The influence of hydric environments during egg incubation on embryonic heart rates and offspring phenotypes in a scincid lizard (Lampropholis guichenoti)

Extensive evidence shows that incubation conditions can affect phenotypic traits of hatchling reptiles, but the relative importance of thermal versus hydric factors, and the proximate mechanisms by which such factors influence hatchling phenotypes, remain unclear for most species. We incubated eggs of an Australian scincid lizard, Lampropholis guichenoti, at four different moisture contents ranging from -500 to 0 kPa. Drier substrates reduced water uptake of eggs and resulted in smaller hatchlings, but other phenotypic traits (incubation periods, hatchling sex, body proportions, running speeds, growth rates post-hatching) were not affected by the hydric environment during incubation. Contrary to our prediction, lower water uptake during incubation (and hence, presumably, more viscous blood) did not affect embryonic heart rates. Thus, as in many other squamate species, hatchling phenotypes and embryonic developmental rates of L. guichenoti are less sensitive to hydric conditions in the nest than to thermal regimes.     

Fluctuations in the incubation moisture environment affect growth but not survival of hatchling lizards

Few studies have collected longitudinal data that follow the complete microevolutionary path of an organism linking sources of variation (e.g. environmental versus genetic) to a trait and its subsequent relationship with fitness. Identifying the links within this pathway is imperative for understanding the ecological relevance of effects found at the phenotypic level. Furthermore, experimental studies that examine parts of the pathway in ectothermic organisms often fail to mimic the complexities of the natural developmental environment. Temperature and moisture conditions in reptile nests, for example, can fluctuate greatly on a seasonal and daily basis. Despite the potential effects of fluctuating environments, the vast majority of studies have held environmental treatments constant during the developmental period. We investigated the effects of fluctuating moisture regimes during incubation on eggs, hatchling phenotypes, and subsequent survival in the eastern fence lizard Sceloporus undulatus. Moisture fluctuations during embryonic development caused water absorption by eggs to follow the environmental availability of moisture. Initial hatchling tail length was affected by the pattern of moisture fluctuations, and hatchling growth rates in fluctuating treatments were significantly faster than those in a constant treatment, resulting in larger hatchlings after 4 weeks. A release–recapture experiment conducted in the field did not detect a treatment effect on survival despite the larger body sizes. In summary, although fluctuations affected water absorption by eggs and some hatchling traits, these effects did not have subsequent fitness consequences. The results obtained suggest that egg and hatchling survival are buffered against natural soil moisture fluctuations during incubation, even when egg and hatchling traits are significantly affected. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 89–102.     

Egg components, egg size, and hatchling size in leatherback turtles

Relationships between egg size, egg components, and neonate size have been investigated across a wide range of oviparous taxa. Differences in egg traits among taxa reflect not only phylogenetic differences, but also interactions between biotic (i.e., maternal resource allocation) and abiotic (i.e. nest environment conditions) factors. We examined relationships between egg mass, egg composition, and hatchling size in leatherback turtles (Dermochelys coriacea) because of the unique egg and reproductive characteristics of this species and of sea turtles in general. Albumen comprised 63.0%+/-2.8% (mean+/-S.D.) of egg mass and explained most of the variation in egg mass, whereas yolk comprised only 33.0%+/-2.7%. Additionally, leatherback albumen dry mass was approximately 16% of albumen wet mass. Whereas hatchling mass increased significantly with egg mass (n = 218 clutches), hatchling mass increased by only approximately 2 g for each 10 g increase in egg mass and was approximately 10-20 g greater than yolk mass. Taken together, our results indicate that albumen might play a particularly significant role in leatherback embryonic development, and that leatherback eggs are both capable of water uptake from the nest substrate and also possess a large reservoir of water in the albumen. Relationships between egg mass and egg components, such as variation in egg mass being largely explained by variation in albumen mass and egg mass containing a relatively high proportion of albumen solids, are more similar to bird eggs than to eggs of other non-avian reptiles. However, hatchling mass correlates more with yolk mass than with albumen mass, unlike patterns observed in bird eggs of similar composition.     

