Impaired Control of Body Cooling during Heterothermia Represents the Major Energetic Constraint in an Aging Non-Human Primate Exposed to Cold

Daily heterothermia is used by small mammals for energy and water savings, and seems to be preferentially exhibited during winter rather than during summer. This feature induces a trade-off between the energy saved during daily heterothermia and the energy cost of arousal, which can impact energy balance and survival under harsh environmental conditions. Especially, aging may significantly affect such trade off during cold-induced energy stress, but direct evidences are still lacking. We hypothesized that aging could alter the energetics of daily heterothermia, and that the effects could differ according to season. In the gray mouse lemur (Microcebus murinus), a non-human primate species which exhibits daily heterothermia, we investigated the effects of exposures to 25 and 12°C on body composition, energy balance, patterns of heterothermia and water turnover in adult (N = 8) and aged animals (N = 7) acclimated to winter-like or summer-like photoperiods.Acclimation to summer prevented animals from deep heterothermia, even during aging. During winter, adult animals at 12°C and aged animals at 25°C exhibited low levels of energy expenditure with minor modulations of heterothermia. The major effects of cold were observed during winter, and were particularly pronounced in aged mouse lemurs which exhibited deep heterothermia phases. Body composition was not significantly affected by age and could not explain the age-related differences in heterothermia patterns. However, aging was associated with increased levels of energy expenditure during cold exposure, in concomitance with impaired energy balance. Interestingly, increased energy expenditure and depth of heterothermia phases were strongly correlated.In conclusion, it appeared that the exhibition of shallow heterothermia allowed energy savings during winter in adult animals only. Aged animals exhibited deep heterothermia and increased levels of energy expenditure, impairing energy balance. Thus, an impaired control of the heterothermic process induced high energy costs in the aging mouse lemur exposed to cold.     

Thermoregulatory responses to variations of photoperiod and ambient temperature in the male lesser mouse lemur: a primitive or an advanced adaptive character?

The lesser mouse lemur, a small Malagasy primate, is exposed to strong seasonal variations in ambient temperature and food availability in its natural habitat. To face these environmental constraints, this nocturnal primate exhibits biological seasonal rhythms that are photoperiodically driven. To determine the role of daylength on thermoregulatory responses to changes in ambient temperature, evaporative water loss (EWL), body temperature (T _b) and oxygen consumption, measured as resting metabolic rate (RMR), were measured in response to ambient temperatures ranging from 5 °C to 35 °C, in eight males exposed to either short (10L:14D) or long (14L:10D) daylengths in controlled captive conditions. In both photoperiods, EWL, T _b and RMR were significantly modified by ambient temperatures. Exposure to ambient temperatures below 25 °C was associated with a decrease in T _b and an increase in RMR, whereas EWL remained constant. Heat exposure caused an increase in T _b and heat loss through evaporative pathways. Thermoregulatory responses to changes in ambient temperature significantly differed according to daylength. Daily variations in T _b and EWL were characterized by high values during the night. During the diurnal rest, lower values were found and a phase of heterothermia occurred in the early morning followed by a spontaneous rewarming. The amplitude of T _b decrease with or without the occurrence of torpor (T _b < 33 °C) was dependent on both ambient temperature and photoperiod. This would support the hypothesis of advanced thermoregulatory processes in mouse lemurs in response to selective environmental pressure, the major external cue being photoperiodic variations. Accepted: 4 August 1998     

Effect of Ambient Temperature on the Body Temperature Rhythm of Male Gray Mouse Lemurs (Microcebus murinus)

In order to cope with the seasonal variations in ambient temperature and food availability in the natural habitat, gray mouse lemurs (Microcebus murinus) exhibit adaptive energy-saving mechanisms similar to those in hibernating species with seasonal and daily heterothermia. To determine thermoregulatory responses, via telemetry we recorded body temperature and locomotor activity variations during the breeding season in three captive male mouse lemurs kept at ambient temperatures (T_a) ranging from 18° to 34°C. Rhythms in body temperature and locomotor activity were clearly exhibited regardless of ambient temperature. As _a increased, mean body temperature increased from 36.5 ± 0.1°C to 37.6 ± 0.3°C, with significant change in the amplitude of the body temperature rhythm when _a rose above 28°C. Effects of _a were mostly due to changes in the fall in body temperature occurring daily at the beginning of the light phase when the subjects entered diurnal sleep. The daily decrease in body temperature was not modified by exposure to ambient temperatures from 18°C to 28°C whereas it disappeared under warmer condition. Changes in locomotor activity levels only delayed the occurrence of thermoregulatory modulation. These results strongly suggest that, during the breeding season, the thermoneutral zone of mouse lemurs is close to 28°C and that the diurnal fall in body temperature could be considered as an important adaptive energy-saving mechanism adjusted to ecological constraints.     

