Palatability of autotrophic dinoflagellates to newly hatched larval crabs

To determine the general palatability of autotrophic dinoflagellates to newly hatched crab larvae and whether there are taxonomic, predator/prey size relationships, or toxicity components to their ability to discriminate among dinoflagellates, larvae of six species of crabs from two families were fed 16 species/strains of dinoflagellates from three orders. Dinoflagellate cell length ranged from 18 to 50 µm, and toxic and non-toxic species/strains were included. Experiments measuring incidence of prey ingestion, grazing rates on individual constituents of selected prey combinations, and development on one toxic species shown to be readily ingested were conducted between 2000 and 2002. Thirteen of sixteen dinoflagellates were palatable to larvae, with no consistent pattern of prey discrimination based on taxonomic affinity, toxicity, larval hatching season, or predator/prey size relationships. Although the three dinoflagellates not ingested were toxic, three other toxic species/strains were ingested, with accelerated mortality occurring in the one case. Ingestion of non-favored prey occurred only at very low rates when mixed with readily ingested prey, indicating selectivity. Larvae hatching in winter generally ingested dinoflagellates as readily as did zoeae hatched in spring and summer. Newly hatched larvae ingested a wide variety of dinoflagellates, while discriminating among related species. Such discrimination will not always prevent larval ingestion of prey that will result in mortality.Communicated by J.P. Grassle, New Brunswick     

Effects on larval crabs of exposure to algal toxin via ingestion of heterotrophic prey

We tested whether ingesting toxic algae by heterotrophic prey affected their nutritional value to crab larval predators, using toxic algal strains that are either ingested directly by larval crabs or rejected by them. Ingestion of toxic strains of the dinoflagellates Alexandrium andersoni and A. fundyense by the rotifer Brachionus plicatilis was confirmed. Rotifers having ingested either algal type for five days were fed to freshly hatched larvae of three crab species, with larval survival and stage durations determined. For both algal/rotifer treatments in all three crab species, larvae fed algae directly died during the first zoeal stage, while those fed rotifers that had been fed either algal strain survived to the experiment’s end (zoeal stage 3). Survival was lower, and stage duration longer, for larvae fed rotifers cultured on toxic algae when compared to those fed non-toxic algae. The role of toxic algae in the planktonic food web may be influenced by its direct or indirect ingestion by larval crabs.     

Experimental evidence of chemokinesis in newly hatched cod larvae (Gadus morhua L.)

The swimming behaviour of laboratory-reared newly hatched cod larvae (Gadus morhua L.) was observed in a control solution of artificial seawater and in seven solutions, each with a different concentration of arginine (10⁹ to 10^-3 M). The behaviour of 20 larvae was analysed in each of the eight solutions; the individual observation time was 1 min. Individual movements were recorded on video and analyzed using a computer-assisted program. The larvae swam in straight lines (a trajectory), rested, moved and started swimming again. For the parameters analyzed, i.e., number of movements, angle between successive trajectories and straightness index, there was no significant difference between the behaviour of the larvae in the different solutions. However, for the larvae in 10^-5, 10^-4 and 10^-3 M arginine solutions, the analyzed parameters, i.e., time active, frequency of trajectories (number of movements exceeding body length), distance swum min^-1, length of individual trajectories and trajectory velocity, were all significantly lower than for the larvae in the control solution of artificial seawater and for larvae in the solutions of 10^-9, 10^-8, 10^-7 and 10^-6 M arginine. The results show that the mean distance swum by cod larvae min^-1 was two to five times longer in artificial seawater without arginine and in the four lower concentrations of arginine than in the three higher concentrations. Scanning micrographs show that newly hatched (pre-feeding) cod larvae possess olfactory organs. It seems reasonalbe to assume that the observed changes in swimming behaviour are mediated by the olfactory sense and are important in the feeding strategy of cod larvae. We suggest that the observed behaviour increases the probability of the larvae localizing patches of prey organisms and remaining in the patch once they have found it. The results show that chemokinesis is a mechanism by which the spatial distribution of fish larvae will be correlated with their prey.     

