Patent Document

FIELD OF THE INVENTION 
   The present invention relates to apparatus for culturing marine animals and more particularly to agitators for culturing invertebrate larvae or juveniles. 
   BACKGROUND OF THE INVENTION 
   Culturing marine invertebrate larvae and other zooplankton is often difficult. In particular, it is difficult to rear large numbers of invertebrate larvae and other zooplankters for gene expression studies, or to establish healthy cultures for exposure to a variety of experimental conditions. 
   Prior art methods for rearing marine invertebrate larvae include a method for stirring larval cultures within closed containers using paddles, as disclosed in Strathmann R R (1971), “The Feeding Behavior of Planktotrophic Echinoderm Larvae: Mechanisms, Regulation and Rates of Suspension-feeding”, J Exp Mar Biol Ecol 6: 109-160. An alternative method in the art uses water droplets, as disclosed in Hadfield as quoted in Strathmann M F (1987), “Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast: Data and Methods for the Study of Eggs, Embryos, and Larvae”, University of Washington Press, Seattle and London. Such methods maintain a high concentration of algal food sources for the planktotrophic larvae, but they are not self-cleaning, as water is not continuously cleared from the culturing vessel. 
   There are techniques in the prior art for rearing marine organisms in an open aquarium having constantly running seawater exiting through a mesh-covered opening. These are inconvenient as more fragile organisms tend to get caught on the mesh-covered opening, due to the excessive force of the water flow. Devices that circumvent this problem are designed to generate gentle water flows, as disclosed in Greve W (1968), “The “Planktonkreisel”, a New Device for Culturing Zooplankton”, Mar Biol 1: 201-203; and Ward W W (1974), “Aquarium Systems for the Maintenance of Ctenophores and Jellyfish and for the Hatching and Harvesting of Brine Shrimp ( Artemia salina ) Larvae”, Chesapeake Sci 15: 116-118, but they are not a good option for culturing more robust lecithotrophic larvae because they provide insufficient agitation to clear out debris and yolk from eggs that fail to develop properly. A system of cages suspended in flowing water, as disclosed in Høeg J T (1984), “A Culture System for Rearing Marine Invertebrate Larvae and its Application to Larvae of Rhizocephalan Barnacles”, J Exp Mar Biol Ecol 84: 167-172, also does not allow for easy manipulation, sampling, stirring, and collection of individuals in culture, nor does it keep some types of buoyant larvae from being forced onto the ceiling of the enclosure. 
   SUMMARY OF THE INVENTION 
   The apparatus according to the invention circulates larvae, provides an easy mode of collection, labeling, and cleaning, is scalable and has many possible applications. The apparatus can be deployed quickly, and as it does not require any electric or mechanical components, is ideal for short and long-term work at marine stations with running seawater, and is especially suited to culturing non-feeding larvae. 
   An apparatus for culturing individuals of a marine species is provided including a base supporting a plurality of culture containers, such as beakers, a portion of each of the culture containers submersible within a sea table, the portion of each of the culture container having a mesh surface; and a seawater supply, the seawater supply providing a flow of seawater from a seawater source to each of the culture containers; wherein the mesh surface allows passage of said seawater through the culture containers but prevents passage of the individuals. 
   The mesh surfaces may be at the bottom of each of the culture containers. The bottom of the culture container may be suspended above a surface of the sea table. The apparatus may include a lid that positions the seawater supply. The lid, itself, may be positioned above the base. 
   The seawater supply may be a tube in fluid communication with and drawing seawater from the seawater source. The tube may have a plurality of apertures, each of the apertures positioned above a culture container. Each of the apertures may hold a water conduit for providing a stream of seawater from the tube to the container. The lid may have a plurality of apertures, each of the apertures on the lid alignable with the apertures on the base, thereby allowing access to the containers. A plurality of clamps below the lid may position the water conduit. 
   An apparatus for culturing individuals of a marine species is provided, including means for holding the individuals within a container positionable within a sea table; means for allowing seawater to enter and pass through the container; and means for allowing waste to exit the container. 
   A method of cultivating individuals of a marine species is provided, including steps of: providing a container for containing said individuals, said container positioned within a sea table, said container having a mesh surface through which said individuals cannot pass; and providing a flow of seawater into and out of said container, said seawater removing waste from said container. The mesh surface may be positioned at the bottom of the container and above a surface of the sea table and the seawater may be provided by a supply tube accessing a seawater source. 

