Woody encroachment of grasslands: Near‐surface thermal implications assessed through the lens of an astronomical event

Temperature has long been understood as a fundamental condition that influences ecological patterns and processes. Heterogeneity in landscapes that is structured by ultimate (climate) and proximate (vegetation, topography, disturbance events, and land use) forces serve to shape thermal patterns across multiple spatio‐temporal scales. Thermal landscapes of grasslands are likely shifting as woody encroachment fragments these ecosystems and studies quantifying thermal fragmentation in grassland systems resulting from woody encroachment are lacking. We utilized the August 21st, 2017, solar eclipse to mimic a rapid sunrise/sunset event across a landscape characterized as a grassland to experimentally manipulate levels of solar radiation in the system. We then quantified changes in near‐surface temperatures resulting from changes in solar radiation levels during the eclipse. Temperatures were monitored across three grassland pastures in central Oklahoma that were characterized by different densities (low, medium, and high) of Juniperus virginiana to understand the impact of woody encroachment on diurnal temperature patterns and thermal heterogeneity in a grassland''s thermal landscape. The largest temperature range across sites that occurred during the eclipse was in the mixed grass vegetation. Similarly, the largest change in thermal heterogeneity occurred in the grassland with the lowest amount of woody encroachment. Thermal heterogeneity was lowest in the highly encroached grassland, which also experienced the lowest overall change in thermal heterogeneity during the eclipse. Time series models suggested that solar radiation was the most influential factor in predicting changes in thermal heterogeneity as opposed to ambient temperature alone. These results suggest that highly encroached grasslands may experience lower diurnal variability of temperatures at the cost of a decrease in the overall thermal heterogeneity of that landscape. It appears that fine‐scale spatio‐temporal thermal variation is largely driven by solar radiation, which can be influenced by vegetation heterogeneity inherent within a landscape.     

Global analysis of thermal tolerance and latitude in ectotherms

A tenet of macroecology is that physiological processes of organisms are linked to large-scale geographical patterns in environmental conditions. Species at higher latitudes experience greater seasonal temperature variation and are consequently predicted to withstand greater temperature extremes. We tested for relationships between breadths of thermal tolerance in ectothermic animals and the latitude of specimen location using all available data, while accounting for habitat, hemisphere, methodological differences and taxonomic affinity. We found that thermal tolerance breadths generally increase with latitude, and do so at a greater rate in the Northern Hemisphere. In terrestrial ectotherms, upper thermal limits vary little while lower thermal limits decrease with latitude. By contrast, marine species display a coherent poleward decrease in both upper and lower thermal limits. Our findings provide comprehensive global support for hypotheses generated from studies at smaller taxonomic subsets and geographical scales. Our results further indicate differences between terrestrial and marine ectotherms in how thermal physiology varies with latitude that may relate to the degree of temperature variability experienced on land and in the ocean.     

Panmixia across elevation in thermally sensitive Andean dung beetles

Janzen's seasonality hypothesis predicts that organisms inhabiting environments with limited climatic variability will evolve a reduced thermal tolerance breadth compared with organisms experiencing greater climatic variability. In turn, narrow tolerance breadth may select against dispersal across strong temperature gradients, such as those found across elevation. This can result in narrow elevational ranges and generate a pattern of isolation by environment or neutral genetic differentiation correlated with environmental variables that are independent of geographic distance. We tested for signatures of isolation by environment across elevation using genome‐wide SNP data from five species of Andean dung beetles (subfamily Scarabaeinae) with well‐characterized, narrow thermal physiologies, and narrow elevational distributions. Contrary to our expectations, we found no evidence of population genetic structure associated with elevation and little signal of isolation by environment. Further, elevational ranges for four of five species appear to be at equilibrium and show no decay of genetic diversity at range limits. Taken together, these results suggest physiological constraints on dispersal may primarily operate outside of a stable realized niche and point to a lower bound on the spatial scale of local adaptation.     