Thermal ecology of five Cnemidophorus species (Squamata: Teiidae) in east coast of Brazil

In this study we compared the body temperature of 16 populations belonging to five species of the genus Cnemidophorus from restinga habitats along the eastern coast of Brazil in order to evaluate the importance of how some environmental factors affect lizard body temperatures. Cloacal body temperatures (T_b) were taken immediately after capture with a quick-reading thermometer (Schultheis). Substrate temperatures (T_s) and air temperatures (T_a; approximately 1 cm above the substrate) were taken as close as possible to the point when each lizard was initially sighted. Most of the mean body temperatures in activity of the different populations and species of Cnemidophorus along the coast of Brazil ranged from 36.5 to 39.3 °C, except for Cnemidophorus lacertoides (T_b=35.2 °C) in the restinga of Joaquina, SC and for Cnemidophorus ocellifer (T_b=34.8 °C ) in the restinga of Praia do Porto, SE. Some studies show that the body temperature of lizards is more related to phylogenetic than ecological factors, suggesting that species of the same genus tend to have similar body temperatures even occurring in different types of environments. In general, regardless of the locality and latitude along the eastern coast of Brazil, the different species of lizards of the genus Cnemidophorus and their respective populations have similar body temperatures in activity and the apparent differences result from the influence of the local thermal environment of each restinga.     

When things get hot: Thermoregulation behavior in the lizard Sceloporus aeneus at different thermal conditions

Rising environmental temperatures have become a global threat for ectotherms, with the increasing risk of overheating promoting population declines. Flexible thermoregulatory behavior might be a plausible mechanism to mitigate the effects of extreme temperatures. We experimentally evaluated thermoregulatory behavior in the bunchgrass lizard, Sceloporus aeneus, at three different environmental temperatures (25, 35 and 45 °C) both with and without a thermal refuge. We recorded themoregulatory behaviors (body posture and movement between hot and cold patches) and compared individual lizards across all experimental temperature and shelter combinations. Behavioral thermoregulation in S. aeneus was characterized by the expression of five body postures, whose frequencies varied based on environmental temperature and microthermal conditions. Behavioral responses allowed lizards to maintain a mean body temperature <40 °C, the critical thermal maximum for temperate species, even at extreme environmental temperatures (45 °C). Although S. aeneus express an array of behavioral postures that provide an effective mechanism to cope with elevating temperatures, the presence of a thermal refuge was important to better achieve this. Together, our study offers a novel method to evaluate microhabitat preference that encompasses both behavioral observations and time-space analysis based on the ambient thermal distribution, a consideration that can aid in the formulation of more accurate predictions on ectotherm vulnerability related to increasing global environmental temperatures.     

Temperature-induced plasticity in membrane and storage lipid composition: thermal reaction norms across five different temperatures

Temperature is a key environmental factor inducing phenotypic plasticity in a wide range of behavioral, morphological, and life history traits in ectotherms. The strength of temperature-induced responses in fitness-related traits may be determined by plasticity of the underlying physiological or biochemical traits. Lipid composition may be an important trait underlying fitness response to temperature, because it affects membrane fluidity as well as availability of stored energy reserves. Here, we investigate the effect of temperature on lipid composition of the springtail Orchesella cincta by measuring thermal reaction norms across five different temperatures after four weeks of cold or warm acclimation. Fatty acid composition in storage and membrane lipids showed a highly plastic response to temperature, but the responses of single fatty acids revealed deviations from the expectations based on HVA theory. We found an accumulation of C_18:2n6 and C_18:3n3 at higher temperatures and the preservation of C_20:4n6 across temperatures, which is contrary to the expectation of decreased unsaturation at higher temperatures. The thermal response of these fatty acids in O. cincta differed from the findings in other species, and therefore shows there is interspecific variation in how single fatty acids contribute to HVA. Future research should determine the consequences of such variation in terms of costs and benefits for the thermal performance of species.     

