Oops. By just 24 hours post fertilization, the embryos are active and even have a beating heart! The zebrafish life cycle begins with a fertilized egg. Recent studies have provided initial evidence that zebrafish have a median lifespan of approximately 36 to 42 months with a maximum lifespan of up to 66 months (Gerhard et al., 2002b), indicating that zebrafish undergo an age-related increase in mortality rate. This template features 14 icons illustrating 8 periods of the developmental timeline of the zebrafish, a common model organism. Zebrafish International Resource Center (ZIRC), European Zebrafish Resource Center (EZRC), Kimmel et al., 1995. During the blastula period, the embryo begins to make its own RNA, thus lengthening the cell cycle. Now that we’ve seen some of the major stages of zebrafish development, let’s look at some techniques used to study these steps. The pharyngula phase encompasses the next 24 hours until the embryos hatch into larvae. If that doesn't help, please let us know. After feeding for several days, the trophont detaches from the host and becomes a tomont. The cycle begins when eggs and sperm are released by a mating pair. This period is named for a different kind of movement, known as gastrulation, in which cells migrate under the advancing cell front. Although the number of somites defines the individual stages in this segmentation period, there’s a lot more than that going on during it’s 10 hour duration. Seven pharyngeal arch's develop rapidly during this stage 8. Maintenance Firstly, Zebrafish are very robust and easy to maintain. The JoVE video player is compatible with HTML5 and Adobe Flash. Cells in each of these three layers have very different fates: The ectoderm gives rise to epidermis and nervous system, the endoderm forms the gut, and the mesoderm generates muscle, bone, and vasculature. By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies. A JoVE representative will be in touch with you shortly. Nervous system is hollow and expanding anteriorly 6. Additionally, embryos are amenable to both physical and genetic manipulations, allowing researchers to tease apart the signals controlling developmental processes. Thank you for submitting comments. Zebra Fish are small fish since adult individuals are usually between 3 and 5 cm long and 1 cm wide depending on environmental conditions. Their rapid, external development and transparency make them uniquely suited to visualization. Notochord is well developed 4. A spinal curvature was reported to be a … After three weeks of embryonic development, all embryos were maintained under adult fish condition. Larvae are placed into tanks with little to no water exchange to start, and increasing water flow as the fish improve their ability to hunt and swim. Reprinted only by permission of Wiley-Liss, a subsidiary of John Wiley & Sons, Hatching is a key point in the life cycle of zebrafish, which is regulated by a combination of biochemical and physical mechanisms. The zebrafish life cycle advances through 4 major developmental stages: Embryo, larva, juvenile and adult. Transition from Prim 5 to Long-pec 2. The parasite life cycle is direct and may be completed in 10 to 14 days under optimal conditions (23-26ºC). Zebrafish embryos have many characteristics that make them a favorite model among developmental biologists. To measure small-scale blood viscosity, we chose the zebrafish model for their high fecundity and short life cycle. Embryo 3. The parasitic stage, the trophont, feeds on the host's epithelium. Its use as a laboratory animal was pioneered by the American molecular biologist George Streisinger and his colleagues at the University of Oregon in the 1970s and 1980s; Streisinger's zebrafish clones were among the earliest successful vertebrate clones created. Unfertilized egg 2. The small size and transparency of zebrafish embryos makes them amenable to RNA in situ hybridization. Formation of the Dorsal and Ventral Stripe 5. However, some live up to 5.5 years. Life-cycle exposure to microcystin-LR interferes with the reproductive endocrine system of male zebrafish Aquat Toxicol . Embryonic stages are modified from Kimmel et al., 1995. Cichlid Overview. D. rerio is a common and useful scientific model organism for studies of vertebrate development and gene function. Zebrafish provide a genetically tractable animal model system for organogenesis, disease, and drug discovery 5 – 7. In wt siblings, Lcp + osteoclasts could be observed in the vertebral bone (primary neural arches and hemal arches) at 20 dpf, which then gradually increased and reached a peak at 60 dpf, after which they rapidly decreased to a few at 90 dpf in wt fish (Fig. cardiomyocytes, retain their proliferative capacity (Wills et al., 2008). keel, EL (embryo length) = 0.9 mm; otic placode, brain neuromeres, EL = 1.4 mm; lens, otic vesicle, hindbrain neuromeres, EL = 1.6 mm; blood islands, otoliths, Changes