Conserved and narrow temperature limits in alpine insects: Thermal tolerance and supercooling points of the ice-crawlers,Grylloblatta (Insecta: Grylloblattodea: Grylloblattidae)

For many terrestrial species, habitat associations and range size are dependent on physiological limits, which in turn may influence large-scale patterns of species diversity. The temperature range experienced by individuals is considered to shape the breadth of the thermal niche, with species occupying temporally and/or geographically stable climates tolerating a narrow temperature range. High-elevation environments experience large temperature fluctuations, with frequent periods below 0 °C, but Grylloblatta (Grylloblattodea: Grylloblattidae) occupy climatically stable microhabitats within this region. Here we test critical thermal limits and supercooling points for five Grylloblatta populations from across a large geographic area, to examine whether the stable microhabitats of this group are associated with a narrow thermal niche and assess their capacity to tolerate cold conditions. Thermal limits are highly conserved in Grylloblatta, despite substantial genetic divergence among populations spanning 1500 m elevation and being separated by over 500 km. Further, Grylloblatta show exceptionally narrow thermal limits compared to other insect taxa with little capacity to improve cold tolerance via plasticity. In contrast, upper thermal limits were significantly depressed by cold acclimation. Grylloblatta maintain coordinated movement until they freeze, and they die upon freezing. Convergence of the critical thermal minima, supercooling point and lower lethal limits point to adaptation to a cold but, importantly, constant thermal environment. These physiological data provide an explanation for the high endemism and patchy distribution of Grylloblatta, which relies on subterranean retreats to accommodate narrow thermal limits. These retreats are currently buffered from temperature fluctuations by snow cover, and a declining snowpack thus places Grylloblatta at risk of exposure to temperatures beyond its tolerance capacity.     

Exposure to solar radiation drives organismal vulnerability to climate: Evidence from an intertidal limpet

Understanding the physiological abilities of organisms to cope with heat stress is critical for predictions of species’ distributions in response to climate change. We investigated physiological responses (respiration and heart beat rate) of the ectotherm limpet Patella vulgata to heat stress events during emersion and the role of seasonal and microclimatic acclimatization for individual thermal tolerance limits. Individuals were collected from 5 microhabitats characterized by different exposure to solar radiation in the high intertidal zone of a semi-exposed rocky shore in winter and summer of 2014. Upper thermal tolerance limits (heat coma temperatures – HCTs, and heart rate Arrhenius break temperatures - ABTs) were determined for individuals from each microhabitat in both seasons under laboratory conditions. While we found a clear seasonal acclimatization, i.e., higher HCTs and ABTs in summer than in winter, we did not find evidence for microhabitat-specific responses that would suggest microclimatic acclimatization. However, operative limpet temperatures derived from in-situ temperature measurements suggest that individuals from sun exposed microhabitats have a much narrower thermal safety margins than those from less exposed surfaces or within crevices. Microhabitat specific thermal safety margins caused by high thermal heterogeneity at small spatial scales and the lack of short term acclimatization will likely shape small scale distribution patterns of intertidal species in response to the predicted increase in the frequency and intensity of heat waves.     

Thermal limits of wild and laboratory strains of two African malaria vector species, <Emphasis Type="Italic">Anopheles arabiensis</Emphasis> and <Emphasis Type="Italic">Anopheles funestus</Emphasis>

ABSTRACT: Malaria affects large parts of the developing world and is responsible for almost 800,000 deaths annually. As climates change, concerns have arisen as to how this vector-borne disease will be impacted by changing rainfall patterns and warming temperatures. Despite the importance and controversy surrounding the impact of climate change on the potential spread of this disease, little information exists on the tolerances of several of the vector species themselves. METHODS: Using a ramping protocol (to assess critical thermal limits - CT) and plunge protocol (to assess lethal temperature limits - LT) information on the thermal tolerance of two of Africa's important malaria vectors, Anopheles arabiensis and Anopheles funestus was collected. The effects of age, thermal acclimation treatment, sex and strain (laboratory versus wild adults) were investigated for CT determinations for each species. The effects of age and sex for adults and life stage (larvae, pupae, adults) were investigated for LT determinations. RESULTS: In both species, females are more tolerant to low and high temperatures than males; larvae and pupae have higher upper lethal limits than do adults. Thermal acclimation of adults has large effects in some instances but small effects in others. Younger adults tend to be more tolerant of temperature changes than older age groups. Long-standing laboratory colonies are sufficiently similar in thermal tolerance to field-collected animals to provide reasonable surrogates when making inferences about wild population responses. Differences between these two vectors in their thermal tolerances, especially in larvae and pupae, are plausibly a consequence of different habitat utilization. CONCLUSIONS: Limited plasticity is characteristic of the adults of these vector species relative to others examined to date, suggesting limited scope for within-generation change in thermal tolerance. These findings and the greater tolerance of females to thermal extremes may have significant implications for future malaria transmission, especially in areas of current seasonal transmission and in areas on the boundaries of current vector distribution.     