Do changes in feed intake or ambient temperature cause changes in cattle rumen temperature relative to core temperature?

A technique was developed to monitor and describe the relationship between core body temperature (T_c) and rumen temperature (T_rum) in cattle. This relationship was assessed in cattle subjected to varying environmental temperatures and subsequent variations in dry matter and water intake. Increasing the environmental wet bulb temperature (WBT) from ambient conditions (approximately 15 °C WBT) to mild heat stress conditions (25 °C WBT) caused an increase in both T_c and T_rum with significant decreases in feed intake and increases in water consumption. Despite increases in both T_c and T_rum, reductions in dry matter intake, and an increase in water consumption, the relationship between T_c and T_rum did not change.     

Chlamydomonas reinhardtii thermal tolerance enhancement mediated by a mutualistic interaction with vitamin B12-producing bacteria

Temperature is one of the most important environmental factors affecting the growth and survival of microorganisms and in light of current global patterns is of particular interest. Here, we highlight studies revealing how vitamin B₁₂ (cobalamin)-producing bacteria increase the fitness of the unicellular alga Chlamydomonas reinhardtii following an increase in environmental temperature. Heat stress represses C. reinhardtii cobalamin-independent methionine synthase (METE) gene expression coinciding with a reduction in METE-mediated methionine synthase activity, chlorosis and cell death during heat stress. However, in the presence of cobalamin-producing bacteria or exogenous cobalamin amendments C. reinhardtii cobalamin-dependent methionine synthase METH-mediated methionine biosynthesis is functional at temperatures that result in C. reinhardtii death in the absence of cobalamin. Artificial microRNA silencing of C. reinhardtii METH expression leads to nearly complete loss of cobalamin-mediated enhancement of thermal tolerance. This suggests that methionine biosynthesis is an essential cellular mechanism for adaptation by C. reinhardtii to thermal stress. Increased fitness advantage of METH under environmentally stressful conditions could explain the selective pressure for retaining the METH gene in algae and the apparent independent loss of the METE gene in various algal species. Our results show that how an organism acclimates to a change in its abiotic environment depends critically on co-occurring species, the nature of that interaction, and how those species interactions evolve.     

Physical studies of RNA involvement in bacteriophage lambda DNA replication and prophage excision

Non-diffusible genetic elements in bacteriophage λ DNA replication and λ prophage excision have been analyzed by the DNA-cutting assay of Freifelder and Kirschner (1971) and Freifelder et al. (1972). The mutant ti12, which affects a unique site for replication in or near the origin of replication (Dove et al., 1971), makes λ DNA partially refractory to replicative DNA-cutting. RNA synthesis in the vicinity of the origin, of replication seems to control the susceptibility of λ DNA to replicative DNA-cutting (Dove et al., 1969). Analogously, RNA synthesis in the vicinity of the left-hand prophage terminus seems to control excisional DNA-cutting of derepressed λ DNA, as predicted by the studies of Davies et al. (1972). These physical studies confirm previous genetic analyses and imply that the elements involved act at a very early stage in replication and in excision.     

High-throughput gender identification of three Columbidae species using melting curve analysis

The objective was to perform high-throughput gender identification of three Columbidae species (Columba livia, Columba pulchricollis, and Streptopelia tranquebarica). Although the chromo-helicase-DNA binding protein (CHD)-based Griffiths P2/P8 primer set resolved the amplicon products of these species in 3% agarose gel electrophoresis, it was unsuitable for molecular gender identification using the melting curve analysis (MCA) curve for high-throughput analysis. After sequencing the CHD-Z and CHD-W genes for these species, we redesigned a female-specific CHD-W primer (dove-W) and a female/male (or CHD-Z/CHD-W)-common primer (dove-ZW) to combine with the Griffiths P2 primer to generate two PCR amplicons with different lengths (P2/dove-W and P2/dove-ZW for 252- and 104-bp, respectively). Melting temperature (Tm) values for P2/dove-W and P2/dove-ZW amplicons were determined and resolved in MCA at approximately 79.0∼79.5 and 77.5 °C, respectively. Accordingly, females contained two Tm peaks, whereas males contained one. In conclusion, melting curve analysis (MCA) using our proposed primer sets was a robust gender identification method for the three Columbidae species tested.     

