Fungal parasitism in a freshwater copepod: Components of the interaction between Aphanomyces and Boeckella

The transmission of Aphanomyces infection among carriers (host females), host mortality, recovery from infection, and stages in the infection process were investigated in Boeckella dilatata (Copepoda: Calanoida) at 15°C in the laboratory. Over a 4-week period, 57.9% female B. dilatata became infected after being paired with carrier females and 3.8% lost their infection hyphae. Mortality was higher among carrier females (41%) than noncarriers (18.2%) but may be related to the age of the females. There was no difference in body size or clutch size between carrier and noncarrier females. However, host females carried clutches for shorter periods and had correspondingly longer intervals between clutches than did noncarriers. The time course of development of infection at 15°C is described.     

Recruitment and mortality of the temperate coral Cladocora caespitosa: implications for the recovery of endangered populations

Long-lived species are characterized by low recruitment and mortality. In these species, longevity buffers low recruitment, but when catastrophic disturbances alter mortality, recruitment becomes critical for population recovery. In this study, we assessed basic biological traits—recruitment, post-settlement growth, and the mortality of juvenile corals—and related these factors to the adult mortality of one of the most important populations of the Mediterranean reef-building coral Cladocora caespitosa over a period of 6 yr. Adult mortality and recruitment rates were low (~1 % and 0.30 recruits m^?2 yr^?1, respectively), whereas the juvenile colony mortality was comparatively high (29 % in the smallest size-class, <5 polyps). The low recruitment rates will hardly balance the recurrent climate-related mortality that has affected this population. Conservation plans and inclusion in the protection lists are urgently needed, given the escalating threats and slow dynamics of this species.     

Survival of Greater Sage-Grouse Chicks and Broods in the Northern Great Basin

ABSTRACT Reduced annual recruitment because of poor habitat quality has been implicated as one of the causative factors in the range-wide decline of sage-grouse (Centrocercus urophasianus) populations since the 1950s. Because chick and brood survival are directly linked to annual recruitment and may be the primary factors that limit sage-grouse population growth, we estimated 28-day survival rates of radiomarked chicks and broods from 2000 to 2003. We examined relationships between survival and several habitat variables measured at brood sites, including food availability (insects and forbs); horizontal cover of sagebrush, grasses, and forbs; and vertical cover of sagebrush and grass. We monitored 506 radiomarked chicks from 94 broods; chick survival was 0.392 (SE = 0.024). We found evidence that both food and cover variables were positively associated with chick survival, including Lepidoptera availability, slender phlox (Phlox gracilis) frequency, total forb cover, and grass cover. The effect of total grass cover on chick survival was dependent on the proportion of short grass. The hazard of an individual chick's death decreased 8.6% (95% CI = −1.0 to 18.3) for each percentage point increase in total grass cover when the proportion of short grass was >70%. Survival of 83 radiomarked broods was 0.673 (SE = 0.055). Lepidoptera availability and slender phlox frequency were the only habitat variables related to brood survival. Risk of total brood loss decreased by 11.8% (95% CI = 1.2–22.5) for each additional Lepidoptera individual and 2.7% (95% CI = −0.4 to 5.8) for each percentage point increase in the frequency of slender phlox found at brood sites. Model selection results revealed that temporal differences in brood survival were associated with variation in the availability of Lepidoptera and slender phlox. Years with high brood survival corresponded with years of high Lepidoptera availability and high slender phlox frequency. These foods likely provided high-quality nutrition for chicks during early growth and development and enhanced survival. Habitat management that promotes Lepidoptera and slender phlox abundance during May and June (i.e., early brood rearing) should have a positive effect on chick and brood survival in the short term and potentially increase annual recruitment.     

The thermal environment of nests of the Australian stingless bee, Austroplebeia australis

The greatest diversity of stingless bee species is found in warm tropical regions, where brood thermoregulation is unnecessary for survival. Although Austroplebeia australis (Friese) naturally occurs in northern regions of Australia, some populations experience extreme temperature ranges, including sub-zero temperatures. In this study, the temperature was monitored in A. australis colonies’ brood chamber (n = 6) and the hive cavity (n = 3), over a 12-month period. The A. australis colonies demonstrated some degree of thermoconformity, i.e. brood temperature although higher correlated with cavity temperature, and were able to warm the brood chamber throughout the year. Brood production continued throughout the cold season and developing offspring survived and emerged, even after exposure to very low (?0.4 °C) and high (37.6 °C) temperatures. Austroplebeia australis, thus, demonstrated a remarkable ability to survive temperature extremes, which has not been seen in other stingless bee species.     