Effect of amino acids on the swimming activity of newly hatched turbot larvae (Scophthalmus maximus)

The swimming behaviour of newly hatched turbot (Scophthalmus maximus L.) larvae was observed in artificial seawater (ASW) and in solutions of 21 l-amino acids at a concentration of 10⁻⁵  M. The behaviour of 20 larvae was analysed in each solution. Each larva was observed for 1 min. Individual movements were recorded on video and analysed using a computer-assisted program. The larvae swam in convoluted, randomised three-dimensional paths, rested and started swimming again. There were large variations in the swimming behaviour of turbot larvae during ontogeny. In ASW the mean frequency of trajectories longer than a body length of 4 mm larva⁻¹ min⁻¹ increased from 1.2 at Day 1, to 10 at Day 4. Analysing the data (Dunnett's method) revealed that the frequency of swimming trajectories increased in the presence of glycine, histidine and glutamine, and decreased in the presence of proline. The total distance swum increased for glycine but decreased for proline. The threshold concentration for glycine detected by turbot larvae was 10⁻⁵  M. The straightness index did not change in the presence of the amino acids. The possible role of these changes in behaviour is discussed. Received: 12 June 1997 / Accepted: 13 January 1998     

Effect of salinity on hypoxia tolerance of resting green crabs, Carcinus maenas, after feeding

Mechanisms that can influence the tolerance of hypoxia in brackish waters were studied in resting and fed crabs, Carcinus maenas, at 15?°C. Mortality, blood oxygenation, acid-base status and lactate concentration were analysed in fed crabs held in full-strength normoxic seawater (32.5‰?S) and then transferred for 24?h to a partial pressure of oxygen (P_o2) of 3?kPa (1.4?mg?l^?1) and various salinities (17, 12.5, 10, 8‰?S). At salinity levels >10‰, fed crabs tolerated P_o2 values as low as 3?kPa in the ambient water and 0.5?kPa in their arterial blood for 24?h without switching to anaerobic metabolism. Only below 10‰?S did their blood-lactate content rise, leading to their death despite the fact that their blood O₂-content was twice the control value measured in full-strength normoxic seawater and their blood P_o2 did not decrease below values recorded at higher salinity levels. Addition of CO₂ to 8‰?S water (CO₂ partial pressure increasing from 0.1 to 0.3?kPa) decreased blood-lactate production and mortality, suggesting that at 10‰?S impairment of the O₂ supply is limited by an excessive blood O₂-affinity. The results are discussed in terms of the distribution (?10‰?S) of C. maenas along salinity gradients in estuaries and bays.     

Food and feeding dynamics of the larval Antarctic fishNototheniops larseni

The feeding dynamics of larvae of the Antarctic fishNototheniops larseni were analyzed from data collected over three years in Bransfield Strait and adjacent waters (Antarctica). Seasonal feeding was examined from 1977/1978 (November–March). The diel feeding cycle was investigated during a 96 h station established in February 1976, while food selection was analyzed using larvae and zooplankton samples collected in February 1982. Hatching occurs in early spring, and larvae fed on eggs of calanoid copepods and on cyclopoid copepods. Copepod eggs were the principal food near the pack ice, and cyclopoids in open waters. Cyclopoids were the staple food in summer. Eggs of the Antarctic krillEuphausia superba were ingested selectively and formed major portions of the larval summer diet in neritic (Joinville Island) and oceanic (Elephant Island) spawning areas ofE. superba. In the fall, copepods predominated in the diets. Most abundant and most frequently ingested prey in summer and fall wereOncaea spp. Feeding commenced at dawn and continued at least until dusk. Krill eggs were taken chiefly during morning hours and egg incidence declined during the day, suggesting that eggs were ingested soon after spawning. Prey size at the onset of feeding was estimated as 0.130 to 0.330 mm. Size-selective feeding was evident in small larvae, while in larger larvae median prey length remained constant. High feeding incidence among yolk-sac larvae in spring, high overall feeding incidence in summer, and size-selective foraging of small larvae suggested favorable feeding conditions in the 1977/1978 season. Yolk-absorption times in Antarctic fish larvae vary on a scale of weeks and may be further retarded due to early feeding. Hence, year-to-year variability of yolk incidence inN. larseni indicated variable biotic environments of early feeding larvae rather than temporal shifts of hatching periods. As hatching periods are constant between years in contrast to the variable retreat of the pack ice and subsequent onset of the production cycle in space and time, maternal yolk reserves are probably utilized to compensate for such variations.     