The Dependence of the Oxygen-Concentrating Mechanism of the Teleost Eye (Salmo gairdneri) on the Enzyme Carbonic Anhydrase

Microoxygen polarographic electrodes were constructed and used to measure oxygen tension (P_OO2) in the eyes of rainbow trout (Salmo gairdneri). The values obtained are compared with arterial blood and environmental water P_OO2 and indicate that there is an oxygen-concentrating mechanism in the eye supplying oxygen to the avascular retina. Anatomically similar retes suggest that the mechanism is similar to the one which exists in the swim bladder. Elimination of the arterial blood supply to the choroidal gland rete mirabile of the eye (through pseudobranchectomy) and the consequent lowering of ocular oxygen tensions implicate the choroidal gland as one of the major components of the oxygen-concentrating mechanism. After pseudobranchectomy the presence of ocular P_OO2 above that of arterial blood is indicative of a secondary structure in the eye capable of concentrating oxygen. Inhibition of carbonic anhydrase, using acetazolamide, is shown to result in complete suppression of the oxygen-concentrating mechanism. A hypothesis is advanced for the participation of retinal-choroidal and erythrocyte carbonic anhydrase in the oxygen-concentrating mechanism.     

Differential temperature dependencies of antioxidative enzymes in two contrasting species: Fagus sylvatica and Coleus blumei

In order to characterise the sensitivity of antioxidative systems to temperature-induced oxidative stress, two species (Coleus blumei and Fagus sylvatica, L.) representative of environments with contrasting temperature characteristics have been exposed to low or high temperatures of 10 or 35 °C, respectively. Beech leaves were harvested in light and darkness. Coleus leaves were separated into green and white leaf tissue. The thermal dependencies of the activities of protective enzymes and chlorophyll fluorescence over a temperature range from 10 to 35 °C were determined. Ascorbate peroxidase activities were activated at low temperatures in vitro and, thereby, may provide an instantaneous protection against H₂O₂ accumulation which is faster than de novo synthesis. Monodehydroascorbate radical reductase was apparently not involved in short-term acclimation to low or high temperature. After short-term acclimation to low temperature, glutathione reductase and glutathione were more diminished in Coleus than in beech. Both species contained higher concentrations of ascorbate and glutathione at high temperatures than at low temperatures whereas glutathione reductase activity increased. Ascorbate peroxidase activity from Coleus leaves, though detectable under standard assay conditions (25 °C), failed at 35 °C in vitro. The results suggest that the higher temperature susceptibility of Coleus than that of beech was associated with a differential loss in glutathione reductase/glutathione at low temperature and an inhibition of ascorbate peroxidase at high temperature. Since the thermal dependencies of antioxidative enzymes were significantly affected by the preceding environmental conditions, the relative enzymatic activities determined under standard assay conditions may not be representative of enzymatic activities in foliage exposed to varying environmental temperatures.     

Energy metabolism and body temperature in 13 sunbird species (Nectariniidae)

• 1.1. Diurnal cycles of body temperature, T_b, and energy metabolism, M, at different ambient temperatures (T_a: +5 −+ 32°C) were tested in 13 sunbird species from various habitats and of different body masses (5.2–14.2 g) including one of the smallest passerines, Aethopyga christinae.
• 2.2. Resting M-level (night) reaches T_a-dependent mean values of 54% (+5°C) and 49% (+25°C) of activity M-levels (day). Expected level is ca 75%.
• 3.3. Resting metabolic rate of sunbirds lies within the range of theoretically expected values for birds.
• 4.4. Mean linear metabolism-weight regression of the night values follows: M = 0.102 × W^0.712 (M = energy metabolism in kJ/hr and W = body mass in g).
• 5.5. Thermal conductances, T_c, are lower (−24%) than the predicted values. This is caused by a decrease of T_b at low T_a. Mean nocturnal T_c is 3.2 J/g × hr × °C, mean day-time value is 4.3 J/g × hr × °C.
• 6.6. The zone of thermoneutrality is, in most species, within a T_a-range of 24–28°C.
• 7.7. Normal day and night levels of T_b are in the same range as reported for other birds of the same weight class. T_b decreases slightly with falling T_a (partial heterothermia). Lowest recorded T_b was 34.2°C.
• 8.8. No species tested showed any sign of torpor at night, independent of T_a, body mass or habitat origin.