Phenotypic responses of hatchlings to constant versus fluctuating incubation temperatures in the multi-banded krait, Bungarus multicintus (Elapidae)

Most studies on egg incubation in reptiles have relied on constant temperature incubation in the laboratory rather than on simulations of thermal regimes in natural nests. The thermal effects on embryos in constant-temperature studies often do not realistically reflect what occurs in nature. Recent studies have increasingly recognized the importance of simulating natural nest temperatures rather than applying constant-temperature regimes. We incubated Bungarus multicintus eggs under three constant and one fluctuating-temperature regimes to evaluate the effects of constant versus fluctuating incubation temperatures on hatching success and hatchling phenotypes. Hatching success did not differ among the four treatments, and incubation temperature did not affect the sexual phenotype of hatchlings. Incubation length decreased as incubation temperature increased, but eggs incubated at fluctuating temperatures did not differ from eggs incubated at constant temperatures with approximately the same mean in incubation length. Of the hatchling phenotypes examined, residual yolk, fat bodies and locomotor performance were more likely affected by incubation temperature. The maximal locomotor speed was fastest in the fluctuating-temperature and 30 degrees C treatments and slowest in the 24 degrees C treatment, with the 27 degrees C treatment in between. The maximal locomotor length was longest in the fluctuating-temperature treatment and shortest in the 24 degrees C and 27 degrees C treatments, with the 30 degrees C treatment in between. Our results show that fluctuating incubation temperatures do not influence hatching success and hatchling size and morphology any differently than constant temperatures with approximately the same mean, but have a positive effect on locomotor performance of hatchlings.     

Influence of incubation temperature on morphology, locomotor performance, and early growth of hatchling wall lizards (Podarcis muralis)

Eggs of wall lizards (Podarcis muralis) were incubated at three temperatures approaching the upper limit of viability for embryonic development in this species (26, 29, and 32 degrees C) to assess the influence of temperature on various aspects of hatchling phenotype likely affecting fitness. The thermal environment affected size and several morphometric characteristics of hatchling lizards. Hatchlings from eggs incubated at 32 degrees C were smaller (snout-vent length, SVL) than those from 26 and 29 degrees C and had smaller mass residuals (from the regression on SVL) as well as shorter tail, head, and femur relative to SVL. Variation in the level of fluctuating asymmetry in meristic and morphometric traits associated with incubation temperatures was quite high but not clearly consistent with the prediction that environmental stress associated with the highest incubation temperatures might produce the highest level of asymmetry. When tested for locomotor capacity in trials developed at body temperatures of 32 and 35 degrees C, hatchlings from the 32 degrees C incubation treatment exhibited the worst performance in any aspect considered (burst speed, maximal length, and number of stops in the complete run). Repeated measures ANCOVAs (with initial egg mass as covariate) of snout-vent length and mass of lizards at days 0 and 20 revealed significant effects of incubation temperature only for mass, being again the hatchlings from eggs incubated at 32 degrees C those exhibiting the smallest final size. All together, our results evidenced a pervasive effect of thermal regime during incubation (and hence of nest site selection) on hatchling phenotypes. However, incubation temperature does not affect hatchling phenotypes in a continuous way; for most of the analysed traits a critical threshold seems to exist between 29 and 32 degrees C, so that hatchlings incubated at 32 degrees C exhibited major detrimental effects. J. Exp. Zool. 286:422-433, 2000.     

两种麻蜥卵和孵出幼体的特征

研究了山地麻蜥和丽斑麻蜥实验条件下的卵及孵出幼体的特征.山地麻蜥产卵雌体的体长大于丽斑麻蜥,窝卵重小于丽斑麻蜥,但平均卵重和相对窝卵重与丽斑麻蜥相似.两种蜥蜴均通过增加卵长径和卵短径来增加卵重,但卵的外形不同,山地麻蜥卵较长.两种蜥蜴卵孵化过程中均吸水增重.相似孵化条件(波动温度、-12 kPa)下,山地麻蜥的孵化期明显比丽斑麻蜥长.山地麻蜥幼体的尾、头部大于丽斑麻蜥,但体重和SVL相似.     