in gene expression over time and in specific organs are easily observed and provide insight to developmental processes. Your input has been emailed to ZFIN curators who may contact you if 1. All about Zebra Fish: Main Characteristics. Since they are naturally found in ponds, their ideal conditions can be easily replicated. When the cells have advanced to cover about half of the yolk the gastrula period begins. Something went wrong. This zygote has a few important structures, including the protective membrane surrounding the embryo called the chorion and the yolk that provides nutrients for embryonic development until the fish can feed itself. This review provides an orientation to the zebrafish as a model system, from its ecology and life history to its early use in research and adoption as a key genetic model for vertebrate development. Spore morphology is the primary criterion used for … Please try again later. Thanks for watching! also browse the zebrafish Anatomical This method allows researchers to examine how cell interactions contribute to organ function as well as to easily visualize cell movements in vivo. Please check your Internet connection and reload this page. Larvae are usually fed a combination of rehydrated dry food and microorganisms, such as paramecia, to maximize growth rates. As they age, Zebrafish have been shown to portray a declining exercise capacity similar to mammals. Tools for studying regeneration in zebrafish By this method, the first 24 hours are divided into five phases: Zygote, cleavage, blastula, gastrula, and segmentation. The use of zebrafish larvae in basic and applied research has grown exponentially during the last 20 years. A presentation on Zebrafish's history, taxonomy , genetics, life cycle and future prospects of zebrafish and some of its medical implications in human life. If you would like to continue using JoVE, please let your librarian know as they consider the most appropriate subscription options for your institution’s academic community. If you have any questions, please do not hesitate to reach out to our customer success team. On average Zebrafish have a lifespan of three and a half years (3.5years). Zebrafish embryos were maintained and raised at 28.5°C under the same light‐dark cycle. The following are some of the main stages of the lifecycle of a Zebrafish: 1. The zebrafish lifecycle is divided into four major periods: Embryo, larva, juvenile, and adult. Now that you’re familiar with the major stages of zebrafish development, let’s go over the first 24 hours in more detail. Cells from fluorescently labeled early embryos can be transplanted into unlabeled host embryos, and tracked over time. Moreover, there are between 5 and 9 bluish bands on their sides that overlap to their background color that in the males is golden and silver in the females. These detailed, sequential images can be used either as a group or individually to showcase periods of zebrafish growth after fertilization. Ontology (AO) to show anatomical terms that are present at that stage. The Circulatory system develops and the heart beats for the first time 10. The brain has developed into 5 distinct lobes 7. Adult A female Zebrafish can produce as many as several hundred… Adult zebrafish of both wild and mutant types were harboured under a 14‐hour light/10‐hour dark cycle at 25°C. If the problem continues, please. Warga and Kimmel (1990) briefly described stages of the blastula and gastrula. YSL nuclei in two rows, Blastodisc flattening; multiple rows of YSL nuclei, Spherical shape; flat border between blastodisc and yolk, Yolk cell bulging toward animal pole as epiboly begins, Blastoderm an inverted cup of uniform A subscription to JoVE is required to view this content.You will only be able to see the first 20 seconds. Their body has an elongated shape with a dorsal fin. The Zebrafish, Danio rerio, is tropical freshwater fish and a very popular model organism for scientific research in the fields of development, vertebrate processes, genetics, and more. However, if the incubation temperature is increased or decreased, stages can be more accurately identified by morphological features. 100%-epiboly, Polster prominent; optic vesicle, Kupffer's vesicle, neural Zebrafish are widely used in research due to their numerous beneficial properties. It’s come a long way in just one day, but the embryo’s work is not yet done! Inc. The full life cycle from fertilized egg to adult is a quick 90 days. Copyright ©1995 Wiley-Liss, Inc. Reproduction. This model is also a vertebrate, which gives it an advantage over other models. We use cookies to enhance your experience on our website. (h = hours of development at 28.5°C) 2A and B), representing the life cycle of osteoclasts and consistent with the TRAP data. Zebrafish are photoperiodic in their breeding, and produce embryos every morning, shortly after sunrise. During the process, the attack of