Effects of the insect growth regulator Dimilin® (TH 6040) on larval development of two estuarine crabs

Effects of Dimilin^® (TH 6040), an insect growth regulator which interferes with the formation of the insect cuticle, were studied on the larval development of Rhithropanopeus harrisii (Gould) and Sesarma reticulatum (Say) (Crustacea: Brachyura). When larvae were exposed to 0.5 (R. harrisii only), 1, 3, 5, 7, and 10 ppb Dimilin from hatching to the first crab stage, survival in both species decreased in relation to increased concentrations of Dimilin. Survival of R. harrisii larvae wa significantly lower at 1 ppb and higher levels compared with control experiments, and in S. reticulatum a significant decrease in survival began at the 3 ppb level. At 10 ppb Dimilin, no larvae survived to the megalopa stage in either of the two species. The results indicate that early stage larvae of R. harrisii are more sensitive to Dimilin than those of S. reticulatum. When R. harrisii larvae were treated with 10 ppb Dimilin during the intermolt period of each of the 4 zoeal stages, nearly all larvae died during molting to the succeeding stage. First zoeal larvae of R. harrisii exposed to 10 ppb Dimilin at various days during the intermolt period were more sensitive to the compound late than early in the period. It is suggested that Dimilin also may interfere with the formation of the cuticle in crab larvae.     

Prey size selection,feeding rates and growth dynamics of heterotrophic dinoflagellates with special emphasis on Gyrodinium spirale

The relationship between size and growth rate in heterotrophic dinoflagellates (collected from the Kattegat, Denmark during 1989 and 1990) was studied. In addition, prey size selection, feeding rates and growth dynamics were studied for the naked heterotrophic dinoflagellate Gyrodinium spirale Bergh. Heterotrophic dinoflagellates have growth rates which are approximately three times lower than that of their potential competitors, the ciliates. G. spirale requires a relatively high prey concentration in order to grow. Ingestion rate at the maintenance level is about half of that of maximum ingestion rate. Consequently, yield is lower than typically found for planktonic protozoa. When exposed to low prey concentrations, the dinoflagellate is able to reduce its metabolism and thus prolong survival. The optimum prey particle size for G. spirale, which feeds by direct engulfment, corresponds to its own size. The ability to ingest relatively large prey may explain why these organisms are competitive in nature.     

Inhibitory effects of copper on marine dinoflagellates

The effect of copper on three species of marine dinoflagellates [Scrippsiella faeroense (Paulsen) Balech et Soares, Prorocentrum micans Ehrenberg, Gymnodinium splendens Lebour] was studied. It inhibited the growth of all species and was lethal to one species in batch cultures. The effect was more pronounced in semicontinuous culture than in batch cultures. Chlorophyll concentrations and rate of uptake of radioactive carbon by cells of S. faeroense were affected in a manner similar to cell numbers. Copper inhibited growth of cells, most probably either by arresting cell division or by penetrating inside the cell and affecting metabolism.     

Parental feeding rate in relation to begging behavior in asynchronously hatched broods of the great tit Parus major

Summary Variations in the begging behaviour of the nestlings of altricial birds can provide the parents with information about the nestlings' nutritional needs and thus influence the parental feeding rate. In a series of four experiments, the stimulus situation encountered by great tits on their feeding visits to the brood was manipulated to explore its effect on feeding rate.A higher feeding rate was observed under the following conditions: (1) after a period of food deprivation, as compared with both normal conditions and satiation through artificial feeding; (2) in periods when recorded begging calls were played during feeding visits, as compared with control periods; (3) after temporary removal from the nest of heavier, as compared with lighter, siblings. The lighter nestlings in the brood benefitted more —in terms of gain in weight — from the increase in parental feeding rate following the playing of begging calls than did the heavier nestlings. Differences in weight spread within broods did not affect the amount of food the parents brought.We conclude that parental feeding rate is affected not simply by the begging of the hungriest nestling, but rather by the behaviour of all the nestlings in the brood, which makes possible an adjustment of the feeding rate to the average hunger level of the brood. The effects of hatching asynchrony and sibling competition on parental feeding rate are discussed.     