     

Upper thermal tolerance plasticity in tropical amphibian species from contrasting habitats: Implications for warming impact prediction

Tropical ectothermic species are currently depicted as more vulnerable to increasing temperatures because of the proximity between their upper thermal limits and environmental temperatures. Yet, the acclimatory capacity of thermal limits has rarely been measured in tropical species, even though they are generally predicted to be smaller than in temperate species. We compared critical thermal maximum (CT_max) and warming tolerance (WT: the difference between CT_max and maximum temperature, T_max), as well as CT_max acclimatory capacity of toad species from the Atlantic forest (AF) and the Brazilian Caatinga (CAA), a semi-arid habitat with high temperatures. Acclimation temperatures represented the mean temperatures of AF and CAA habitats, making estimates of CT_max and WT more ecologically realistic. CAA species mean CT_max was higher compared to AF species in both acclimation treatments. Clutches within species, as well as between AF and CAA species, differed in CT_max plasticity and we discuss the potential biological meaning of these findings. We did not find a trade-off between absolute CT_max and CT_max plasticity, indicating that species can have both high CT_max and high CT_max plasticity. Although CT_max was highly correlated to T_max, CT_max plasticity was not related to T_max or T_max coefficients of variation. CAA species mean WT was lower than for AF species, but still very high for all species, diverging from other studies with tropical species. This might be partially related to over-estimation of vulnerability due to under-appreciation of realistic acclimation treatments in CT_max estimation. Thus, some tropical species might not be as vulnerable to warming as previously predicted if CT_max is considered as a shifting population parameter.     

Early life-history processes in benthic octopus: Relationships between temperature, feeding, food conversion, and growth in juvenile Octopus pallidus

Initial growth in cephalopods is exponential, making early life-history critical in determining growth trajectories. Few captive studies have however examined the early life-history of cephalopods in fluctuating temperatures as would be encountered in the wild. This study investigates the relationship between early growth and the significant factors affecting growth, namely food intake, food conversion and fluctuating environmental temperatures. Pale octopus (Octopus pallidus) hatchlings were reared in captivity under either a warming or cooling temperature regime. Individual variations and periodicity in feeding rates (F_r), food conversion rates (C_r), growth rates (G_r), and the relationship between these variables and temperature were examined weekly. Food conversion rates were variable between individuals but also within individual octopus, which exhibited large fluctuations in C_r over time, exceeding 100% d^− 1 in one instance. Although individual F_r, C_r and G_r displayed fluctuations over time, there was no evidence of periodicity for any of the variables. Changes in temperature were not significantly correlated to changes in F_r, C_r or G_r. Feeding rate did not appear to influence growth rate or food conversion rate. Food conversion rate was negatively correlated to feeding rate in the same week, and positively correlated to growth rate. Short periods of low or no food consumption were common and the high values obtained for food conversion rate for some individuals suggest that octopus can grow substantially with little or no food intake. Individual variability observed in octopus growth may be dependent on the growth mechanism involved, specifically a fine balance between the continuous hyperplasic and hypertrophic growth found in cephalopods.     

Effects of low environmental pH and temperature on hatching and metabolic rates in embryos of Anax junius drury (Odonata: Aeshnidae) and the role of hypoxia in the hatching process

• 1.1. Studies were conducted in order to determine the combined effects of low environmental pH and temperature on embryonic survival capacity and metabolic rates in the dragonfly, Anax junius Drury. Studies were also conducted to assess the effects of hypoxia on hatching success as well as to investigate the role of hypoxia as a possible physiological triggering mechanism for hatching.
• 2.2. At water temperatures of 10–30°C, an environmental pH value of 3.0 was extremely limiting and significantly reduced hatching success.
• 3.3. Over a pH range of 3.0–5.0, a water temperature of 30°C was found to be severely limiting. Over a pH range of 6.0–7.0, hatching success was greater than 80% at test temperatures ranging from 10 to 25°C.
• 4.4. Embryos of A. junius exhibited a greater tolerance to markedly low environmental pH (3.0) than that previously reported for fish and amphibians, although survival capacity was less than 10%.
• 5.5. An environmental pH value of 3.0 has a significant detrimental effect on embryonic development. Survivorship and developmental rate increase significantly over a pH range of 4.0–5.0.
• 6.6. Oxygen consumption rates were lowest for fertilized eggs exposed to a pH of 3.0 at all test temperatures (10–30°C). Metabolic rates increased significantly at pH 4.O.
• 7.7. Embryos hatch successfully under hypoxic conditions in both aqueous and nonaqueous media. Results suggest that hypoxia acts as a triggering mechanism for hatching in this aquatic insect.