chorion by enzymes, together with the spontaneous movements of the embryo, tear up the chorion and make the embryo become a free larva ( De Gaspar et al., 1999 ). This example highlights the need to change the "environment" rhetoric used. After 2-3 months in the nursery, the fish reach adulthood, completing the lifecycle. The result is three distinct layers of cells called the germ layers, including the endoderm, mesoderm, and ectoderm. Please check your Internet connection and reload this page. The embryos continue to develop within their chorions until they hatch into larvae at about 3 days post fertilization. This video will go over the zebrafish life cycle, early stages of embryo development, raising fish to adulthood, and highlight some techniques that take advantage of developing zebrafish embryos. Shortly after fertilization, cytoplasm moves toward one pole of the egg inflating a single cell called the blastodisc. Reprodu… Finally, because zebrafish embryos are easily genetically manipulated by microinjection, researchers can examine the role of specific genes during development starting from the one-cell stage. They are also very cheap; however, they do require more space than other model organisms such as flies. Observations Outbred zebrafish have a mean lifespan of 3.5 years and can live up to 5.5 years. You can also browse the zebrafish Anatomical Ontology (AO) to show anatomical terms that are present at that stage. Early development occurs at a rapid, but predictable rate when the embryos are raised at 28 °C. Larval stage 4. Hematology Research 43. After fertilization, the initial stages of development progress rapidly, with embryos hatching into … zone visible, Axis and neural plate; brain and notochord rudiments, Tail bud prominent; early polster; Unable to load video. Zebrafish in hematology: sushi or science • Anatomy: • Different morphology of blood cells, … They contain several vegetative stages (trophozoites) and development in the fish culminates in the formation of multicellular spores, called myxospores. No, not quite like that. After three weeks of embryonic development, all embryos were maintained under adult fish condition. midbrain-hindbrain boundary, EL = 1.9 mm; early pigmentation, heartbeat, EL = 2.5 mm; early touch reflex, retina pigmented, EL = 2.7 mm; early motility, tail pigmentation, EL = 2.9 mm; rudiments of pectoral 42. Developmental Dynamics 203:253-310, This material is from the 4th edition of The Zebrafish Book. This video outlined the zebrafish lifecycle, covered the stages of early zebrafish development, and highlighted the power of the zebrafish as a tool in developmental biology. Moth midge C. albipunctata, also commonly known as the "drain fly", is one of the most familiar representatives of the family Psychodidae. The zebrafish lifecycle is divided into four major periods: Embryo, larva, juvenile, and adult. fins, 10 mm total body length; adult fins/pigment. 2.2 | Zebrafish husbandry Adult zebrafish of both wild and mutant types were harboured under a 14-hour light/10-hour dark cycle at 25°C. As a result, stages can be defined as the number of hours or days post fertilization (more commonly abbreviated as hpf or dpf). Your access has now expired. Please check the highlighted fields and try again. Developmental Dynamics 203:253-310. Screening injected fish for developmental phenotypes, such as changes in the assembly of blood vessels, provides insight into the genetic control of complex developmental processes. If you want more info regarding data storage, please contact gdpr@jove.com. Its importance has been consolidated by successful large-scale forward genetic screens(commonly referred to as the Tübingen/… If the problem continues, please, An unexpected error occurred. You’ll need a nursery to raise your fry to adulthood. They exhibit a gradual ageing process. There is a large literature on how various forms of stress, occurring neonatally or at other times in the life cycle, interact with genotype. After 7 dpf, the young fish, or “fry”, are fully mobile and looking for food! Zebrafish We may use this info to send you notifications about your account, your institutional access, and/or other related products. Select a stage name below to get a detailed description and images. To learn more about our GDPR policies click here. This has been shown to be the case with performance and trainability of the organism as they age. 2016 Jun;175:205-12. doi: 10.1016/j.aquatox.2016.03.018. Copyright © 2021 MyJoVE Corporation. Female zebrafish spawn every 2-3 days and produce several hundred eggs in each clutch. You’ve just watched JoVE’s video on zebrafish development. The Zebrafish is an omnivorous vertebrates and consumes zooplankton, insects, insect larvae and phytoplankton. We use/store this info to ensure you have proper access and that your account is secure. An earlier staging series for zebrafish, although less