to 29 °C, and their rate of attacks on fish increased steeply and progressively above an apparent threshold lying between roughly 19 °C and 24 °C, up to at least 29 °C. Temperature may limit T. melanogaster's foraging at the pond to the hours after roughly 0900 h and to the period after roughly March, despite evidence that prey abundance is maximal in March.     

Changes in the activity of enzymes of phenylpropanoid metabolism in tomatoes stored at low temperatures

A large increase in the activity of an enzyme involved in chlorogenic acid metabolism, hydroxycinnamyltransferase occurs in tomatoes stored at low temperatures. In contrast, the activity of the enzyme remains constant or falls slightly during normal ripening at 20°. The rise in activity occurs at temperatures below 10° and fails to occur at 15° or 20°. This increase in activity during low temperature storage occurs with fruit at all stages of ripening from mature green to fully ripe. The hydroxycinnamyltransferase of chilled tomatoes falls rapidly on transfer to 20° with a lag of about 4–8 hr and within 48 hr returns to that of unchilled fruit. The effects of such warming treatments are reversible since when a chilling period is resumed following warming to 20°, the rise in hydroxycinnamyltransferase activity is also resumed. Of the 5 other enzymes of phenylpropanoid metabolism studied, only PAL shows a similar increase in activity during low temperature storage although the activity of the other enzymes was maintained at higher levels in fruit at 2° than at 20°. The possible relationship between the behaviour of hydroxycinnamyltransferase activity at various temperatures and the known susceptibility of tomatoes to chilling injury is discussed.     

Effects of developmental and growth history on metamorphosis in the gray treefrog, Hyla versicolor (Amphibia, Anura)

In ecological models, the timing of amphibian metamorphosis is dependent upon rate of larval growth, e.g., tadpoles that experience a decrease in growth rate can initiate metamorphosis early. Recent authors have suggested that this plasticity may be lost at some point during the larval period. We tested this hypothesis by exposing groups of tadpoles of the gray treefrog, Hyla versicolor, to different growth schedules. In endocrine models, metamorphosis is dependent on thyroxine levels and thyroxine is antagonized by prolactin (amphibian larval growth hormone), consistent with the idea that a rapidly growing tadpole can delay metamorphosis. Thus, we also manipulated the rate of development by supplementing or maintaining natural thyroxine levels for half of the tadpoles in each growth treatment. All tadpoles that received thyroxine supplements metamorphosed at the same time regardless of growth history. They also metamorphosed earlier than tadpoles not treated with thyroxine. Tadpoles not given thyroxine supplements metamorphosed at different times: those growing rapidly during day 15-34 metamorphosed earlier than tadpoles growing slowly. Growth rate before day 15 and after day 34 had no effect on metamorphic timing. The difference in larval period between these rapidly growing tadpoles and their sisters given thyroxine treatments was less than the same comparison for tadpoles that grew slowly during the same period. This apparent prolactin/thyroxine antagonism did not exist after day 34. These results are consistent with the hypothesis of a loss of plasticity in metamorphic timing.     

Cooler temperatures slow the repair of DNA damage in tadpoles exposed to ultraviolet radiation: Implications for amphibian declines at high altitude

Ultraviolet B radiation (UVBR) damages the DNA of exposed cells, causing dimers to form between adjacent pyrimidine nucleotides. These dimers block DNA replication, causing mutations and apoptosis. Most organisms utilize biochemical or biophysical DNA repair strategies to restore DNA structure; however, as with most biological reactions, these processes are likely to be thermally sensitive. Tadpoles exposed to elevated UVBR at low environmental temperatures have significantly higher rates of mortality and developmental deformities compared with tadpoles exposed to the same levels of UVBR at higher environmental temperatures. We hypothesized that low environmental temperatures impair the primary enzymatic (photolyase) DNA repair pathway in amphibians, leading to the accumulation of DNA damage. To test this hypothesis, we compared DNA repair rates and photolyase gene expression patterns in Limnodynastes peronii. Tadpoles were acutely exposed to UVBR for 1 hr at either 20 or 30°C, and we measured DNA damage and photolyase expression levels at intervals following this exposure. Temperature had a significant effect on the rate of DNA repair, with repair at 30°C occurring twice as fast as repair at 20°C. Photolyase gene expression (6‐4 PP and CPD) was significantly upregulated by UVBR exposure, with expression levels increasing within 6 hr of UVBR exposure. CPD expression levels were not significantly affected by temperature, but 6‐4 PP expression was significantly higher in tadpoles in the 30°C treatment within 12 hr of UVBR exposure. These data support the hypothesis that DNA repair rates are thermally sensitive in tadpoles and may explain why enigmatic amphibian declines are higher in montane regions where UVBR levels are naturally elevated and environmental temperatures are lower.     