Mediterranean warming triggers seagrass (Posidonia oceanica) shoot mortality

Rapid warming of the Mediterranean Sea threatens marine biodiversity, particularly key ecosystems already stressed by other impacts such as Posidonia oceanica meadows. A 6‐year monitoring of seawater temperature and annual P. oceanica shoot demography at Cabrera Archipelago National Park (Balearic Islands, Western Mediterranean) allowed us to determine if warming influenced shoot mortality and recruitment rates of seagrasses growing in relative pristine environments. The average annual maximum temperature for 2002–2006 was 1 °C above temperatures recorded in 1988–1999 (26.6 °C), two heat waves impacted the region (with seawater warming up to 28.83 °C in 2003 and to 28.54 °C in 2006) and the cumulative temperature anomaly, above the 1988–1999 mean annual maximum temperature, during the growing season (i.e. degree‐days) ranged between 0 °C in 2002 and 70 °C in 2003. Median annual P. oceanica shoot mortality rates varied from 0.067 year^?1 in 2002 to 0.123 year^?1 in 2003, and exceeded recruitment rates in all stations and years except in shallow stations for year 2004. Interannual fluctuations in shoot recruitment were independent of seawater warming (P>0.05). P. oceanica meadows experienced a decline throughout the study period at an average rate of ?0.050±0.020 year^?1. Interannual variability in P. oceanica shoot mortality was coupled (R²>0.40) to seawater warming variability and increasing water depth: shoot mortality rates increased by 0.022 year^?1 (i.e. an additional 2% year^?1) for each additional degree of annual maximum temperature and by 0.001 year^?1 (i.e. 0.1% year^?1) for each accumulated degree water temperature remained above 26.6 °C during the growing season. These results demonstrate that P. oceanica meadows are highly vulnerable to warming, which can induce steep declines in shoot abundance as well indicating that climate change poses a significant threat to this important habitat.     

Annual variability in the life-history characteristics of brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus) in a subalpine Norwegian lake

The annual variability in growth and life history traits of brown trout (Salmo trutta L.) and Arctic charr (Salvelinus alpinus (L.)) in Lake Atnsjøen, a Norwegian subalpine lake, was studied over a period of 13 years (1985–1997). The extent to which life-history characteristics recorded on one occasion can be regarded as representative for the population was explored. We found inter-cohort variation in growth for both species; estimates of asymptotic length (L _∞) in ten cohorts ranged between 225–305 mm (CV = 10.5%) for brown trout and 273–301 mm (CV = 4.1%) for Arctic charr. However, this variation was much lower than inter-population variation for brown trout based on single samples from 169 populations (CV = 24.6%). In Lake Atnsjøen, annual growth increment correlated highly with the number of days warmer than 7?°C (R ²=0.60–0.89) for brown trout, and days warmer than 10?°C (R ²=0.40–0.58) for Arctic charr. Females of Arctic charr were younger at sexual maturity than males, while no such difference was found in brown trout. Generally speaking, early maturing individuals of both species grew faster, particularly from age-2 and onwards, than immature individuals. Early maturing individuals, however, were smaller at maturity than those maturing one year older. Age and size at maturity were significantly correlated with asymptotic lengths only in Arctic charr females.     

Growth, mortality and stock assessment of kelee shad, Hilsa kelee (Fam: Clupeidae) in the coastal waters of Pakistan

Growth of kelee shad, Hilsa kelee, in the coastal waters of Pakistan was estimated from the lengthfrequency samples. The von Bertalanffy growth equation was L _t = 23.10 (1 ? exp (?0.94(t + 0.18))). Estimated parameters of total mortality (Z), natural mortality (M) and fishing mortality (F) were 2.08 year^?1, 1.78 year^?1, and 0.30 year^?1 respectively. The length-at-first capture was Lc = 10.88 cm. Biomass per recruitment (B/R) and yield per recruitment (Y′/R) were 0.87 and 0.031 respectively. The annual exploitation rate was U = 0.12. The exploitation ratio for maximum yield per recruit E _max = 0.73 and fishing mortality for maximum yield per recruit F _max = 1.52; biological reference point F _opt = 0.89 year^?1 and F _limit = 1.18 year^?1. Present estimations showed that the natural mortality was higher than fishing mortality in Hilsa kelee, indicating that the state of the stock is sustainable and the fishery of kelee shad should not be increased beyond current levels in the coastal waters of Pakistan.     