Incidence,reproduction and feeding of Stylochus zebra,a polyclad turbellarian symbiont of hermit crabs

The hermit crab Pagurus pollicaris is the common host for the polyclad turbellarian Stylochus zebra in the Atlantic coastal waters from Massachusetts to North Carolina, USA. S. zebra is reported for the first time from two other hermit crabs, Pagurus impressus and Petrochirus diogenes. It was not found with Clibanarius vittatus or Pagurus longicarpus, although the latter serves as a host along the Gulf coast of Florida. The incidence of the polyclad with P. pollicaris in Massachusetts is more than twice that in populations to the south. Multiple infestations (up to 7 worms/crab) occur in over 50% of the infested crabs from Massachusetts, but they are rare elsewhere. Worms living in clear plastic shells with crabs orient in the body whorl dorsal to the host. Egg masses of worms are cemented in the same position within gastropod shells harboring the crabs. In the laboratory, the worm feeds on the gastropod Crepidula plana, a common inhabitant of shells occupied by P. pollicaris. Its feeding behavior is described. The relationship between S. zebra and P. pollicaris appears to be generally commensalistic, but it could be more complicated if the predation of crab embryos observed in the laboratory is confirmed under natural conditions.Contribution No. 755 from the Virginia Institute of Marine Science, Gloucester Point, Virginia, USA.     

Effect of delayed first feeding on larval performance of the spider crab Maja brachydactyla assessed by digestive enzyme activities and biometric parameters

The effect of food deprivation on larval performance of the spider crab Maja brachydactyla was studied in terms of survival, moulting capacity, size, weight and enzymatic activities. Five feeding treatments that differed in the initial age of first feeding larvae (fed from hatching, 2, 4 and 6 days post-hatching and unfed) were tested for 20 days. Newly hatched larvae kept without food supply lasted for 10 days and did not moult; with 50% survival observed at 6 days post-hatching. Larvae (zoea I stage) were only able to tolerate 2 days of food deprivation after the onset of exogenous feeding without their performance being compromised. Multivariate analyses suggest that digestive enzyme activities may be good indicators of the nutritional condition of larvae.     

The ecological significance of lipid/fatty acid synthesis in developing eggs and newly hatched larvae of Pacific cod (Gadus macrocephalus)

The lipid/fatty acid composition of marine fish eggs and larvae is linked with buoyancy regulation, but our understanding of such processes is largely restricted to species with pelagic eggs. In this study, we examined developmental changes in the lipid/fatty acids of eggs and embryos of Pacific cod (Gadus macrocephalus), a species that spawns demersal eggs along coastal shelf edges, but as larvae must make a rapid transition to the upper reaches of the water column. Adult Pacific cod were collected in the Gulf of Alaska during the spawning season and eggs of two females were artificially fertilized with sperm from three males for each female. The eggs were subsequently reared in the laboratory to determine (1) how lipids/fatty acids were catabolized during egg and larval development, and (2) whether lipid/fatty acid catabolism had measurable effects on egg/embryo density. Eggs incubated at 4°C began hatching after 3-weeks and continued to hatch over a 10-day period, during which there was a distinct shift in lipid classes (phospholipids (PL), triacyglycerols (TAG), and sterols (ST)) and essential fatty acids (EFAs: 22:6n-3 (DHA), 20:5n-3 (EPA), and 20:4n-6 (AA)). In the egg stage, total lipid content steadily decreased during the first 60% of development, but just prior to hatch we observed an unexpected 2–3-fold lipid increase (~6–9 μg individual⁻¹) and a significant drop in egg density. The increase in lipids was largely driven by PL, with evidence of long-chained fatty acid synthesis. Late-hatching larvae had progressively decreasing lipid and fatty acid reserves, suggesting a shift from lipogenesis to lipid catabolism with continued larval development. Egg density measures suggest that lipid/fatty acid composition is linked to buoyancy regulation as larvae shift from a demersal to a pelagic existence following hatch. The biochemical pathway by which Pacific cod are apparently able to synthesize EFAs is unknown, therefore representing a remarkable finding meriting further investigation.     