Temporal relationships between leaving food, ecdysone release, mucoprotein extrusion and puparium formation in Drosophila virilis

The time sequence of various developmental processes at the end of larval life in Drosophila virilis larvae is reported. If reared at 25·3°C the larvae leave their food about 140 hr after oviposition; 6·6 hr later ecdysone release occurs, while 8·5 to 9 hr after leaving food the mucoprotein, synthesized and stored in the salivary gland cells, is extruded into the lumen of the gland. Puparium formation takes place 11·2 hr after leaving food. Changes in the puffing activity are correlated with these processes.     

The transmission of European wheat striate mosaic virus by Javesella pellucida (Fabr.) injected with extracts of plants and plant-hoppers

Virus-free individuals of the plant-hopper Javesella pellucida (Fabr.) infected plants with European wheat striate mosaic virus (EWSMV) after being injected at 5° C. with extracts of either plants or hoppers, but extracts of hoppers provided a better inoculum. Hoppers were unable to infect plants until at least 8 days at 20–25° C. after they were injected, and nymphs fed on infected plants similarly required 8 days before they gave infective extracts. Few hoppers survived more than a week after injection with untreated extracts of hoppers or with material sedimented from them by centrifuging the extracts at 8000g, but 60–70% survived injection with purer virus preparations. Injection of the virus seemed harmless, because as many hoppers survived CO₂ anaesthesis + injection, whether or not they later infected plants, as survived anaesthesis without injection. Attempts to determine the properties of the virus in vitro gave inconsistent results, but virus from hoppers was still infective after 10 min. at 30° C, 36 hr. at 5° C, precipitation at pH 4.0, storage for several months at -15° C, or at a dilution equivalent to 0.0014 g. hopper/ml. The best extraction medium contained 0.2 M-Na₂HPO₄+ ascorbic acid + 0.01 M-DIECA at pH 7.0–7.3. In sucrose density-gradients, EWSMV sedimented more slowly than tobacco mosaic virus. No specific particle with which infectivity could be correlated was seen by electron microscopy.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Synergy Between Low Temperature and Dessication in Breaking Gemmule Diapause of Eunapius fragilis (Leidy)

Summary

Gemmules of Eunapius fragilis collected during the fall and kept at 20° C for up to 6 months did not germinate. Freshly collected gemmules, which were dried at 20° C for 7 days and then rehydrated, also exhibited a very low capacity for germination. However, gemmules, stored at 20° C for several months and then dried, showed a much higher level of germination (but usually no more than 50%) after they were returned to pond water. Gemmules, stored at 4–5° C for 4 to 6 weeks, exhibited at most very little germination when they were tested at 20° C. On the other hand, gemmules, which were chilled at 4–5° C for 4 to 6 weeks and then dried for 7 days, underwent rapid and nearly complete germination upon rehydration. These results provide clear evidence for a synergistic effect between low temperature and desiccation in breaking gemmule diapause. It is suggested that in temporary habitats where E. fragilis often survives the dry summer as gemmules, drying may be the primary agent releasing the gemmules from diapause so that they germinate in the fall upon the return of water. A brief exposure of the gemmules to low temperatures before and/or during the dry period may enhance the effect of desiccation.     

Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos

The effect of temperature of imbibition on the synthesis and turnover of membrane phosphatidyl choline was studied. Pea seeds (Pisum sativum cv. Alaska) were imbibed in [U-¹⁴C]glycerol and then germinated. Seeds were kept constantly either at 5° or 25°, or were imbibed at one temperature and then germinated at the other one. Glycerol incorporation into phosphatidyl choline in the ER and the plasma membrane, obtained from the embryonic axes after germination, and the glycerol pool were measured. Embryos from seeds kept constantly at 25° showed a rapid incorporation of glycerol into membranes followed by a loss of label; in embryos from seeds kept at 5° incorporation was much lower. Embryos from seeds transferred from 25° to 5° behaved as if continuously kept at 25°, while the behaviour of the embryos from seeds transferred from 5° to 25° resembled embryos from seeds maintained at 5°. The glycerol content of the axes rose during imbibition and fell thereafter. The activities of phospholipases C and D also responded to the initial temperature of imbibition, but the two activities changed differently. The results are discussed in relation to the effect of transient exposure to temperature changes in the seed membranes and the possible way in which such changes are sensed.     

Reproductive and behavioral control in the male and female cat with progestins: long-term field observations in individual animals.