Effects of incubation temperature on the energetics of embryonic development and hatchling morphology in the Brisbane river turtle Emydura signata

Incubation temperature and the amount of water taken up by eggs from the substrate during incubation affects hatchling size and morphology in many oviparous reptiles. The Brisbane river turtle Emydura signata lays hard-shelled eggs and hatchling mass was unaffected by the amount of water gained or lost during incubation. Constant temperature incubation of eggs at 24 °C, 26 °C, 28 °C and 31 °C had no effect on hatchling mass, yolk-free hatchling mass, residual yolk mass, carapace length, carapace width, plastron length or plastron width. However, hatchlings incubated at 26 °C and 28 °C had wider heads than hatchlings incubated at 24 °C and 31 °C. Incubation period varied inversely with incubation temperature, while the rate of increase in oxygen consumption during the first part of incubation and the peak rate of oxygen consumption varied directly with incubation temperature. The total amount of oxygen consumed during development and hatchling production cost was significantly greater at 24 °C than at 26 °C, 28 °C and 31 °C. Hatchling mass and dimensions and total embryonic energy expenditure was directly proportional to initial egg mass. Accepted: 18 March 1998     

孵化水热环境对渔异色蛇孵化卵和孵出幼体的影响

渔异色蛇卵孵化时能从环境中吸收水分导致质量增加,卵质量的增加与初始卵质量和孵化基质湿度有关。较大幅度的孵化基质湿度变化对孵化期、孵化成功率、胚胎动用孵内物质和能量、孵出幼体的性比、大小和质量无显著影响。孵化期随温度升高而缩短,并显示极强的窝间差异。温度对孵出幼体的性别无影响,但显著影响孵化成功率、胚胎对卵内物质和能量的动用、幼体的大小和质量、躯干和剩余卵黄的质量。孵出幼体总长的两性差异不显著,但雌体体长大于雄体而尾长小于雄体。３２℃不适于孵化渔异色蛇卵,该温度下孵出的幼体躯干发育不良,剩余孵黄较多,尾部均呈畸形,孵化过程中能量转化率较低。２４℃和２６℃中孵出的幼体躯干发育良好,孵化过程中能量转化率较高,各项被测定的幼体特征指标均极相似。     

Do changing moisture levels during incubation influence phenotypic traits of hatchling snakes (Tropidonophis mairii, Colubridae)?

Phenotypic traits (e.g., size, strength, speed) of hatchlings in many reptile species are influenced by hydric conditions in the nest. Previous experiments have focused on comparisons between eggs maintained under constant (but different) conditions, but eggs in natural nests frequently experience strong temporal shifts in soil water content during incubation. Keelback snakes (Tropidonophis mairii) in the Australian wet-dry tropics nest over most of the year, so early nests experience decreasing water availability during development, late nests experience increases, and others (midyear) remain stable in this respect. We mimicked these three conditions and incubated 54 eggs (nine from each of six clutches) in a split-clutch design to maintain the same average water content but with differing trajectories through incubation. The experimental treatments significantly affected the total amount of water taken up by the eggs (and thus final egg mass), but incubation periods were unaffected. Hatchling size but not strength showed minor but statistically significant effects of incubation regimes. The ability of keelback eggs to take up excess water whenever it becomes available (either early or late in development) and to retain it even when conditions change buffers embryogenesis effectively (but not completely) against fluctuations in soil water conditions.     

Gold climates and the evolution of viviparity in reptiles: cold incubation temperatures produce poor-quality offspring in the lizard, Sceloporus virgatus

Evolutionary origins of viviparity among the squamate reptiles are strongly associated with cold climates, and cold environmental temperatures are thought to be an important selective force behind the transition from egg-laying to live-bearing. In particular, the low nest temperatures associated with cold climate habitats are thought to be detrimental to the developing embryos or hatchlings of oviparous squamates, providing a selective advantage for the retention of developing eggs in utero, where the mother can provide warmer incubation temperatures for her eggs (by actively thermoregulating) than they would experience in a nest. However, it is not entirely clear what detrimental effects cold incubation temperatures may have on eggs and hatchlings, and what role these effects may play in favouring the evolution of viviparity. Previous workers have suggested that viviparity may be favoured in cold climates because cold incubation temperatures slow cmbryogenesis and delay hatching of the eggs, or because cold nest temperatures are lethal to developing eggs and reduce hatching success. However, incubation temperature has also been shown to have other, potentially long-term, effects on hatchling phcnotypcs, suggesting that cold climates may favour viviparity because cold incubation temperatures produce offspring of poor quality or low fitness. We experimentally incubated eggs of the oviparous phrynosomatid lizard, Sceloporus virgatus, at temperatures simulating nests in a warm (low elevation) habitat, as is typical for this species, and nests in a colder (high elevation) habitat, to determine the effects of cold incubation temperatures on embryonic development and hatchling phenotypes. Incubation at cold nest temperatures slowed embryonic development and reduced hatching success, but also affected many aspects of the hatchlings' phenotypes. Overall, the directions of these plastic responses indicated that cold-incubated hatchlings did indeed exhibit poorer quality phenotypes; they were smaller at hatching (in body length) and at 20 days of age (in length and mass), grew more slowly (in length and mass), had lower survival rates, and showed greater fluctuating asymmetry than their conspecifics that were incubated at warmer temperatures. Our findings suggest that cold nest temperatures are detrimental to S. virgatus, by delaying hatching of their eggs, reducing their hatching success, and by producing poorer quality offspring. These negative effects would likely provide a selective advantage for any mechanism through which these lizards could maintain warmer incubation temperatures in cold climates, including the evolution of prolonged egg retention and viviparity.     