Characterizing temperate rocky shores from the perspective of an early juvenile snail: the main threats to survival of newly hatched Nucella emarginata

Mortality factors most likely to constitute substantial selective pressures for early juvenile gastropods on temperate rocky shores were identified by examining the vulnerability of hatchlings of an intertidal snail, Nucella emarginata, to heat stress, desiccation, and predation in 1992 and 1993. The highest temperature of substrata measured at tidal heights colonized by N. emarginata in Barkley Sound, British Columbia, Canada, was 28.5°C. This temperature was not lethal to hatchlings in laboratory tests. In laboratory and field desiccation experiments, all hatchlings died within 6 h of emersion. Early juveniles could not survive direct exposure to even moderate drying conditions for the duration of a low tide. Hence, intertidal microhabitats which dry up even for short periods during low tides would prove lethal. Of 45 intertidal animal species to which hatchlings were exposed in the laboratory, small decapod crustaceans were the only organisms to cause substantial hatchling mortality. Of these, Pagurus hirsutiusculus and Hemigrapsus nudus were by far the most abundant in the field, and are probably the only important predators of early juvenile N. emarginata at most sites. Total predator densities in the field were as high as 438 individuals m^–2, suggesting that predation pressure may be intense. Desiccation and predation by decapod crustanceans appear to be the most significant threats to early juvenile N. emarginata. These factors commonly occur on most temperate rocky shores and undoubtedly constitute major selective agent influencing population parameters and shaping life-history strategies and early juvenile traits of intertidal invertebrates.     

The effects of feeding or addition of dissolved inorganic nutrients in maintaining the symbiosis between dinoflagellates and a tropical marine cnidarian

The availability of solid food (Artemia nauplii) and dissolved inorganic nutrients (ammonium, nitrate, phosphate) to the shallow-water marine hydroid Myrionema amboinense was manipulated for 1-8 days in order to investigate their role in the growth of intracellular symbiotic dinoflagellates (zooxanthellae) of the genus Symbiodinium. Symbionts from hydroids collected from the field or maintained under laboratory conditions (25°C, 12 h:12 h light:dark cycle, 80 µE m^-2 s^-1 fluorescent lighting) always exhibited a single peak in mitotic index (MI) at dawn. Symbionts in freshly collected field animals had an MI peak of about 15%. Symbiotic dinoflagellates in hydroids fed Artemia nauplii twice daily in the laboratory maintained this dawn peak of MI between 10% and 15%, but in the absence of feeding or added inorganic nutrients, this peak declined to less than 1% within 2-4 days. In contrast, when hydroids were placed in solutions containing ammonium (20 µM NH₄Cl), nitrate (10 µM NaNO₃), and a combination of ammonium and phosphate (2 µM Na₂HPO₄) immediately after collection, the algal MI remained between 5% and 15% for 4-7 days; the addition of 2 µM phosphate did not increase MI relative to unfed rates. When unfed animals were placed in dissolved nitrogen or fed Artemia, the symbiont MI increased from <1% to 10-17% within 2-3 days; P alone had no effect. However, the increase resulting from added inorganic nutrients was temporary, lasting only 5-7 days. These observations suggest that algal division in the host is maintained indefinitely in the field or by feeding particulate foods twice daily in the laboratory, but the addition of inorganic nutrients alone (ammonium, nitrate and ammonium/phosphate) appeared to support the completion of a maximum of one additional round of cell division. Nutrients required for continued growth and division of symbiotic dinoflagellates are linked to host feeding and host growth; without external food, neither host nor symbiont continue to grow. The same phenomenon is seen in zooxanthellate anemones, clams and corals, where total numbers of symbionts appear to be linked to changes in host-tissue biomass (protein), achieving relatively stable densities in M. amboinense, corals and other cnidarian symbioses, depending on their local environmental conditions. The results of the present study help explain the cellular responses of algal symbionts in reef-dwelling invertebrates to additions of dissolved inorganic nutrients to coral-reef ecosystems.     

Release of nitrite by marine dinoflagellates: development of a mathematical simulation

