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Culture optimization for the emergent zooplanktonic model organism Oikopleura dioica.

Bouquet JM, Spriet E, Troedsson C, Otterå H, Chourrout D, Thompson EM - J. Plankton Res. (2009)

Bottom Line: This urochordate, has a simplified anatomical organization, remains transparent throughout an exceptionally short life cycle of less than 1 week and exhibits high fecundity.At 70 Mb, the compact, sequenced genome ranks among the smallest known metazoan genomes, with both gene regulatory and intronic regions highly reduced in size.The organism occupies an important trophic role in marine ecosystems and is a significant contributor to global vertical carbon flux.

View Article: PubMed Central - PubMed

Affiliation: Sars International Centre for Marine Molecular Biology, Thormøhlensgate 55, N-5008 Bergen , Norway.

ABSTRACT
The pan-global marine appendicularian, Oikopleura dioica, shows considerable promise as a candidate model organism for cross-disciplinary research ranging from chordate genetics and evolution to molecular ecology research. This urochordate, has a simplified anatomical organization, remains transparent throughout an exceptionally short life cycle of less than 1 week and exhibits high fecundity. At 70 Mb, the compact, sequenced genome ranks among the smallest known metazoan genomes, with both gene regulatory and intronic regions highly reduced in size. The organism occupies an important trophic role in marine ecosystems and is a significant contributor to global vertical carbon flux. Among the short list of bona fide biological model organisms, all share the property that they are amenable to long-term maintenance in laboratory cultures. Here, we tested diet regimes, spawn densities and dilutions and seawater treatment, leading to optimization of a detailed culture protocol that permits sustainable long-term maintenance of O. dioica, allowing continuous, uninterrupted production of source material for experimentation. The culture protocol can be quickly adapted in both coastal and inland laboratories and should promote rapid development of the many original research perspectives the animal offers.

No MeSH data available.


Collection and culture of Oikopleura dioica. (A) Animals are collected using a customized plankton net with a transparent, large, plexiglass cod end. (B) Overview of the culture system diagrammed in E. (C) Animals are cultured in 6 L of seawater in 8-L polycarbonate beakers and maintained in suspension by a polyvinylcarbonate (PVC) paddle rotating at 15 rpm. (D) The beakers are supplemented with grains of activated charcoal to regulate water quality over time. The paddles are made with a 3 mm thick PVC plate attached to an 8 mm diameter PVC rod using PVC glue. Dimensions (cm) of the paddle (inset) are adjusted according to developmental stage. H×L×W = 25×30×8 for spawning, days 1 and 2. The dimensions for other developmental stages are 25×30×7. (E) Schematic illustration of physical organization of the culture system. The fluorescent lighting mounted in the bench top greatly facilitates working with the transparent animals without the need to displace beakers to light boxes.
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FBN132F4: Collection and culture of Oikopleura dioica. (A) Animals are collected using a customized plankton net with a transparent, large, plexiglass cod end. (B) Overview of the culture system diagrammed in E. (C) Animals are cultured in 6 L of seawater in 8-L polycarbonate beakers and maintained in suspension by a polyvinylcarbonate (PVC) paddle rotating at 15 rpm. (D) The beakers are supplemented with grains of activated charcoal to regulate water quality over time. The paddles are made with a 3 mm thick PVC plate attached to an 8 mm diameter PVC rod using PVC glue. Dimensions (cm) of the paddle (inset) are adjusted according to developmental stage. H×L×W = 25×30×8 for spawning, days 1 and 2. The dimensions for other developmental stages are 25×30×7. (E) Schematic illustration of physical organization of the culture system. The fluorescent lighting mounted in the bench top greatly facilitates working with the transparent animals without the need to displace beakers to light boxes.

Mentions: The culture protocol for O. dioica is based on a system originally developed by Fenaux and Gorsky (Fenaux and Gorsky, 1985). The animals are raised at 15.0 ± 0.5°C in 8 L polycarbonate beakers (Cambro camwear® RFSCW8), containing 6 L of seawater (Fig. 4). Animals and algal feed are maintained in suspension by the rotation of a polyvinylcarbonate paddle connected to an electric motor (Synchromotor Crouzet, 15 rpm; 82.334.5 15 rpm AIG; Tufvassons PFS 150S transformer, 6124-0080, 230-24 V, 50–60 Hz, 6.3 A). Motors are mounted in parallel, and are individually protected by a fuse (1/4 A 250 V). The animals in the beakers with their forage organisms are cultivated in 10–12 h light and 14–12 h dark cycles. Under these conditions the life cycle is 6 days. To manipulate the animals, we use polypropylene beakers for dilution at days 1 and 2, and plastic Sterilin pipettes with blunt cut tips to transfer the animals to fresh beakers on days 3–6. The inside diameter of the transfer pipettes varies from 6 to 10 mm as a function of animal house size. To maintain water quality in beakers over a 24 h period, ∼10 g of pre-rinsed activated charcoal pellets (VWR charcoal 1–3 mm gradient, 12 700-5), are added to each beaker, with the exception of beakers used to accomplish the spawn. Charcoal pellets can be washed under running seawater and re-used up to four times. To facilitate routine observation and work with the animals, counter-illumination (OSRAM light tubes cool white 36 W) is installed in the tables supporting the temperature-controlled aquaria. Portable light boxes made of wood covered with an 8 mm plexiglass plate can be used for manipulating animals outside the wet lab or in the field. The inside of the boxes are painted black to accentuate the light/shadow transition, and they are equipped with a circular light bulb (OSRAM Circolux 24W/827, Lumilux warm white 220-240 V-E27).