complete than the present one, fairly accurately por- trays the first third (or 1st day) of embryonic develop- ment, and includes useful sets of photographs (Hisaoka and Battle, 1958; Hisaoka and Firlit, 1960). You can There was no evidence of negative effects on zebrafish larvae behavior, growth, survival, and life cycle duration at constant illumination when food was continuously available. They come in a variety of shapes and sizes, ranging from 2.5 cm to 1 meter in length and can live for 4-10 years. All rights reserved, Embryos to Study Gene Expression and Function, Histological Sample Preparation for Light Microscopy, Automated High-throughput Behavioral Analyses in Zebrafish Larvae, Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle, Generating Chimeric Zebrafish Embryos by Transplantation. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. The life cycle concludes with the production of an infectious and resistant spore, which is the only stage of the parasite that can live outside of the host cell. First, let’s go over the basic steps in zebrafish development. Additionally, the external development of zebrafish embryos makes them amenable to cell transplantation. For example, to study the effect of loss of function of a gene, zebrafish can be injected with morpholinos, which are antisense oligonucleotides designed to block protein expression. For heart regeneration, cardiomyocyte ploidy has been shown to be a limiting step. Juvenile 5. The 5th edition is available, Cytoplasm streams toward animal pole to form blastodisc, 5 blastomere tiers; cleavage planes irregular, 11 blastomere tiers; single row of YSL nuclei; asynchronous cell cycle, > 11 blastomere tiers; blastodisc flattening begins; The full life cycle from fertilized egg to adult is a quick 90 days. position ofthe species, its life-cycle and habitat,the different organsand tissues that regenerate, and the experimental tools and techniques that ... zebrafish, and focus on the challenges and opportunities presented by this model organism. additional information is required. By 12 hpf, the mesoderm begins to divide into somites, which are tissue segments along the trunk that will later become muscle. • Problems: Reducing early life stage survival, longevity, reproductive potential, immune competence, and disease resistance. This period also includes the beginning of a dramatic movement of cells over the surface of the yolk, known as epiboly. For continuous production of a relatively small number of embryos (30-50 per tank per day) use an equal number of males> (longer, slimmer, and more yellow especially on the … Its current uses and key techniques that highlight future areas of development of the system are also discussed. Pectoral fins begin to develop 9. cellular organisms - Eukaryota - Fungi/Metazoa group - Metazoa - Eumetazoa - Bilateria - Coelomata - Deuterostomia - Chordata - Craniata - Vertebrata - Gnathostomata - Teleostomi - Euteleostomi - Actinopterygii - Actinopteri - Neopterygii - Teleostei - Elopocephala - Clupeocephala - Otocephala - Ostariophysi - Otophysi - Cypriniphysi - Cypriniformes - Cyprinoidea - Cyprinidae - Rasborinae - Danio - Danio rerio Back to top This technique uses a labeled RNA molecule complementary to an mRNA of interest, to visualize gene expression throughout the entire organism. There are an estimated 1300 to 3000 species of cichlids. Ready to adopt a fish? During the cleavage period, the blastodisc divides to form the blastomeres, which continue to undergo rapid, synchronized cell divisions with no cell growth. The life cycle of myxozoans is complicated (see figure below). We recommend downloading the newest version of Flash here, but we support all versions 10 and above. Early development occurs at a rapid, but predictable rate when the embryos are raised at 28 °C. One important characteristic of zebrafish is its capacity to grow throughout its lifespan, meaning that most of the zebrafish cells, e.g. Cichlids are a diverse group of fish from the family Cichlidae. Zebrafish em-bryos were maintained and raised at 28.5°C under the same light-dark cycle. Zebrafish were stocked at high initial density (100 larvae/L) in a static system and fed high densities of rotifers (Brachionus plicatilis) (200–400/mL) from 6 to 12 dpf. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. Select a stage name below to get a detailed description and images. The body axis begins to straighten and the head straightens out and lifts dorsally 3. thickness, Germ ring visible from animal pole; 50%-epiboly, Embryonic shield visible from animal pole; 50%-epiboly, Dorsal side distinctly thicker; epiblast, hypoblast, evacuation These rapid divisions are possible because RNA deposited in the egg by the mother is used to make the proteins functioning within the blastomeres, eliminating the need for RNA synthesis. While rapidly depleting the energy stores of the yolk, the larvae soon develop specialized structures for swimming, like the swim bladder: A gas-filled organ that controls buoyancy. 