One female and two male cats, kept for two years at N.19°20′ and for three and six years at N.37°25′ (one of the males and the same female) were observed daily for their reproductive and social behavior and the control of both behaviors by progestins. In the female, which showed puberty at the age of four months and cycled at N.19°20′ in regular 29.77 (24–35)-day intervals, single oral applications of 5 mg megestrol acetate (MA) or chlormadinone acetate (CAP) 24 hr after onset of heat (full receptivity and multiple matings) completely prevented gestation in 15 heat episodes. Loss of the tablet or lack of medication resulted in normal pregnancies. Later, at N.37°25′, cycle stopped during early winter and/or absence of overt behavioral signs developed which prevented further medications; two unwanted pregnancies required spaying. Three and four years thereafter, three periods of subclinical heat-like conditions developed, which were completely controlled with a single administration of 2.5 mg delmadinone acetate (DMA).In two toms complete control of sexual interest and sex-related behavior was achieved by a once-a-week oral dose of 5 mg of either MA, CAP or DMA. DMA, but not CAP or MA, was also effective at 2.5 mg. Simultaneously, their social rank within a larger group of cats was lost, but regained after the drug effect had expired. Full drug effects were observed in less than 36 hr; reinstatement of sexual activity, behavior and social status 8 to 10 days later followed a reversed pattern again over approximately 36 hr. At N.19°20′ medication was essential year around, while at N.37°25′ treatment could be ceased from September through January. Contrary to the drug effect in one tom, castration did not result in the loss of social status, while his sexual interest and behavior was entirely diminished.     

The effects of low temperature on myelin formation in optic nerves of Xenopus tadpoles

To test the effect of cold on CNS myelin formation, optic nerves of stages 52–55 Xenopus tadpoles were examined electron microscopically after maintenance at 15, 10, 7 and 4 °C for 1–7 days. Nerves from tadpoles maintained at 15 °C resembled 22 °C (room temperature) controls. After 3 days at 10, 7, or 4 °C, tongue processes and perikarya of many myelin forming oligodendrocytes were swollen and filled with vesicular membrane profiles. The number of axonal microtubules was decreased in affected fibers but the lamellar structure of their myelin sheaths remained normal. Astrocytes were hypertrophic and contained large aggregates of filaments. Longer exposure to 10 or 7 °C increased the number of affected fibers but the changes were not more severe or associated with degeneration. The delayed onset, lack of progression and reversibility of the changes indicated that cold has a direct metabolic effect on myelin forming oligodendrocytes. Alterations produced by nerve transection or exposure to mitotic inhibitors differed, suggesting that cold induced changes were not due primarily to either axonal degeneration or reduced axonal transport.     

Development of endothermy in a tasmanian marsupial, Bettongia gaimardi and its response to cold and noradrenaline

Marsupials at birth are ectothermic and gradually attain the ability to change their metabolic heat production during pouch life. How this process occurs in the bettong has been measured on 13 pouch young from week 1 until 3 weeks after pouch vacation (week 18). Oxygen consumption was measured at 35 °C (pouch temperature) and at 22 °C. The results at 35 °C showed an increase in metabolic rate from week 1 until week 12 when there was a decrease to near adult levels after pouch vacation. At 22 °C young bettongs had a lower metabolic rate (compared with measurements made at 35 °C) until week 9 after which there was an increase above measurements made at 35 °C. Noradrenaline had little effect until week 10 after which the metabolic rate (although measured at 35 °C) paralleled the levels measured at 22 °C. The free thyroxine level was low in early pouch life, increased to a peak at week 12 then decreased. Thermal conductance increased until week 10 after which it decreased, reaching values similar to those of adult bettongs by week 20. The results indicate that non-shivering thermogenesis occurs in this macropodoid marsupial. This phenomenon may be a phylogenetic difference between macropodid and non-macropodid marsupials as also suggested by Nicol et al. (1997). Accepted: 19 February 1998     

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, <Emphasis Type="Italic">Euphlyctis cyanophlyctis</Emphasis>

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32°C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32°C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32°C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32°C) during stages 7–42 produced male-biased sex ratio. Rearing of tadpoles at 32°C during stages 25–42 produced male-biased sex ratio, while exposure during stages 7–25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22°C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.     

Low temperature slowing and cold-block of fast axoplasmic transport in mammalian nerves in vitro

1) Fast axoplasmic transport in mammalian nerve in vitro was studied using an isotope labeling technique. The rate of outflow in cat sciatic nerve fibers of 410 mm/day in vitro was reduced at temperatures below 38°C with a Q₁₀ of 2.0 in the range 38–18°C and a Q₁₀ of 2.3 at 38–13°C. 2) At a temperature of 11°C a partial failure of transport occurred. At temperatures below 11°C a complete block of fast axoplasmic transport occurred, a phenomenon termed “cold-block.” No transport at all was seen over the temperature range of 10–0°C for times lasting up to 48 hr. 3) Transport was resumed after a period of cold-block lasting up to 22 hr when the nerves were brought back to a temperature of 38°C. Some deleterious effects due to cold-block were seen in the recovery phase as indicated by a reduction in crest amplitude, change in its form, and slowed rate. 4) The ∼P level (combined ATP and creatine phosphate) remained near control level in nerves kept at low or cold-block temperatures for times as long as 64 hr. The reduction in fast axoplasmic transport rate seen at low temperatures for times up to 22 hr was therefore considered due to a decrease in the utilization of ATP, a concept in accord with the “transport filament” model proposed to account for fast axoplasmic transport. 5) The sloping of the front of the crest over the temperature range of 18–13°C suggests an additonal factor at the lower temperatures. A disassembly of microtubules is discussed as a possible explanation of the cold-block phenomenon.