Thermal limits to survival and activity in two life stages of false codling moth Thaumatotibia leucotreta (Lepidoptera,Tortricidae)

The present study examines life stage‐related variation in the thermal limits to activity and survival in an African pest, the false codling moth Thaumatotibia leucotreta (Lepidoptera, Tortricidae). Thermal tolerance, including the functional activity limits of critical thermal maxima and minima (CT_max and CT_min respectively), upper and lower lethal temperature, and the effect of heat and cold hardening (short‐term acute plasticity), is measured across a diverse range of low or high temperature stress conditions in both larvae and adults. We also report the sum of inducible and cognate forms of the amounts of heat shock protein 70 (HSP70) as an explanatory variable for changes in thermotolerance. The results show that the larvae have high variability in CT_max and CT_min at different ramping rates and low levels of basal (innate) thermal tolerance. By contrast, the adults show high basal tolerance and overall lower variability in CT_max and CT_min, indicating lower levels of phenotypic plasticity in thermotolerance. HSP70 responses, although variable, do not reflect these tolerance or survival patterns. Larvae survive across a broader range of temperatures, whereas adults remain active across a broader range of temperatures. Life stage‐related variation in thermal tolerance is most pronounced under the slowest (most ecologically‐relevant) ramping rate (0.06 °C min^–1) during lower critical thermal limit experiments and least pronounced during upper thermal limit experiments. Thus, the ramping rate can hinder or enhance the detection of stage‐related variation in thermal limits to activity and survival of insects.     

Differential thermal tolerance and energetic trajectories during ontogeny in porcelain crabs,genus Petrolisthes

Thermal tolerance limits of marine intertidal zone organisms are elevated compared to subtidal species, but are typically just slightly higher than maximal habitat temperatures. The small thermal safety margins maintained by intertidal zone organisms suggest that high thermal tolerance is associated with a physiological cost. If true, we hypothesize that species that transition between intertidal zone and planktonic habitats during ontogeny, will adjust their thermal tolerance accordingly to capitalize upon potential energy savings while in a thermally benign habitat. We tested this hypothesis in porcelain crabs that transition between the thermally stressful, intertidal zone as embryos, to the thermally benign pelagic zone as larvae, and back at settlement. We found the more thermally tolerant, mid-intertidal zone species, Petrolisthes cinctipes, and the less thermally tolerant, subtidal zone species, Petrolisthes manimacilis, exhibited reduced thermal tolerance (LT₅₀) in the transition from embryos to larvae. This was associated with an increased oxygen consumption rate in both species, though P. cinctipes exhibited a significantly greater increase in oxygen consumption. P. cinctipes also showed an increase in thermal tolerance in settled juveniles compared to pelagic zoea I larvae, resulting in an overall V-shaped thermal tolerance relationship during ontogeny, while in P. manimaculis thermal tolerance was significantly lower in juveniles compared to zoea I. In neither species were these changes (zoea I to juvenile) associated with a significant change in metabolism. While embryos and juveniles of P. cinctipes have thermal tolerance limits near intertidal habitat thermal maxima (∼32.5 °C), all three life-history stages in P. manimaculis (especially embryos and larvae) exhibit considerable thermal safety margins. The mechanisms underlying this “excess” thermal tolerance in P. manimacilis embryos are unknown, but suggest that patterns of thermal tolerance in early life history stages are species-specific.     

Male and female meiosis in grasshoppers

Male meiosis in the grasshopper Stethophyma grossum is well known as an example of proximal chiasma localisation. An investigation of female meiosis in oocytes of this species shows that both the frequency and distribution of chiasmata are quite different from the male situation. Mean chiasma frequency per cell (14.98) in considerably higher in females than in males (11.28) which agrees with the trend established in other comparative studies of male and female meiosis. More strikingly, males and females also show not only different but quite opposite patterns of chiasma distribution. In spermatocytes of males, chiasmata are strictly localised proximally in most bivalents, but in oocytes of females very few chiasmata form in proximal regions and nearly all chiasmata form either in distal or interstitial regions. The genetical significance of these findings is considered.     