Phenotypic variation in an oviparous montane lizard (Bassiana duperreyi): the effects of thermal and hydric incubation environments

Recent studies have shown that incubation temperatures can profoundly affect the phenotypes of hatchling lizards, but the effects of hydric incubation environments remain controversial. We examined incubation-induced phenotypic variation in Bassiana duperreyi (Gray, 1938; Sauria: Scincidae), an oviparous montane lizard from south-eastern Australia. We incubated eggs from this species in four laboratory treatments, mimicking cool and moist, cool and dry, warm and moist, and warm and dry natural nest-sites, and assessed several morphological and behavioural traits of lizards after hatching. Incubation temperature influenced a lizard's hatching success, incubation period, tail length and antipredator behaviour, whereas variation in hydric conditions did not engender significant phenotypic variation for most traits. However, moisture affected incubation period slightly differently in males and females, and for a given snout-vent length moisture interacted weakly with temperature to affect lizard body mass. Although incubation conditions can substantially affect phenotypic variation among hatchling lizards, the absence of strong hydric effects suggests that hatchling lizards react less plastically to variation in moisture levels than they do to thermal conditions. Thus, our data do not support the generalization that water availability during embryogenesis is more important than temperature in determining the phenotypes of hatchling reptiles.     

Communal egg-laying behaviour and the consequences of egg aggregation in the brown anole (Anolis sagrei)

Communal nesting is a behaviour that involves multiple females laying eggs in the same nest or nesting site. This behaviour may be a consequence of a shortage of preferable nest sites (constraint hypothesis) or an adaptation generated by fitness benefits associated with egg aggregation (adaptive hypothesis). Experimental tests of these hypotheses require information about maternal nest site choice and its fitness consequences. To address these, we studied a lizard (brown anole; Anolis sagrei) that produces single-egg clutches, but often aggregates eggs in nest sites. In a lab study, females were given the option of nesting in (a) soil previously used as nest substrate vs. fresh soil and (b) soil with eggshells vs. without eggshells. We also experimentally examined the effects of egg aggregation by incubating eggs singly, in groups of four, and in groups of nine. We recorded egg surface temperature, water uptake, and hatchling morphology. Females were more likely to nest in pots with used soil and with eggshells than in pots with fresh soil or without eggshells. We observed no effects of egg aggregation on egg survival, egg temperature, or most measures of hatchling morphology. However, singly-incubated eggs absorbed more water than eggs incubated in the four and nine egg aggregations and this resulted in offspring with greater body condition (i.e. heavier for their length) at hatching. The behavioural experiment demonstrates that females actively choose nest sites that have been used previously (as expected under the adaptive hypothesis), but the egg-aggregation experiment shows no benefits to offspring based on the variables measured. Thus, results of the behaviour study support the adaptive hypothesis; however, results from our egg-incubation study do not. Likely, the adaptive and constraint hypotheses are not mutually exclusive, and a diversity of factors influence the evolution of communal nesting behaviour.     

