The influence of androgens on hibernation phenology of free-living male arctic ground squirrels.

Free-living ground squirrel species are sexually dimorphic in hibernation phenology. The underlying causes of these differences are not yet known. Androgens, testosterone (T) in particular, inhibit hibernation. To determine the influence of endogenous androgens on annual timing of hibernation we first measured circulating levels of T and dehydroepiandrosterone (DHEA), an adrenal androgen implicated in non-mating season aggression in other species, in free-living male arctic ground squirrels (Urocitellus parryii, AGS). We also manipulated endogenous androgen levels by surgical castration, and consequently compared body temperature records from intact (n=24) and castrated (n=9) males to elucidate the influence of endogenous androgens on annual body temperature cycles. The highest T levels (0.53±0.10ng/mL) were in reproductively mature male AGS in spring; whereas, both immature males in spring and all males in late summer had T levels an order of magnitude lower (0.07±0.01 and 0.06±0.00ng/mL, respectively). DHEA levels were higher in males during the late summer compared to reproductively mature males in spring (120.6±18.9 and 35.9±2.3pg/mL, respectively). Eliminating gonadal androgens via castration resulted in males delaying euthermy by extending heterothermy significantly in spring (Apr 22 ±2.9) than reproductive males (Mar 28 ±3.9) but did not change the timing of hibernation onset (castrate: Oct 12 ±1.0 vs. intact: Oct 3 ±3.1). We conclude that while androgens play a significant role in spring hibernation phenology of males, their role in fall hibernation onset is unclear.

Deep supercooling, vitrification and limited survival to -100{degrees}C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae.

Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of -35 to -42 degrees C, with the lowest supercooling point recorded for an individual of -58 degrees C. We previously noted that some larvae did not freeze when cooled to -80 degrees C, and we speculated that these larvae vitrified. Here we present evidence through differential scanning calorimetry that C. c. puniceus larvae transition into a glass-like state at temperatures<-58 degrees C and can avoid freezing to at least -150 degrees C. This novel finding adds vitrification to the list of insect overwintering strategies. While overwintering beneath the bark of fallen trees, C. c. puniceus larvae may experience low ambient temperatures of around -40 degrees C (and lower) when microhabitat is un-insulated because of low snow cover. Decreasing temperatures in winter are correlated with loss of body water from summer high levels near 2.0 to winter lows near 0.4 mg mg(-1) dry mass and concomitant increases in glycerol concentrations (4-6 mol l(-1)) and thermal hysteresis. Finally, we provide direct evidence that Cucujus from Wiseman, Alaska, survive temperatures to -100 degrees C.

Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator.

Hibernating arctic ground squirrels, Spermophilus parryii, were able to adopt and spontaneously arouse from core body temperatures as low as -2.9 degrees C without freezing. Abdominal body temperatures of ground squirrels hibernating in outdoor burrows were recorded with temperature-sensitive radiotransmitter implants. Body temperatures and soil temperatures at hibernaculum depth reached average minima during February of -1.9 degrees and -6 degrees C, respectively. Laboratory-housed ground squirrels hibernating in ambient temperatures of -4.3 degrees C maintained above 0 degree C thoracic temperatures but decreased colonic temperatures to as low as -1.3 degrees C. Plasma sampled from animals with below 0 degree C body temperatures had normal solute concentrations and showed no evidence of containing antifreeze molecules.

Metrology for the next generation of semiconductor devices.

The semiconductor industry continues to produce ever smaller devices that are ever more complex in shape and contain ever more types of materials. The ultimate sizes and functionality of these new devices will be affected by fundamental and engineering limits such as heat dissipation, carrier mobility and fault tolerance thresholds. At present, it is unclear which are the best measurement methods needed to evaluate the nanometre-scale features of such devices and how the fundamental limits will affect the required metrology. Here, we review state-of-the-art dimensional metrology methods for integrated circuits, considering the advantages, limitations and potential improvements of the various approaches. We describe how integrated circuit device design and industry requirements will affect lithography options and consequently metrology requirements. We also discuss potentially powerful emerging technologies and highlight measurement problems that at present have no obvious solution.

mRNA stability and polysome loss in hibernating Arctic ground squirrels (Spermophilus parryii).

All small mammalian hibernators periodically rewarm from torpor to high, euthermic body temperatures for brief intervals throughout the hibernating season. The functional significance of these arousal episodes is unknown, but one suggestion is that rewarming may be related to replacement of gene products lost during torpor due to degradation of mRNA. To assess the stability of mRNA as a function of the hibernation state, we examined the poly(A) tail lengths of liver mRNA from arctic ground squirrels sacrificed during four hibernation states (early and late during a torpor bout and early and late following arousal from torpor) and from active ground squirrels sacrificed in the summer. Poly(A) tail lengths were not altered during torpor, suggesting either that mRNA is stabilized or that transcription continues during torpor. In mRNA isolated from torpid ground squirrels, we observed a pattern of 12 poly(A) residues at greater densities approximately every 27 nucleotides along the poly(A) tail, which is a pattern consistent with binding of poly(A)-binding protein. The intensity of this pattern was significantly reduced following arousal from torpor and undetectable in mRNA obtained from summer ground squirrels. Analyses of polysome profiles revealed a significant reduction in polyribosomes in torpid animals, indicating that translation is depressed during torpor.