Mitigating thermal effect of behaviour and microhabitat on the intertidal snail Littorina saxatilis (Olivi) over summer

High shore intertidal ectotherms must withstand temperatures which are already close, at or beyond their upper physiological thermal tolerance. Their behaviour can provide a relief under heat stress, and increase their survival through thermoregulation. Here, we used infrared imaging to reveal the thermoregulatory behavioural strategies used by the snail Littorina saxatilis (Olivi) on different microhabitats of a high shore boulder field in Finistère (western France) in summer. On our study site, substrate temperature is frequently greater than L. saxatilis upper physiological thermal limits, especially on sun exposed microhabitats. To maintain body temperatures within their thermal tolerance window, withdrawn snails adopted a flat posture, or elevated their shells and kept appended to the rock on the outer lip of their aperture with dried mucous (standing posture). These thermal regulatory behaviours lowered snail body temperatures on average by 1–2 °C. Aggregation behaviour had no thermoregulatory effect on L. saxatilis in the present study. The occupation of biogenic microhabitats (barnacles) was associated with a 1 °C decrease in body temperatures. Barnacles and microhabitats that experienced low sun exposure, low thermal fluctuations and low thermal maxima, could buffer the heat extremes encountered at high shore level especially on sun exposed microhabitats.     

Thermal tolerance ranges and climate variability: A comparison between bivalves from differing climates

The climate variability hypothesis proposes that in variable temperate climates poikilothermic animals have wide thermal tolerance windows, whereas in constant tropical climates they have small thermal tolerance windows. In this study we quantified and compared the upper and lower lethal thermal tolerance limits of numerous bivalve species from a tropical (Roebuck Bay, north western Australia) and a temperate (Wadden Sea, north western Europe) tidal flat. Species from tropical Roebuck Bay had higher upper and lower lethal thermal limits than species from the temperate Wadden Sea, and Wadden Sea species showed an ability to survive freezing temperatures. The increased freezing resistance of the Wadden Sea species resulted in thermal tolerance windows that were on average 7 °C greater than the Roebuck Bay species. Furthermore, at a local-scale, the upper lethal thermal limits of the Wadden Sea species were positively related to submersion time and thus to encountered temperature variation, but this was not the case for the Roebuck Bay species. A review of previous studies, at a global scale, showed that upper lethal thermal limits of tropical species are closer to maximum habitat temperatures than the upper lethal thermal limits of temperate species, suggesting that temperate species are better adapted to temperature variation. In this study, we show for the first time, at both local and global scales, that the lethal thermal limits of bivalves support the climate variability effect in the marine environment.     

Comparative thermal ecology parameters of the mexican dusky rattlesnake (Crotalus triseriatus)

Montane habitats exhibit a high degree of thermal heterogeneity, and thus provide considerable thermoregulatory challenges for ectotherms. Comparative analyses provide an opportunity to understand how variation in abiotic factors (e.g., operative temperatures, thermal quality) can affect life history traits within species. We studied the thermal ecology of three populations of the rattlesnake Crotalus triseriatus inhabiting different volcanoes in the central region of Mexico using the Hertz et al. (1993) protocol. The average body temperature of dusky rattlesnakes from the three study sites was 22.4 °C; mean active body temperature was higher in site 2 than in sites 1 and 3, but no differences between females, males and juveniles nor an interaction among site and sex was found. The thermal quality was low in the three sites, particularly in sites 1 and 3. Thermoregulation accuracy statistically differed among populations: individuals from site 2 were more accurate thermoregulating, while individuals from site 1 were the least accurate. Compared to other snakes, dusky rattlesnakes can be considered as a eurythermic species, which can often be active at relatively low body temperatures.     