     

Do female newts modify thermoregulatory behavior to manipulate egg size?

Reproductive females manipulate offspring phenotypes by modifying conditions during embryogenesis. In ectotherms, the environmental control over embryogenesis is often realized by changes in maternal thermoregulation during gravidity. To determine if reproduction influences thermoregulatory behavior in species where females lay eggs shortly after fertilization (strict oviparity), we compared preferred body temperatures (T_p) between reproductive (egg-laying) and non-reproductive female newts, Ichthyosaura alpestris. Next, we exposed reproductive females to temperatures mimicking T_p ranges of reproductive and non-reproductive individuals to find out whether the maternally modified thermal regime influences ovum and jelly coat volume, and early cleavage rates at the time of oviposition. In the thermal gradient, reproductive females maintained their body temperatures within a narrower range than non-reproductive individuals. The exposure of ovipositing females to temperatures preferred during their reproductive and non-reproductive period had a negligible influence on egg size and early cleavage rates. We conclude that the modification of maternal thermoregulatory behavior provides a limited opportunity to manipulate egg traits in newts.     

A test of the thermal coadaptation hypothesis with ultimate measures of fitness in flour beetles

Whole-organism performance of ectotherms depends on body temperature, which is tightly linked to environmental temperatures. Individuals attempting to optimize fitness must thus select appropriate temperatures. The thermal coadaptation hypothesis posits that T_o for traits closely linked to fitness should match temperatures selected by a species (T_set) and should coevolve with T_set. T_o may mismatch T_set if the thermal reaction norm for fitness is asymmetric. In this study, we examined six traits related to fitness in red and in confused flour beetles (Tribolium castaneum and T. confusum, respectively), including longevity, lifetime reproductive success, reproductive rate, and development time at four temperatures between 23 and 32 °C. For reproductive traits, T_o matched T_set whereas for longevity T_o was lower than T_set. Tribolium species have a strongly r-selected life history strategy, therefore reproductive traits are likely more tightly linked to fitness than longevity due to high predation rates at early life stages. We therefore provide support for the thermal coadaptation hypothesis for reproductive traits that are tightly linked to fitness. Our results highlight the importance of knowing the relationships of traits to fitness when studying thermal physiology.     

Response of effective quantum yield of photosystem 2 to <Emphasis Type="Italic">in situ</Emphasis> temperature in three alpine plants

The response of effective quantum yield of photosystem 2 (ΔF/F_m’) to temperature was investigated under field conditions (1 950 m a.s.l.) in three alpine plant species with contrasting leaf temperature climates. The in situ temperature response did not follow an optimum curve but under saturating irradiances [PPFD >800 µìmol(photon) m^?2s^?1] highest ΔF/F_m’ occurred at leaf temperatures below 10°C. This was comparable to the temperature response of antarctic vascular plants. Leaf temperatures between 0 and 15°C were the most frequently (41 to 56%) experienced by the investigated species. At these temperatures, ΔF/F_m’ was highest in all species (data from all irradiation classes included) but the species differed in the temperature at which ΔF/F_m’ dropped below 50% (Soldanella pusilla >20°C, Loiseleuria procumbens >25°C, and Saxifraga paniculata >40°C). The in situ response of ΔF/F_m’ showed significantly higher ΔF/F_m’ values at saturating PPFD for the species growing in full sunlight (S. paniculata and L. procumbens) than for S. pusilla growing under more moderate PPFD. The effect of increasing PPFD on ΔF/F_m’, for a given leaf temperature, was most pronounced in S. pusilla. Despite the broad diurnal leaf temperature amplitude of alpine environments, only in S. paniculata did saturating PPFD occur over a broad range of leaf temperatures (43 K). In the other two species it was half of that (around 20 K). This indicates that the setting of environmental scenarios (leaf temperature×PPFD) in laboratory experiments often likely exceeds the actual environmental demand in the field.