Effects of egg size on Double-crested Cormorant (Phalacrocorax auritus) egg composition and hatchling phenotype

Maternal investment of yolk and albumen in avian eggs varies with egg mass and contributes to variation in hatchling mass. Here we use the natural variation in mass and composition of Double-crested Cormorant (Phalacrocorax auritus) eggs to examine consequences of variation in yolk and albumen mass on hatchling phenotype. The Double-crested Cormorant, a large bird with altricial young, lays eggs ranging in mass from 40 to 60 g and containing an average of 82% albumen and 18% yolk. Variation in Cormorant egg mass arises primarily from variation in the amount of albumen and water in the eggs; yolk mass remains relatively constant, contributing only 10% to egg mass variation. Likewise, variation in hatchling mass correlates positively with albumen mass and albumen solids contribute to hatchling dry mass. Thus, variation in Cormorant egg mass is primarily the result of variation in the amount of egg albumen, which contributes most to variation in hatchling mass. Similarities in egg composition of altricial birds, along with data presented here, suggest that variation in hatchling mass of all altricial birds may depend most on the amount of egg albumen, unlike species with precocial young that hatch from eggs with substantially more yolk.     

The effects of thermal and hydric environments on hatching success, embryonic use of energy and hatchling traits in a colubrid snake, Elaphe carinata.

We examined the effects of thermal and hydric environments on hatching success, the embryonic use of energy and hatchling traits in a colubrid snake, Elaphe carinata. The eggs were incubated at four temperatures ranging from 24 to 32 degrees C on substrates with water potentials of 0 and -220 kPa using a 4x2 factorial design. Both thermal and hydric environments affected the water exchange between eggs and their surroundings. Eggs incubated in wetter substrates gained mass throughout the course of incubation, whereas eggs in drier substrates gained mass during the first half of incubation and lost mass thereafter. Hatching success was noticeably higher at 26 and 30 degrees C than at 24 and 32 degrees C, but among treatments, differences in hatching success were not significant. Temperature significantly affected the duration of incubation and most hatchling traits examined. Deformed hatchlings were found in all temperature treatments, with more deformities observed at 32 degrees C. Hatchlings from eggs incubated at different temperatures differed in wet body mass, but the differences stemmed mainly from variation in water contents. Embryos at different temperatures completed development at nearly the same expenditure of energy and catabolized nearly the same amount of lipids, but hatchlings from different temperatures differed in the development condition of carcass at hatching. Hatchlings from eggs incubated at 26 degrees C were larger in SVL than those from other higher or lower incubation temperatures, characteristically having larger carcasses; hatchlings from 32 degrees C eggs were smaller in SVL and had smaller carcasses but larger residual yolks than those from lower incubation temperatures. Hatchlings from eggs incubated at 24 degrees C were shorter in tail length but greater in size (SVL)-specific body wet mass than those from higher incubation temperatures. Within the range from -220 to 0 kPa, the substrate water potential did not affect hatching success, the embryonic use of energy and all hatchling traits examined, and the effects of temperature were independent of the effects of substrate water potential. Therefore, our data add evidence showing that embryonic development in reptiles with pliable-shelled eggs is relatively insensitive to variation in hydric environments during incubation.     

EGG COMPOSITION AND HATCHLING PRECOCITY IN SEABIRDS*

This paper presents analyses of the composition (shell, yolk, albumen) and of the quality (lipid, protein and water contents) of yolk and albumen of eggs of 25 species of seabirds belonging to the Sphenisciformes, Procellariiformes, Pelecaniformes, and Lari of the Charadriiformes. These data are compared with those published for species in the same orders and suborders, and the combined seabird data are compared with published data for non-seabirds. Variation in the proportionate weight of eggshell is not related to hatchling precocity but is probably due to adaptive differences in nest substratum and the agility of the incubating birds. Lipid and protein contents of yolk, and water content of albumen, are uniform in eggs of all examined species. Protein levels of albumen and water content of yolk and of entire egg contents vary greatly between taxa. Contrary to previous claims there is no simple relationship between egg composition and hatchling precocity. Differences in egg composition will probably only be clearly understood when the nutritional needs of the embryo, and of the hatchling in the time before it regularly obtains food, are known.     