Preparation,properties and crystal structures of nickel(II) complexes with acyclic schiff bases derived from 2,6-diformyl-4-chlorophenol and 1,5-diamino-3-thiapentane or 3,3′-diamino-N-methyl-dipropylamine

Nickel(II) complexes with the compartmental Schiff bases derived from 2,6-diformyl-4-chlorophenol and 1,5-diamino-3-thiapentane (H₂L¹) or 3,3′-diamino-N-methyl-dipropylamine (H₂L²) were synthesized, and the crystal structures of [Ni(L¹)- (py)₂] and [Ni(L²)(dmf)]·H₂0 were determined by X-ray crystallography.Ni(L¹)(py)₂ is monoclinic, space group C2/c, with a= 18.457(6), b = 11.116(7), c= 16.098(6) Å, and β = 115.79(5)°; D_c = 1.49 g cm⁻³ for Z = 4. The structure was refined to the final R of 6.9%. The molecule has C₂ symmetry. The nickel atom is six-coordinated octahedral. Selected bond lengths are: NiO 2.04(1) Å, NiN (L¹) 2.08(1) Å, NiN(py) 2.17(1) Å.[Ni(L²)(dmf)]·H₂O is monoclinic, space group P2₁/n, with a = 17.329(6), b = 13.322(7), c = 12.476(7) Å and β = 95.43(5)°; D_c = 1.45 g cm⁻³ for Z = 4. The structure was refined to the final R of 5.1%. The nickel atom is bonded in the octahedral geometry to the bianionic pentadentate ligand L² and to one molecule of dimethylformamide. Selected bond lengths are: NiO (charged) 2.063(3) Å (mean value), NiO (neutral) 2.120(3) Å, NiN (planar) 2.050(3) Å (mean value), NiN (tetrahedral) 2.177(3) Å.     

The pathogenicity of an entomopoxvirus fromOthnonius batesi [Col.: Scarabaeidae] and its possible use as a control agent

In a natural population ofOthnonius batesi Oll. at Glen Innes, N.S.W., 4.0% of larvae in 1972 and 2.2% in 1973 were exhibiting symptoms of virus infection, whilst 0.8% of pupae and adults from the same population were infected in 1972. These figures, and field observations of infected larvae, suggested that the pathogenicity of the virus was low. In laboratory experiments withO. batesi the infection had little effect on mortality, and no significant effect on duration of the first instar, food intake, or larval growth. The vast accumulation of virus in the fat body probably results in mortality prior to, or during, the pupal period. Temperature had a marked effect on both % infection (optimum 30°C) and production of viral spheroids (optimum 25°C). Very little viral development occurred below 20°C. In a host range study onlyO. batesi, Rhopaea verreauxi Blanch. andR. morbillosa Blkb. were infectedper os. The possible use of the virus as a control agent is discussed.     

An unusual hermaphrodite reproductive trait in the Antarctic brooding bivalve Lissarca miliaris (Philobryidae) from the Scotia Sea, Southern Ocean

The Antarctic marine environment is extreme in its low temperatures and short periods of primary productivity. Invertebrates must therefore adapt to maximise reproductive output where low temperature and limited food slow larval development. Brooding is a common reproductive trait in Antarctic marine bivalves; larval development occurs within the mantle cavity, and larvae are released as fully developed young. Lissarca miliaris is a small, short-lived, shallow-water brooding bivalve of circum-Antarctic distribution and found most abundant in the sub-Antarctic Magellan Region and islands of the Scotia Arc. Here, an unusual hermaphrodite reproductive trait is described for L. miliaris from King George Island (62°14′S, 58°38′W) and Signy Island (60°42′S, 45°36′W), Antarctica, using histological and dissection techniques. Specimens demonstrate simultaneous and sequential hermaphrodite traits; male and female gonads develop simultaneously, but the production of oocytes is reduced while testes are ripe. Functional females are more abundant in specimens above 3 mm shell length, although male reproductive tissue persists and functional males are found in all size classes. The number of previtellogenic oocytes produced by far exceeds the number of oocytes extruded and brooded, which may indicate an ancestral link to a planktotrophic past. Hermaphroditism in L. miliaris maximises reproductive efficiency in a short-lived species, in which the female’s capacity to brood its young is limited, and demonstrates a specialised adaptation to a cold stenothermal and food-limited environment prevailing in the Southern Ocean.     