Enabling Quantitative Optical Imaging for In-die-capable Critical Dimension Targets.

Dimensional scaling trends will eventually bring semiconductor critical dimensions (CDs) down to only a few atoms in width. New optical techniques are required to address the measurement and variability for these CDs using sufficiently small in-die metrology targets. Recently, Qin et al. [Light Sci Appl, 5, e16038 (2016)] demonstrated quantitative model-based measurements of finite sets of lines with features as small as 16 nm using 450 nm wavelength light. This paper uses simulation studies, augmented with experiments at 193 nm wavelength, to adapt and optimize the finite sets of features that work as in-die-capable metrology targets with minimal increases in parametric uncertainty. A finite element based solver for time-harmonic Maxwell's equations yields two- and three-dimensional simulations of the electromagnetic scattering for optimizing the design of such targets as functions of reduced line lengths, fewer number of lines, fewer focal positions, smaller critical dimensions, and shorter illumination wavelength. Metrology targets that exceeded performance requirements are as short as 3 µm for 193 nm light, feature as few as eight lines, and are extensible to sub-10 nm CDs. Target areas measured at 193 nm can be fifteen times smaller in area than current state-of-the-art scatterometry targets described in the literature. This new methodology is demonstrated to be a promising alternative for optical model-based in-die CD metrology.

Effect of winter high temperatures on reproduction and circannual rhythms in hibernating ground squirrels.

We tested whether prevention of hibernation in ground squirrels by midwinter exposure to high ambient temperatures influenced timing of the spring phase of reproductive maturation and the phase and period of subsequent circannual rhythms of reproduction and body mass. Exposing hibernating adult male Spermophilus lateralis to 30 degrees C for 6 weeks beginning December 4 advanced the timing of testicular recrudescence by 4-5 weeks, compared to controls left at 4 degrees C. Males exposed to 30 degrees C for 6 weeks beginning at the average time of spontaneous end of hibernation (January 15) reached reproductive maturation at a time intermediate to those of controls and of the December 4 experimental group. However, neither the date of the subsequent fall's body mass peak, the date of the next year's reproductive maturation, nor the periods of circannual rhythms of body mass and reproduction differed among groups. Premature interruption of hibernation appears to allow early expression of reproduction, but does not affect the underlying timing mechanism.

Thermogenic capacity at subzero temperatures: how low can a hibernator go?

Abstract Hibernation in mammals is a physiological and behavioral adaptation to survive intervals of low resource availability through profound decreases in metabolic rate (MR), core body temperature (Tb), and activity. Most small mammalian hibernators thermoconform, with Tb approximating ambient temperature (Ta); arctic species are an exception, since they must actively defend what can be large thermal gradients between Tb and Ta. Here we compare the thermogenic capacity of the arctic ground squirrel (Urocitellus parryii) to that of the golden-mantled ground squirrel (Callospermophilus lateralis), a temperate-zone montane hibernator. We allowed animals to reenter torpor at sequentially lower Ta's and found that arctic ground squirrels maintained steady state torpor at Ta's as low as -26°C, through a 36-fold increase in torpid MR (TMR), compared to their minimum TMR, exhibited at a Ta of 0°C. Golden-mantled ground squirrels are able to maintain steady state torpor at Ta's at least as low as -8°C, through a 13.5-fold increase in MR, compared to their minimum TMR at a Ta of 2°C. In a second experiment, torpid animals were exposed to continuously decreasing Ta's (0.25°C/30 min); individuals of both species increased their metabolism while remaining torpid at low Ta's (as low as -30°C for arctic ground squirrels and -10°C for golden-mantled ground squirrels). Although the capacity to hibernate at subfreezing Ta's is not unique to arctic ground squirrels, their large body size, greater torpid metabolic scope, and previously ascribed capacity to supercool allow them to occupy much colder hibernacula for prolonged seasons of hibernation.

Warming up for sleep? Ground squirrels sleep during arousals from hibernation.

Hypothermia during mammalian hibernation is periodically interrupted by arousals to euthermy, the function of which is unknown. We report that arctic ground squirrels (Spermophilus parryii) consistently sleep during these arousals, and that their EEG shows the decrease in slow wave activity (delta power) that is characteristic of a declining requirement for sleep. These results are consistent with the novel hypothesis that the need for sleep slowly accumulates during torpor, and that returning to euthermy is periodically required to allow sleep. Sleep thus seems to be energetically expensive for a hibernating mammal, and cannot be considered solely a strategy for saving energy.