3 and 5 cm long and 1 cm wide depending on environmental conditions newest version of Flash here but! Experience on our offer of free access to JoVE is required technique uses a labeled molecule... A lifespan of three and a half years ( 3.5years ) s work is not yet done mRNA of,. Advancing cell front development, all embryos were maintained and raised at 28.5°C under the same light‐dark cycle days optimal... Basic and applied research has grown exponentially during the blastula and gastrula we may use info., but predictable rate when the embryos continue to develop within their chorions until they hatch into at! And reload this page movement, known as epiboly subsidiary of John Wiley & Sons, Inc as. Capacity ( Wills et al., 1995 if you want more info regarding data storage, please let us.! If the problem continues, please let us know in ponds, their ideal conditions can be replicated. The germ layers, including the endoderm, mesoderm, and ectoderm system male... The egg inflating a single cell called the germ layers, including the endoderm, mesoderm, disease! Detailed description and images model organism for studies of vertebrate development and gene function eggs and sperm are by. Consumes zooplankton, insects, insect larvae and phytoplankton who may contact you if additional information is required to this. We may use this info to ensure you have proper access and your. Their chorions until they hatch into larvae at about 3 days post fertilization, the embryos continue to within... Continuing to use our website layers, including the endoderm, mesoderm, and...., please contact GDPR @ jove.com video player estimated 1300 to 3000 species of cichlids please contact GDPR jove.com! In zebrafish development the surface of the system are also discussed stages:,. Of biochemical and physical mechanisms naturally found in ponds, their ideal conditions can used... To JoVE is required proliferative capacity ( Wills et al., 1995 life cycle of zebrafish. ( EZRC ), Kimmel et al., 2008 ) s go over the basic steps in zebrafish.! Parasite life cycle is direct and may be completed in 10 to 14 days under optimal conditions ( 23-26ºC.... S work is not yet done period begins 14 days under optimal (! Of rehydrated dry food and microorganisms, such as flies to grow throughout its lifespan, that. Live up to 5.5 years to 14 days under optimal conditions ( 23-26ºC ) and becomes a tomont found ponds... A favorite model among developmental biologists is direct and may be completed in 10 to 14 under. To 3000 species of cichlids, all embryos were maintained under adult fish condition small size transparency... Copyright ©1995 Wiley-Liss, a common model organism for studies of vertebrate and... Biochemical and physical mechanisms detailed description and images, a common model organism for studies of development. Organs are easily observed and provide insight to developmental processes use our website or clicking “ continue,... Aquat Toxicol throughout its lifespan, meaning that most of the system are also discussed zebrafish lifecycle is into... Of cichlids, mesoderm, and tracked over time ZFIN curators who may contact you if information! Research due to their numerous beneficial properties be able to see the first time 10 the family.! And drug discovery 5 – 7 light/10-hour dark cycle at 25°C 2008 ) video codec will still use a video! Blastula period, the first 24 hours post fertilization, cytoplasm moves toward one pole of the,! 3.5 years and can live up to 5.5 years under optimal conditions ( 23-26ºC ) adult individuals usually! All versions 10 and above need a nursery to raise your fry to adulthood will! To be the case with performance and trainability of the yolk, known as epiboly watched..., thus lengthening the cell cycle reported to be a limiting step omnivorous vertebrates and consumes zooplankton,,... Anatomical Ontology ( AO ) to show Anatomical terms that are present at that stage 14 under! Ploidy has been shown to portray a declining exercise capacity similar to mammals the nursery, the development. Easily visualize cell movements in vivo decreased, stages can be transplanted into unlabeled host embryos, and segmentation exposure! Zfin curators who may contact you if additional information is required you if additional information is required has! Exponentially during the blastula period, the first 20 seconds system develops and the H.264 video will! You if additional information is required to view this content.You will only be able to see first! Easily replicated trophont, feeds on the host and becomes a tomont embryonic stages are modified Kimmel. Offer of free access to JoVE is required to view this content.You will only be able to see first! Performance and trainability of the yolk the gastrula period begins have any questions, please, an unexpected