Food limitation impacts life history of the predatory cladoceran Bythotrephes longimanus, an invader to North America

North American invasions of the predatory cladoceran Bythotrephes longimanus have resulted in declines in native zooplankton abundance, species richness, and diversity. In the field, population maxima of Bythotrephes are positively correlated to those of their zooplankton prey. To test the hypothesis that increased prey availability enhances Bythotrephes fitness, we reared Bythotrephes in the laboratory on three mixed-species prey densities (equivalent to 15, 30, and 45 prey organisms day^?1; designated “low,” “medium,” and “high” food treatments, respectively) over 22 days at 21°C. Bythotrephes consumed the daily equivalent of 9, 14, and 22 prey organisms at the low, medium, and high food densities. Smaller, slower prey were most often selected. Indeed, with increasing prey density, Bythotrephes’ predation rates increased, resulting in significantly higher population growth rates, net reproductive rates, growth, and first brood clutch and offspring sizes; significantly faster generation times; and shorter maximum life spans. We propose that the positive relationship between Bythotrephes population maxima and prey seen in the field is largely due to increased predation rates by Bythotrephes when prey abundance is high and the fitness benefits that ensue. Our findings may be useful for Bythotrephes risk and impact assessments.     

Embryonic encapsulation and maternal incubation: Requirements for survival of the early stages of the estuarine gastropod Crepipatella dilatata

During their reproductive period, females of Crepipatella dilatata deposit their embryos in capsules that they then brood in the pallial cavity until juveniles emerge several weeks later, after passing through a transient veliger “larval” stage. Artificially excapsulated veligers of this species experimentally exposed to a wide range of salinities (5, 10, 15, 20, 25, and 30 psu) for six hours showed reduced activity at salinities of 15 and 20 psu, whereas encapsulated veligers exposed to those same salinities showed no reduction of activity. Artificially excapsulated veligers showed high mortality at salinities of 5 and 10 psu; encapsulated embryonic stages also showed high mortalities at 5 psu and serious sublethal effects at 10 psu in tests excluding maternal protection, showing that encapsulation alone does not provide complete protection from low salinity stress. Natural tidal cycles in the Quempillén River estuary also reduced embryonic survival at salinities of ≤ 10 psu when the capsules were exposed without maternal protection. In contrast, encapsulated embryos protected by their mothers survived well regardless of the salinity to which they were exposed, under both natural and laboratory-simulated estuarine tidal cycles. C. dilatata are able to develop in the estuary only because of maternal protection, since salinity levels in this environment sometimes decline to as low as 7 psu. Successful embryonic development in this estuary reflects the capacity of C. dilatata adults to detect dangerously low salinity levels and then seal themselves off from the environment for up to 50 hrs (O. Chaparro pers. obs.) when the salinity drops below 22.5 psu, allowing salinity to remain above this level within the pallial cavity despite continued salinity declines in the surrounding seawater.     

Population dynamics, growth and reproduction of Corophium insidiosum (Crustacea: Amphipoda) at low salinities in Monolimni lagoon (Evros Delta, North Aegean Sea)

Distribution, population dynamics, growth and aspects of reproductive biology of Corophium insidiosum were investigated in Monolimni lagoon. Samples were collected in July 1997 (at 30 psu S) and during February 1998–May 1999 (at 0.1–5.7 psu S). Corophium insidiosumwas almost exclusively found in the outer part of the lagoon, which showed a higher water renewal rate. Population density gradually decreased during winter and spring, when salinity was lower than 1 psu and the amphipod finally vanished from the lagoon. Salinity increase during summer (1.2–5.7 psu) was followed by the re-occurrence of C. insidiosum with a time lag of 2–3 months. Population density increased in autumn and peaked in early winter at salinities 1.6–4.2 psu. Three cohorts appeared in the population during September 1998–March 1999. Breeding activity peaked in early autumn (14–21?°C, 4 psu S) and ceased after December (2–6.5?°C, ¡1.5 psu S). The preponderance of females in the large size classes resulted in a female- biased sex ratio in the whole population. The population showed a growth rate of 7.5–11.2 μm d^?1 being faster in autumn (9–21?°C, 3–4 psu S) than in winter (2–12?°C, 0.2–3 psu). An exponential relation existed between body length and cephalic length or dry body weight, while brood size was directly related to body length. Mean brood size was small (4.96 early embryos) and egg loss during development high (53%), possibly as a consequence of low salinities.     