Antifreeze proteins in Alaskan insects and spiders.

Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).

Leptin prevents posthibernation weight gain but does not reduce energy expenditure in arctic ground squirrels.

In mammals, leptin reduces energy intake and may increase energy expenditure as a means to maintain body weight and/or adiposity at an appropriate level. Hibernating mammals seasonally alter body mass, food intake, and body composition and, therefore, represent an attractive model for investigating the physiological regulation of changing body mass and adiposity. Previous experiments in our laboratory demonstrated that administration of mouse recombinant leptin reduces food intake and body weight in arctic ground squirrels during prehibernation fattening. In addition, leptin appeared to reduce metabolic efficiency (weight gain per unit of energy intake). This result suggests that reduced food intake alone may not account for the observed weight loss. Here, we describe the effect of a 3-week constant infusion of leptin given to posthibernation arctic ground squirrels on food consumption and energy expenditure. Mouse recombinant leptin (1 mg/ml) was administered through subcutaneously implanted mini-osmotic pumps (10 microliters/hr flow rate). Resting metabolic rate was monitored before and during the 3-week leptin administration period by indirect calorimetry. Body temperature and locomotory activity were monitored continuously by abdominal radiotransmitters. At the end of the leptin administration period, thermogenic capacity was evaluated by measuring brown fat uncoupling protein-1 mRNA and protein levels. Leptin administration resulted in reduced food intake and prevented posthibernation weight gain, but it did not alter any of the measured parameters of energy expenditure.

Probability of freezing in the freeze-avoiding beetle larvae Cucujus clavipes puniceus (Coleoptera: Cucujidae) from interior Alaska.

Freeze-avoiding insects must resist freezing or die. A suite of adaptations to low temperatures, including the production of antifreeze proteins, colligative antifreezes (polyols), and dehydration allows most individuals to prevent freezing below the lowest ambient temperatures experienced in situ; however, there can be a wide variance in the minimum temperatures that individuals of freeze-avoiding species reach before freezing. We used logistic regression to explore factors that affect this variance and to estimate the probability of freezing in larvae of the freeze-avoiding beetle Cucujus clavipes puniceus. We hypothesized that water content ≤0.5 mg mg(-1) dry mass would lead to deep supercooling (avoidance of freezing below -58°C). We found a significant interaction between water content and ambient below-snow temperature and a significant difference between individuals collected from two locations in Alaska: Wiseman and Fairbanks. Individuals collected in Wiseman deep supercooled with greater water content and to a greater range of ambient temperatures than individuals collected in Fairbanks, leading to significantly different lethal water contents associated with 50% probability of freezing.

Simultaneous freeze tolerance and avoidance in individual fungus gnats, Exechia nugatoria.

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean +/- SEM: -31.5 +/- 0.2 degrees C, 70% survival and -50.7 +/- 0.4 degrees C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (-30.1 +/- 1.1 degrees C) and head/thorax freezing (-48.7 +/- 1.3 degrees C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 +/- 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 +/- 0.8% water).

Annual cycles of gonadotropins and androgens in the hibernating golden-mantled ground squirrel.

Ground squirrels, captured in the field, were housed at ambient temperatures of 23 degrees (photoperiod = 10L:14D) for 13 months. Plasma was sampled at 3 to 4-week intervals and measured for gonadotropin and androgen levels. Testis size was examined monthly by laparotomy. Male ground squirrels showed clear circannual cycles in body mass, testis size, and levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), and dihydrotestosterone (DHT). During summer and fall, FSH, LH and androgen levels were low, testes were undeveloped, and body mass was increasing. Testes began to rapidly enlarge in January and reached maximum size in February. A rise in FSH preceded gonadal growth but LH remained low until near the time of testis mass peak. LH remained elevated during spring while FSH levels fell and testes regressed. Plasma T and DHT levels generally paralleled LH concentrations; DHT levels were approximately one-fifth those of T levels. During winter animals lost weight but were only occasionally found in a slightly hypothermic condition. Females had elevated plasma LH levels (greater than 1 ng/ml) predominantly in the spring but displayed no cycle in plasma FSH levels. A second group of males held at 4 degrees for 8 months (photoperiod = stimulated natural for 47 degrees N) were regularly torpid during a hibernation season that lasted between November and May. Most (15/21) of these males did not show gonadal development by spring; these non-reproductive males had had restricted body mass gains the previous fall. Plasma FSH was low in both reproductive and non-reproductive males during fall and winter but increased in March while animals were still hibernating. FSH levels continued to increase in April only in reproductive males and reached maximal levels after hibernation was spontaneously terminated. LH titers were elevated in individual males in winter during torpor and were greater in reproductive than in non-reproductive males in May. Androgen levels were undetectable in torpid squirrels, elevated in animals sampled during periodic arousals, and elevated in most males within 3 weeks after terminating hibernation.