Thermal acclimation capacity of Jack Beardsley mealybug (Pseudococcus jackbeardsleyi) to survive in a warming world

The study of thermal tolerance and acclimation capacity in Jack Beardsley mealybug, Pseudococcus jackbeardsleyi Gimpel and Miller is the crucial step in determining their abilities to cope with climate change. Thus, the aim of this research was to determine the effects of acclimation temperatures on the changes in thermal tolerance of P. jackbeardsleyi. The influences of acclimation temperature at moderate (25?°C) and high (35?°C) temperatures on their lower and upper thermal limits were measured composed of critical thermal minimum (CT_min), maximum (CT_max), chill coma temperature (CCT) and heat coma temperature (HCT) for first instar nymphs and adults. The important information derived from this study revealed that the upper thermal limits of adults are constrained to a relative narrow range that will make them sensitive to relative small changes in temperatures, whilst all mean upper thermal indices at 35?°C were significantly higher than at 25?°C for nymphs. For this highlight notice, nymphs have more potential to change their upper thermal limits which will allow them to withstand high temperatures in the field. These results are a sign to warn us that P. jackbeardsleyi could become highly noxious which cause severe outbreaks damage to the crops in the tropics under global warming.     

Experimental evaluation of nest shelter effects on weight loss in incubating common eiders Somateria mollissima

Evaluating consequences of habitat selection is an important step in understanding life history strategies and behavioural decisions of animals. Kilpi and Lindström (1997) found that incubating common eiders Somateria mollissima on exposed, treeless islands lost weight faster than females nesting on wooded islands and proposed that this difference was due to adverse incubation conditions at exposed nests. Therefore, we tested whether common eiders gained an advantage when nesting in sheltered habitats by placing artificial shelters over randomly-selected females after the onset of incubation within an eider colony in arctic Canada. We predicted that sheltered females would be heavier on completion of incubation than control hens lacking shelters. Females nesting in artificial shelters experienced a more moderate thermal environment at both cold and warm temperature extremes. Eiders nesting in shelters were heavier than control females during mid incubation, consistent with habitat-specific rates of weight loss reported by Kilpi and Lindström (1997) . Natural overhead cover was available at potential nests but few eiders used those sites. We suspect that microclimatic advantages offered by sheltered sites may be offset by costs of increased female vulnerability to predators. Further work is needed to test this hypothesis, and to determine mechanisms responsible for lower weight loss in eiders attending well concealed nests.     

Effects of dehydration on thermoregulatory behavior and thermal tolerance limits of Rana catesbeiana ()

Predicting the effects of high environmental temperatures and drought on populations requires understanding how these conditions will influence the thermoregulatory behavior and thermal tolerance of organisms. Ectotherms show proportional (fine-tuned) and all-or-none (abrupt) responses to avoid overheating. Scattered evidence suggests that dehydration alters these behavioral responses and thermal tolerance, but these effects have not been evaluated in an integrative manner. We examined the effects of hydration level on the behavioral thermoregulation and behavioral and physiological thermal limits of the “bullfrog” (Rana catesbeiana), a well-studied and important invasive species. To examine the effects of dehydration on proportional responses, we compared the Preferred Body Temperatures (PBT) of frogs with restricted and unrestricted access to water. To assess the effect of dehydration on all-or-none responses, we measured and compared the Voluntary Thermal Maximum (VTMax) at different hydration levels (100%, 90%, 80% of body weight at complete hydration). Finally, to understand the effect of dehydration on physiological thermal tolerance, we measured the Critical Thermal Maximum (CTMax) of frogs at matched hydration levels. PBT, VTMax, and CTMax all decreased in response to higher dehydration levels. However, bullfrogs changed their PBT more than their VTMax or CTMax in response to dehydration. Moreover, some severely dehydrated individuals did not exhibit a VTMax response. We discuss the implications of our results in the context of plasticity of thermoregulatory responses and thermal limits, and its potential application to mechanistic modeling.     

Predation risk on incubating adults constrains the choice of thermally favourable nest sites in a plover

Birds are thought to choose nest sites that meet two main functions: providing security to both nest contents and incubating adults, and providing an appropriate microclimate for incubation. Many shorebirds nest in sites with no or little cover. In a lake in southern Spain, nearly 70% of the nests of Kentish plovers, Charadrius alexandrinus, were in sites with little or no cover, where ambient temperatures might be more than 50°C during very hot days, thus causing the incubating adults to suffer from heat stress. We tested the hypothesis that Kentish plovers nest mainly in exposed sites because this may allow the incubating birds to detect approaching predators early, and thus to reduce predation risk. When we occluded the view that incubating adults had from their nests, they took longer to detect approaching predators than when the view was unrestricted. Incubating adults were also more frequently killed by mammals in covered than in exposed nests. Females that nested in covered sites were in lower body condition than those nesting in exposed sites, possibly because they were unable to withstand the high ambient temperatures in exposed sites. Thus, the benefits of thermally favourable nest sites are reduced by the constraints of predation risk.     