The dinoflagellates Scrippsiella trochoidea (Stein) and Alexandrium minutum (Halim) were grown in a light–dark cycle with nitrate or nitrate plus ammonium under three different nutrient-supply regimes (dilution with fresh media in dark phase only or during the entire light–dark cycle at the same daily dilution rate, or with a faster continuous dilution). When supplied with nitrate + ammonium, A. minutum released a proportion (as much as 100% from dark-fed cells) of the nitrate taken up during the dark phase as nitrite, reflecting a rate-limiting step at nitrite reduction and poor regulation of inorganic-N uptake and assimilation. S. trochoidea released much smaller amounts of nitrite, if any. Nitrate and ammonium were not accumulated to any extent by either species in darkness, and the transient increases in the size of the free amino acid pool were too small to explain the fate of the newly assimilated N. Thus uptake through to incorporation of N into macromolecules appeared to be coupled in these species, even in darkness when increasing glutamine:glutamate (Gln:Glu) ratios suggested rising C-stress. A mechanistic model was developed from an earlier ammonium–nitrate interaction model (ANIM) by the inclusion of an internal nitrite pool, with control over the supply of reductant for nitrite reduction linked to photosynthetic and respiratory components. The model can reproduce the release of nitrite seen in the experiments, and also the release of nitrite in response to nitrate-feeding of N-stressed cells reported elsewhere. Received: 22 August 1997 / Accepted: 26 September 1997     

Rhythmicity of larval release in three species of intertidal brachyuran crabs (Crustacea: Brachyura) from Inhaca Island (Mozambique)

 A study of rhythmicity of larval release in three species of intertidal brachyuran crabs, based on laboratory and field experiments, was undertaken at Inhaca Island, southern Mozambique, using Leptodius exaratus and Macrophthalmus grandidieri from December 1994 to January 1995, and Arcotheres palaensis from April to July 1995. L. exaratus and M. grandidieri showed a semi-lunar cycle in larval release. The release of larvae for L. exaratus, a species having conspicuous larvae, occurred in the first half of the night, after the post-crepuscular high tide, which suggests maximisation of protection of larvae from visual predation. The larval release activity matched the late spring and early neap tides. Results from the field were similar to those from the laboratory. M. grandidieri, having inconspicuous larvae, did not show a pattern related to the light–dark cycle and hatched during spring tides (around full and new moons) to maximise larval dispersion. A. palaensis, living inside the host mussel which inhabits the lowest section of the intertidal zone, did not show a relation with moon phase, tidal or light–dark cycles. Received: 16 February 1999 / Accepted: 8 December 1999     

Changes in the feeding behavior of the deposit-feeding polychaete<Emphasis Type="Italic"> Laeonereis acuta</Emphasis> on soft sediments inhabited by burrowing crabs

In this paper, we evaluate the influence of the burrowing crab Chasmagnathus granulatus on the feeding behavior, body condition, burrowing activity, and fecal production of the deposit-feeding polychaete Laeonereis acuta. Previous results and ours show that, due to crab activity, sediment organic matter decreases with depth outside a crab bed, but there were no differences inside. Also, particle sizes were smaller inside the crab bed. Polychaetes showed better body condition inside the crab bed, suggesting higher energy gain. They fed on the surface more frequently outside the crab bed than inside. However, feeding rate was higher inside the crab bed, which is consistent with the larger weight and volume of fecal pellets found inside crab bed. In both areas, the bedload sediment transport was not similar to the sediment ingested by this polychaete. A 1-month exclusion/inclusion experiment performed outside and inside the crab bed showed that the body condition of polychaetes changed between areas but that this was not directly due to crab manipulation. Thus, our results suggest that the higher sediment quality inside a crab bed can positively affect the feeding behavior of the deposit-feeding polychaete L. acuta, increasing its feeding rate and consequently enhancing its body condition.Communicated by P.W. Sammarco, Chauvin     

Compression of home ranges in ghost crabs on sandy beaches impacted by vehicle traffic

Animal movement is a pivotal element of many ecological processes, and on ocean-exposed sandy shores, ghost crabs (genus Ocypode) undertake extensive nocturnal forays on the beach surface. Because crab populations are also threatened by vehicle traffic, indicators that can detect sublethal effects before population declines are manifest are important. To this end, we tested on a barrier island in Eastern Australia whether movement patterns of crabs respond predictably to disturbance by vehicles; this was done by tracking (using the spool-and-line technique) crabs at night in beach sections open and closed to traffic. Beach traffic not only halved population densities of crabs on the unvegetated beach seawards of the dunes, but it also fundamentally changed crab behaviour and movement: individuals from beach areas rutted by tyre tracks travelled shorter distances in a more erratic zigzag pattern, and they had significantly compressed home ranges. Such behavioural changes linked to human pressures could be well suited as an early warning signal for wider negative ecological impacts (as demonstrated by reduced abundances). They also emphasize the need to incorporate sublethal effects into the assessment and management of ecological changes resulting from beach recreation.