Effects of seasonally varying hydric conditions on hatchling phenotypes of keelback snakes (Tropidonophis mairii, Colubridae) from the Australian wet-dry tropics

Does water availability during incubation significantly affect the phenotypes of hatchling reptiles in natural nests? Two obstacles to obtaining any general answer to this question are the scarcity of studies on tropical species, and the difficulty of comparing experimental treatments to actual hydric conditions in nature. We used a split‐clutch design to incubate 102 eggs (eight clutches) of a colubrid snake species (the keelback, Tropidonophis mairii), from a floodplain in the Australian wet‐dry tropics. This species breeds over most of the year, and highly seasonal rainfall regimes generate strong shifts in water content of the soil over this period. We measured soil water content in a natural nest, and incubated eggs in both soil and vermiculite (the usual medium for experimental studies) at a range of water contents. These calibration trials let us compare our experimental ‘wet’ and ‘dry’ incubation treatments to conditions in natural nests, in terms of actual water uptake by eggs. Hatchlings from dry incubation were unable to resorb their desiccated yolk and thus were smaller (17% in mass, 12% in body length) than their siblings from moist substrates. Incubation conditions also affected the hatchlings’ muscular strength and locomotor speed: even at the same body length, dry‐incubated hatchlings were weaker and slower than their wet‐incubated siblings. Incubation moisture affected strength differently in males and females. We conclude that seasonal variation in water content of the soil in natural nests can generate strong phenotypic variation in hatchling snakes. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 76 , 339–347.     

孵化温度对赤链蛇胚胎代谢和幼体行为的影响

研究了赤链蛇(Dinodon rufozonatum)在孵化过程中卵的生长、孵化期、胚胎代谢和孵出幼体行为表现的热依赖性。结果显示:孵化温度对孵化期、卵增重、孵化过程中消耗的总能量和孵出幼体的运动表现有显著影响,但不影响胚胎代谢率、孵化成功率和幼体吐信频次。孵化期随着孵化温度的升高而缩短,孵化过程中,24℃终末卵重和胚胎代谢率显著大于30℃,而27℃与其他两个温度没有差异;27℃孵出幼体游速较24℃快,30℃孵出幼体与其他两个温度孵出幼体的游速无显著差异。上述结果显示:24—30℃是赤链蛇适合的孵化温度范围,与赤链蛇所处的生境温度相近。     

Changes in mass of eggs of softshell turtless (Trionyx spiniferus) incubated under hydric conditions simulating those of natural nests

Rigid-shelled eggs of softshell turtles ( Trionyx spiniferus ) incubated at 29C under hydric conditions simulating those at the interior of natural nests (i.e. eggs not contacting the substrate) declined in mass during incubation owing to the continuous escape of water vapour to air trapped inside the chambers, with eggs incubated in relatively dry chambers declining more in mass than eggs held in relatively wet chambers. Eggs held under hydric conditions approximating those at the periphery of natural nests (i.e. eggs partly buried in the substrate) also declined in mass during incubation, but at lower rates than characterized eggs not contacting the substrate, indicating that absorption of liquid water from the substrate compensated for part of the water vapour escaping to the air inside the chambers. There was no relationship between hatching success and either the position of eggs in the chambers or the wetness of substrates, nor was there any variation among experimental groups in the size of hatchlings. Thus, development of embryos of Trionyx spiniferus seems not to be closely coupled with the hydric environment of the nest, a situation contrasting with that of embryos of those species of turtles producing pliable-shelled eggs.     

Larger eggs at lower water temperature as a measure to assure effective hatchling size in the landlocked form of Ayu, <Emphasis Type="Italic">Plecoglossus altivelis</Emphasis>

Egg size is a critical life-history trait in which maternal investment is optimized to maximize maternal fitness. The adaptive significance of variable egg size among spawning groups of Ayu (Plecoglossus altivelis) landlocked in the Lake Biwa system was examined through field investigations and rearing experiments. Observed egg size variations were explained by the water temperature around spawning grounds established near the mouths of inlet streams. Two typical streams with different incubation temperatures showed similar maternal body sizes and hatchling sizes, but eggs attached to the stream bed were larger in the colder stream. An experiment that used eggs from a single clutch showed that a smaller hatchling size was obtained with a lower incubation temperature, indicating that the effect of differences in egg size on hatchling size can be canceled out by variations in incubation temperature. In general, larvae that are less than a certain threshold of effective body size are not expected to be assured of early success among conspecifics competing for foods. It is proposed that environments in which the incubation temperature varies favor variability in egg size to ensure that sufficient food is accessible to larvae.