Coevolution of color pattern and thermoregulatory behavior in polymorphic pygmy grasshoppers Tetrix undulata

Ectothermic organisms, such as insects and reptiles, rely on external heat sources to control body temperature and possess physiological and behavioral traits that are temperature dependent. It has therefore been hypothesised that differences in body temperature resulting from phenotypic properties, such as color pattern, may translate into selection against thermally inferior phenotypes. We tested for costs and benefits of pale versus dark coloration by comparing the behaviors (i.e., basking duration and bouts) of pygmy grasshopper (Tetrix undulata) individuals exposed to experimental situations imposing a trade-off between temperature regulation and feeding. We used pairs consisting of two full-siblings of the same sex that represented different (genetically coded) color morphs but had shared identical conditions from the time of fertilization. Our results revealed significant differences in behavioral thermoregulation between dark and pale individuals in females, but not in males. Pale females spent more time feeding than dark females, regardless of whether feeding was associated with a risk of either hypothermia or overheating. In contrast, only minor differences in behavior (if any) were evident between individuals that belonged to the same color morph but had been painted black or gray to increase and decrease their heating rates. This suggests that the behavioral differences between individuals belonging to different color morphs are genetically determined, rather than simply reflecting a response to different heating rates. To test for effects of acclimation on behaviors, we used pairs of individuals that had been reared from hatchlings to adults under controlled conditions in either low or high temperature. The thermal regime experienced during rearing had little effect on behaviors during the experiments reported above, but significantly influenced the body temperatures selected in a laboratory thermal gradient. In females (but not in males) preferred body temperature also varied among individuals born to mothers belonging to different color morphs, suggesting that a genetic correlation exists between color pattern and temperature preferences. Collectively, these findings, at least in females, are consistent with the hypothesis of multiple-trait coevolution and suggest that the different color morphs represent alternative evolutionary strategies.     

Reproductive behaviour of female Chorthippus biguttulus grasshoppers

Female grasshoppers of acoustically communicating species assume series of reproductive states that are associated with particular behaviours. Studies on laboratory populations of Chorthippus biguttulus (L.) revealed that females of this species lack the period of ‘passive copulatory readiness’, increase their attractiveness to males by sound production and mate multiple times before their first oviposition. In particular, female Ch. biguttulus display a period of ‘primary rejection’ after their imaginal moult during which they reject male mating attempts followed by a period of ‘active copulatory readiness’ in which they produce acoustic signals and may copulate with courting males. Female stridulation generally stimulated male mating activity and stridulating females attracted more male mating attempts than mute females in the same cage, indicating that males preferentially court females that signal ‘active copulatory readiness’. After receipt of a spermatophore, Ch. biguttulus females displayed periods of ‘secondary rejection’ followed by re-establishment of ‘active copulatory readiness’. Acoustic responses of females to male songs, an indicator of reproductive readiness, were significantly reduced until 2 days after mating and remained slightly reduced in comparison to pre-mating levels. Some females mated multiple times before their first oviposition and cycled between ‘secondary rejection’ and ‘active copulatory readiness’.     

Can Tropical Insects Stand the Heat? A Case Study with the Brown Planthopper Nilaparvata lugens (St?l)

The brown planthopper Nilaparvata lugens (Stål) is the most serious pest of rice across the world, especially in tropical climates. N. lugens nymphs and adults were exposed to high temperatures to determine their critical thermal maximum (CT_max), heat coma temperature (HCT) and upper lethal temperature (ULT). Thermal tolerance values differed between developmental stages: nymphs were consistently less heat tolerant than adults. The mean (± SE) CT_max of nymphs and adult females and males were 34.9±0.3, 37.0±0.2 and 37.4±0.2°C respectively, and for the HCT were 37.7±0.3, 43.5±0.4 and 42.0±0.4°C. The ULT₅₀ values (± SE) for nymphs and adults were 41.8±0.1 and 42.5±0.1°C respectively. The results indicate that nymphs of N. lugens are currently living at temperatures close to their upper thermal limits. Climate warming in tropical regions and occasional extreme high temperature events are likely to become important limiting factors affecting the survival and distribution of N. lugens.