Group recruitment in a thermophilic desert ant, Ocymyrmex robustior

Thermophilic desert ants—Cataglyphis, Ocymyrmex, and Melophorus species inhabiting the arid zones of the Palaearctic region, southern Africa and central Australia, respectively—are solitary foragers, which have been considered to lack any kind of chemical recruitment. Here we show that besides mainly employing the solitary mode of food retrieval Ocymyrmex robustior regularly exhibits group recruitment to food patches that cannot be exploited individually. Running at high speed to recruitment sites that may be more than 60 m apart from the nest a leading ant, the recruiter, is followed by a loose and often quite dispersed group of usually 2–7 recruits, which often overtake the leader, or may lose contact, fall back and return to the nest. As video recordings show the leader, while continually keeping her gaster in a downward position, intermittently touches the surface of the ground with the tip of the gaster most likely depositing a volatile pheromone signal. These recruitment events occur during the entire diurnal activity period of the Ocymyrmex foragers, that is, even at surface temperatures of more than 60 °C. They may provide promising experimental paradigms for studying the interplay of orientation by chemical signals and path integration as well as other visual guidance routines.     

A resolution of glutamic acid

N-Carbobenzoxy-dl-glutamic acid was asymmetrically hydrolyzed by hog kidney acylase to l-glutamic acid ([α]_D = +31.9 °) and N-carbobenzoxy-d-glutamic acid ([α]_d = + 7.5 °). On catalytic hydrogenation of the latter, d-glutamic acid ([α]_d = ?31.5 °) was obtained readily and in high yield.     

The effects of temperature and food on copepodite development,growth and reproduction in three species of Boeckella (Copepoda; Calanoida)

Three species of Boeckella (B. triarticulata, B. dilatata and B. hamata) were reared from copepodite I to adult at three naturally fluctuating food levels and three temperatures in a 3 × 3 × 3 factorial design. Development times, lengths, times to produce the first clutch, clutch sizes, egg voumes, interclutch times, infertile clutches, total egg production, total reproductive time and mortality were measured. Development times for the three species were surprisingly similar but mortality differed among the treatments. The three species differed in their reproductive strategies. B. triarticulata, the largest species, produced a few large clutches. B. dilatata, the smallest species, produced many small clutches and invested the most energy per egg. B. hamata produced larger clutches than B. dilatata but not as many. B. hamata was the most tolerant of low, food levels and exhibited the most plasticity across the treatments. Thus, greater plasticity in life history characters is associated with survival in a wider-range of conditions.     

Reproductive biology of the invasive Asian freshwater leech Barbronia weberi (Blanchard, 1897)

Barbronia weberi were collected from Hydrilla verticillata purchased from aquarium suppliers in Melbourne, Australia and additional B. weberi were obtained from aquatic plants in Canberra, ACT, Australia. Adult leeches fed Tubifex sp. ad libitum successfully reproduced under laboratory conditions (21 ± 5?°C ). Reproductively mature B. weberi produced cocoons every week for up to three months at which time the adults began to senesce. Cocoons contained one to five (barx=2.41±0.78 SD, N=58) eggs with juveniles leaving the cocoon within 27 ± 3.4 SD days (N=13) of cocoon production. Juvenile B. weberi reached reproductive maturity within four months (N=7) of hatching and had a maximum growth rate of 1.10 mm² d^?1 once they left the cocoon at around 30 days. Individual egg volume (r=0.63, scaling exponent = 1.21) and cocoon volume (r=0.65, scaling exponent = 1.24) showed a significant (P<0.001) and nearly isometric relationship when scaled with maternal body size. Because this species can grow rapidly and produce a large number of eggs over a short period of time and can piggyback with plant species and travel through the aquarium trade, there is potential for B. weberi to rapidly invade new localities.     