Effects of ambient temperature on metabolic rate, respiratory quotient, and torpor in an arctic hibernator.

Arctic ground squirrels (Spermophilus parryii) overwinter in hibernaculum conditions that are substantially below freezing. During torpor, captive arctic ground squirrels displayed ambient temperature (T(a))-dependent patterns of core body temperature (T(b)), metabolic rate (TMR), and metabolic fuel use, as determined by respiratory quotient (RQ). At T(a) 0 to -16 degrees C, T(b) remained relatively constant, and TMR rose proportionally with the expanding gradient between T(b) and T(a), increasing >15-fold from a minimum of 0.0115 +/- 0.0012 ml O(2). g(-1). h(-1). At T(a) 0-20 degrees C, T(b) increased with T(a); however, TMR did not change significantly from T(b) 0 to 12 degrees C, indicating temperature-independent inhibition of metabolic rate. The overall change in TMR from T(b) 4 to 20 degrees equates to a Q(10) of 2.4, but within this range of T(b), Q(10) changed from 1.0 to 14.1. During steady-state torpor at T(a) 4 and 8 degrees C, RQ averaged 0.70 +/- 0.013, indicating exclusive lipid catabolism. At T(a) -16 and 20 degrees C, RQ increased significantly to >0.85, consistent with recruitment of nonlipid fuels. RQ was negatively correlated with maximum torpor bout length. For T(a) values <0 degrees C, this relationship supports the hypothesis that availability of nonlipid metabolic fuels limits torpor duration in hibernating mammals; for T(a) values >0 degrees C, hypotheses linked to body temperature are supported. Because anterior body temperatures differ from core, overall, the duration torpor can be extended in hibernating mammals may be dependent on brain temperature.

Environmental and endogenous control of reproductive function in the Great Basin pocket mouse Perognathus parvus.

Pocket mice captured in the field at various times of year were introduced into laboratory experiments to examine the short-term sensitivity of reproductive function to environmental factors, principally day length, and the tendency of the reproductive system to become active spontaneously over longer durations. Spontaneous enlargement and partial activation of the gonads occurred over the course of 4-5 mo in continuous darkness during the hibernation season. Males held for 13 mo in 12L:12D showed patterns of testicular enlargement, but with only partial regression; the degree of endogeneity in the reproductive control system of P. parvus is therefore considerably below that of the pronounced and persistent "endogenous circannual rhythms" shown by certain rodents of the squirrel family. Responses to day length varied seasonally. The partially activated reproductive system of mice that emerged from hibernation in spring was further stimulated by long days (16L:8D); in summer gonadal growth was insensitive to differences in day length, and in autumn the gonads remained undeveloped in short days (8L:16D) but were sensitive to stimulation by long days. This "photoperiodic" response of P. parvus is based on an endogenous circadian rhythm of photosensitivity as proposed by Bünning (1936). We also found that reproductive function of P. parvus is somewhat retarded by low temperature and reduced availability of water. We discuss the general nature of environmental sensitivity of reproductive function and the ways in which the photoperiodic response and spontaneous pattern of winter gonadal development in P. parvus are likely to interact with environmental factors that lead to fine-tuning the final reproductive response.

Plasma androgen and gonadotropin levels during hibernation and testicular maturation in golden-mantled ground squirrels.

The 4-5-mo hibernation season of golden-mantled ground squirrels consists of extended torpor bouts interspersed with brief, periodic intervals of normothermic arousal. Plasma levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and degree of scrotal pigmentation were measured in torpid and aroused male ground squirrels throughout a season of hibernation and in active animals after the termination of torpor. T was basal in torpid animals; beginning 3 weeks before torpor ended, T was elevated in normothermic males during the first half of periodic arousals but returned to basal levels before animals reentered torpor. After the last (terminal) arousal from torpor, T levels were moderately elevated for 4 wk and maximal for the next 6 wk before they returned to basal values. LH patterns were similar to those of T; however, levels of T and LH were positively correlated only in aroused or posthibernation males. FSH levels remained constant and low during most of the heterothermic season but increased in several torpid males within 3 days of terminal arousal. FSH levels peaked 2 wk after the end of heterothermy. Scrotal pigmentation developed over the first 4 wk after terminal arousal. Maturation of reproductive function occurs during the 4 wk after termination of heterothermy, but elevated levels of T during arousals and variable levels of FSH in the last days of torpor suggest that activation or increased sensitivity of the hypothalamic-pituitary-gonadal axis is important in the termination of heterothermy in ground squirrels.