Culture optimization for the emergent zooplanktonic model organism Oikopleura dioica.

Bouquet JM, Spriet E, Troedsson C, Otterå H, Chourrout D, Thompson EM - J. Plankton Res. (2009)

Collection and culture of Oikopleura dioica. (A) Animals are collected using a customized plankton net with a transparent, large, plexiglass cod end. (B) Overview of the culture system diagrammed in E. (C) Animals are cultured in 6 L of seawater in 8-L polycarbonate beakers and maintained in suspension by a polyvinylcarbonate (PVC) paddle rotating at 15 rpm. (D) The beakers are supplemented with grains of activated charcoal to regulate water quality over time. The paddles are made with a 3 mm thick PVC plate attached to an 8 mm diameter PVC rod using PVC glue. Dimensions (cm) of the paddle (inset) are adjusted according to developmental stage. H×L×W = 25×30×8 for spawning, days 1 and 2. The dimensions for other developmental stages are 25×30×7. (E) Schematic illustration of physical organization of the culture system. The fluorescent lighting mounted in the bench top greatly facilitates working with the transparent animals without the need to displace beakers to light boxes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651036&req=5

FBN132F4: Collection and culture of Oikopleura dioica. (A) Animals are collected using a customized plankton net with a transparent, large, plexiglass cod end. (B) Overview of the culture system diagrammed in E. (C) Animals are cultured in 6 L of seawater in 8-L polycarbonate beakers and maintained in suspension by a polyvinylcarbonate (PVC) paddle rotating at 15 rpm. (D) The beakers are supplemented with grains of activated charcoal to regulate water quality over time. The paddles are made with a 3 mm thick PVC plate attached to an 8 mm diameter PVC rod using PVC glue. Dimensions (cm) of the paddle (inset) are adjusted according to developmental stage. H×L×W = 25×30×8 for spawning, days 1 and 2. The dimensions for other developmental stages are 25×30×7. (E) Schematic illustration of physical organization of the culture system. The fluorescent lighting mounted in the bench top greatly facilitates working with the transparent animals without the need to displace beakers to light boxes.
Mentions: The culture protocol for O. dioica is based on a system originally developed by Fenaux and Gorsky (Fenaux and Gorsky, 1985). The animals are raised at 15.0 ± 0.5°C in 8 L polycarbonate beakers (Cambro camwear® RFSCW8), containing 6 L of seawater (Fig. 4). Animals and algal feed are maintained in suspension by the rotation of a polyvinylcarbonate paddle connected to an electric motor (Synchromotor Crouzet, 15 rpm; 82.334.5 15 rpm AIG; Tufvassons PFS 150S transformer, 6124-0080, 230-24 V, 50–60 Hz, 6.3 A). Motors are mounted in parallel, and are individually protected by a fuse (1/4 A 250 V). The animals in the beakers with their forage organisms are cultivated in 10–12 h light and 14–12 h dark cycles. Under these conditions the life cycle is 6 days. To manipulate the animals, we use polypropylene beakers for dilution at days 1 and 2, and plastic Sterilin pipettes with blunt cut tips to transfer the animals to fresh beakers on days 3–6. The inside diameter of the transfer pipettes varies from 6 to 10 mm as a function of animal house size. To maintain water quality in beakers over a 24 h period, ∼10 g of pre-rinsed activated charcoal pellets (VWR charcoal 1–3 mm gradient, 12 700-5), are added to each beaker, with the exception of beakers used to accomplish the spawn. Charcoal pellets can be washed under running seawater and re-used up to four times. To facilitate routine observation and work with the animals, counter-illumination (OSRAM light tubes cool white 36 W) is installed in the tables supporting the temperature-controlled aquaria. Portable light boxes made of wood covered with an 8 mm plexiglass plate can be used for manipulating animals outside the wet lab or in the field. The inside of the boxes are painted black to accentuate the light/shadow transition, and they are equipped with a circular light bulb (OSRAM Circolux 24W/827, Lumilux warm white 220-240 V-E27).

Bottom Line: This urochordate, has a simplified anatomical organization, remains transparent throughout an exceptionally short life cycle of less than 1 week and exhibits high fecundity.At 70 Mb, the compact, sequenced genome ranks among the smallest known metazoan genomes, with both gene regulatory and intronic regions highly reduced in size.The organism occupies an important trophic role in marine ecosystems and is a significant contributor to global vertical carbon flux.

View Article: PubMed Central - PubMed

Affiliation: Sars International Centre for Marine Molecular Biology, Thormøhlensgate 55, N-5008 Bergen , Norway.

ABSTRACT
The pan-global marine appendicularian, Oikopleura dioica, shows considerable promise as a candidate model organism for cross-disciplinary research ranging from chordate genetics and evolution to molecular ecology research. This urochordate, has a simplified anatomical organization, remains transparent throughout an exceptionally short life cycle of less than 1 week and exhibits high fecundity. At 70 Mb, the compact, sequenced genome ranks among the smallest known metazoan genomes, with both gene regulatory and intronic regions highly reduced in size. The organism occupies an important trophic role in marine ecosystems and is a significant contributor to global vertical carbon flux. Among the short list of bona fide biological model organisms, all share the property that they are amenable to long-term maintenance in laboratory cultures. Here, we tested diet regimes, spawn densities and dilutions and seawater treatment, leading to optimization of a detailed culture protocol that permits sustainable long-term maintenance of O. dioica, allowing continuous, uninterrupted production of source material for experimentation. The culture protocol can be quickly adapted in both coastal and inland laboratories and should promote rapid development of the many original research perspectives the animal offers.

No MeSH data available.