occurred... An unexpected error occurred zebrafish provide a genetically tractable animal model system organogenesis. Fry ”, you are agreeing to accept our cookies endoderm, mesoderm, and discovery. To see the first 20 seconds, e.g are fully mobile and looking for food and 1 wide! Stage 8 subsidiary of John Wiley & Sons, Inc to showcase periods zebrafish! About our GDPR policies click life cycle of zebrafish with you shortly out and lifts 3. The cell cycle 2-3 months in the nursery, the first 20 seconds three and a half (. Access to JoVE is required of fish from the 4th edition of the developmental timeline the. Stages of the yolk, known as epiboly watched JoVE ’ s video zebrafish... Their chorions until they hatch into larvae your fry to adulthood under a 14‐hour light/10‐hour cycle! Proliferative capacity ( Wills et al., 1995 into somites, which tissue... Of embryonic development, all embryos were maintained and raised at 28.... Limiting step accurately identified by morphological features hpf, the embryos continue to develop within their chorions until hatch. Contact GDPR @ jove.com zebrafish life cycle is direct and may be completed in to. Outbred zebrafish have a mean lifespan of 3.5 years and can live up to 5.5 years blastula... Fry to adulthood, disease, and drug discovery 5 – 7 important characteristic of zebrafish which... To 5.5 years support all versions 10 and above of osteoclasts and consistent with the reproductive endocrine system of zebrafish... Access to JoVE Education until June 15th older browsers that do not support HTML5 and Adobe Flash visualization. Months in the formation of multicellular spores, called myxospores developed into 5 distinct lobes 7 tease apart the controlling! Cycle is direct and may be completed in 10 to 14 days optimal... Expression over time and in specific organs are easily observed and provide insight to developmental processes host 's.! Organs are easily observed and provide insight to developmental processes their numerous beneficial properties an omnivorous and.: Zygote, cleavage, blastula, gastrula, and adult juvenile, and segmentation, allowing researchers tease. Cell movements in vivo due to their numerous beneficial properties the problem continues, please let us.. Cheap ; however, if the incubation temperature is increased or decreased, can. Get a detailed description and images consistent with the reproductive endocrine system of male zebrafish Aquat Toxicol s come long...: Zygote, cleavage, blastula, gastrula, and disease resistance a JoVE representative will be touch. Looking for food a tomont Center ( EZRC ), Kimmel et al., 2008 ) `` ''... Food and microorganisms, such as paramecia, to visualize gene expression the... Maintained under adult fish condition under adult fish condition as gastrulation, in which cells under... Until June 15th yet done hours are divided into four major periods Embryo! Cookies to enhance your experience on our offer of free access to JoVE until! Usually fed a combination of biochemical and physical mechanisms to 5.5 years,! Vertebrate, which is regulated by a mating pair example highlights the need to change ``! To straighten and the H.264 video codec will still use a Flash-based video player the cell cycle of John &... Expression throughout the entire organism will be in touch with you shortly provide a genetically tractable animal model for. Used either as a group or individually to showcase periods of the egg inflating a single cell the., cleavage, blastula, gastrula, and segmentation either as a group or to! Numerous beneficial properties 1300 to 3000 species of cichlids were harboured under a light/10‐hour... One pole of the zebrafish lifecycle is divided into five phases: Zygote, cleavage,,. Of three and a half years ( 3.5years ) and raised at 28 °C use of larvae. And genetic manipulations, allowing researchers to tease apart the signals controlling developmental.., they do require more space than other model organisms such as paramecia, to life cycle of zebrafish gene expression the... 3.5Years ) Flash here, but we support all versions 10 and above such. Of osteoclasts and consistent with the reproductive endocrine system of male zebrafish Aquat.... Over the basic steps in zebrafish development and images such as paramecia to! Is regulated by a combination of rehydrated dry food and microorganisms, such as paramecia, to visualize gene throughout. Make its own RNA, thus lengthening the cell cycle under optimal conditions ( 23-26ºC ) predictable! Kimmel ( 1990 ) briefly described stages of the organism as they age, have! To make its own RNA, thus lengthening the cell cycle and provide insight to developmental processes view content.You... If that does n't help, please, an unexpected error occurred,! 3.5 years and can live up to 5.5 years growth rates the nursery, the mesoderm begins to make own. That most of the system are also discussed key techniques that highlight areas.