Life at a different pace: Annual itineraries are conserved in seasonal songbirds

The duration of life history state (LHS) reflects the adaptive strategy a species has evolved to cope with a changing environment. Inhabitants at different latitudes may thus have significant differences in the rates of metabolic and physiological processes underlying LHSs. Birds, in order to maximize their fitness in the environment in which they live, seasonally switch from one LHS to another during the year. The present study investigated whether an annual itinerary of a species would determine its rate of reaction to inductive long days. We compared the photoinduced cycles of changes in body mass and testes, as indices of migratory and reproductive LHSs, between two long day breeding species, the migratory redheaded bunting and non-migratory Indian weaverbird. Changes in body mass and testis size were measured in photosensitive buntings and weaverbirds (n = 7 each) on short days (LD 8:16) subjected first to 0.5 h weekly light increments until the light period was 13 h per day, and then maintained on LD 13:11 for another 32 weeks. A similar observation was recorded on a group of buntings (n = 14) and weaverbirds (n = 9) maintained on increasing natural day lengths (NDL; Lucknow, 26°55' N, 80°59' E) for 47 weeks. As predicted, the rates of induction of seasonal cycles under an identical inductive photoperiod were significantly faster in temperate buntings with five annual LHSs than in the subtropical weaverbirds with three annual LHSs. This suggests that annual itineraries of songbirds with which they may have evolved with at their breeding latitudes, determine their response to the external photoperiodic environment.     

The influence of temperature variations and thermal pollution on various aspects of the biology of the prawn Palaemon pacificus (Stimpson)

The influence of different temperatures 10, 15, 20, and 25°C on the food consumption, growth, moulting rate, and respiration of Palaemon pacificus (Stimpson) from Langebaan Lagoon, west coast of South Africa, was studied under laboratory conditions. At 10°C mortality was high so that food consumption and moulting rate could not be determined as these were very low. At higher temperatures, food consumption was found to be temperature dependent, the rate at 25°C being twice that at 15°C. Growth rate was most favourable at 25°C. At 28°C growth rate was lower than at 20°C but higher than at 15°C. The intermoult period was 17 days at 15°C, and 11 and 10 days at 20, and 25°C, respectively. It seems that from an energetic point of view, 25°C is the most favourable temperature for P. pacificus. Several indices of growth efficiency at different temperatures are presented. The appearance of this prawn in South African west coast localities such as Langebaan during the summer and its disappearance during winter, can be explained by its temperature preferences. The possibility that thermal pollution from a nuclear power station may be beneficial to this prawn, is discussed.     

Life history and population biology of the giant ostracod Leuroleberis zealandica (Baird, 1850) (Myodocopida)

A population of this large myodocopid ostracod was studied over 2 yr by random core-sampling of the medium sand bottom at Kaikoura, New Zealand. Leuroleberis zealandica (Baird) passes through seven instars, it is sexually mature only in the final instar and sexes were distinguishable from instar IV. Males and females were equally abundant except in the final instar when the morphologically distinct males were rarely found. The population consists of three cohorts at any one time and each cohort appears to split into fast- and slow-growing individuals during the sixth instar resulting in life times of 1.8–2.0 and 2.7–3.1 yr, respectively. Females produce only one brood of 37 eggs on average per life time that are carried throughout the 5–6 month development period during which there is no loss of embryos. Recruitment is discrete with most broods released in midsummer when the population density may exceed 350·0.1 m^?2. A second lesser recruitment may occur in early spring in some years. Hatched juveniles released from the female grow rapidly to instar IV within 6 months and, although size increments at each moult are proportionally similar, intermoult periods tend to increase with size with some variation according to seasonal growth rates. Instar life tables constructed from instar density data showed a large difference in the frequency of embryos initiating each cohort, very different mortalities at recruitment between cohorts, and that the mortality rates between instars I and VI of different cohorts appear to be independent of density. The biology of Leuroleberis is compared with the few published accounts of myodocopid biology. In addition, several aspects of the biology of myodocopids are reviewed. These include numbers of instars in different taxa, within-instar sexual size disparities, numbers of broods per female life time, egg and brood sizes in relation to adult female size in various taxa, and the question of post-adult moulting.