   
     DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a perspective view of the apparatus according to the invention positioned within a sea table; 
       FIG. 2  is a perspective view of the lid and base therein, showing the lid and base separated; 
       FIG. 3  is a bottom view of the lid thereof; 
       FIG. 4  is a top view of the lid thereof; 
       FIG. 5  is a partial side view of the lid thereof showing a clamp fitted around a main supply hose and mounted to the underside of the lid; 
       FIG. 6  is a cross sectional side view of a portion of the apparatus according to the invention; 
       FIG. 7  is a perspective cutaway view of a clamp and piping piece; 
       FIG. 8  is an alternate view of a clamp; and 
       FIG. 9  is a view of a culture container and supply tube. 
   

   DETAILED DESCRIPTION OF THE INVENTION 
   The apparatus according to the invention, includes agitator  1 , which uses the supply of seawater typically found at a location such as a marine field station, and stirs delicate organisms by gently and constantly directing a stream of water into a culture of organisms. Agitator  1  is particularly useful for culturing lecithotrophic echinoderm larvae with long developmental times at low ambient temperatures through metamorphosis. Such larvae represent relatively large, buoyant, fragile organisms that are prone to fouling in a standing culture, because the death and subsequent disintegration of a few individuals may result in bacterial overgrowth and anoxic conditions. The apparatus according to the invention provides a space efficient solution in which decaying debris is prevented from accumulating, while healthy individuals are easily collectable, and are able to make contact with the culturing container&#39;s walls so that settlement and metamorphosis can occur. While this document discloses, as an example, the culturing of lecithotrophic echinoderm larvae, other types of marine life, particularly invertebrate larvae may also be cultured using the method and apparatus described herein. 
   Agitator  1  is designed for placement in sea table  100 , as seen in  FIG. 1 . A sea table is an enclosure into which running seawater is collected and evacuated at a constant rate and is available at many marine stations and aquaculture facilities. Sea table  100  includes a water conduit, that provides supply tube  15  with seawater. The seawater may be obtained through supply tube  15 , from a seawater source, such as the sea or large reservoir, to table  100 . Table  100  includes raised edges  127  to hold a volume of seawater. As the water level rises due to the flow of water arriving from water supply tube  15 , excess water exits sea table  100  by draining from the bottom of sea table  100  using a stand pipe to maintain an adequate water level. Draining occurs at the same flow rate as seawater enters table  100 . In alternative embodiments of the invention, rather than placing agitator  1  within a sea table  100 , agitator  1  may be placed within seawater, using flotation devices or the like. In yet another alternative, the water level of sea table  100  may be maintained by allowing the seawater to pass over edges  127 . 
   Agitator  1  may be sized to fit within sea table  100 . Agitator  1  includes a plurality of open culture containers  10 . In a preferred embodiment of the invention, culture containers  10  are plastic beakers. Each culture container  10  is preferably positioned within sea table  100  so that rim  17  of culture container  10  rests just above the water level  125  of sea table  100 . Water is distributed into the plurality of open culture containers  10 , and as the pressure decrease in the main water supply tube  15  is less than the pressure decrease in the individual outlet conduits  20 , which lead water from water supply tube  15  to each culture container  10  as seen in  FIG. 1 , the water flow to culture containers  10  is approximately constant, laminar and gentle. Alternatively, a pump could be used to maintain the water flow. 
   As seen in  FIGS. 2 and 6 , agitator  1  includes three components: a plurality of culture containers (also referred to herein as beakers)  10  having mesh surface bottoms  30 , for holding the organisms (as seen in  FIG. 6 ), a base  60  to position rim  17  of beakers  10  above the water level  125  of sea table  100 , and lid  80  to position outlet conduit  20  carrying seawater to culture containers  10  from main supply tube  15 . Seawater enters each culture container  10  via main supply tube  15  and outlet conduits  20 . Water then exits culture containers  10  via mesh surface  30 . Such water flow allows for self-cleaning of culture containers  10  as waste is removed. As shown in the embodiment represented in the Figures, a mesh bottom  30  is used, although more of, or other portions of, culture containers  10  may be mesh. Mesh bottoms  30  allow seawater to pass into the surrounding sea table  100  wherein water level  125  is maintained. 
   Agitator  1  thereby focuses a stream of seawater into each culture container  10 , from through main supply tube  15 , above the culture container  10 , in the directions indicated by the arrows in  FIG. 6 , such as downward direction  50 , which shows the direction of the water leaving main supply tube  15  via the outlet tube  20  and entering culture container  10 . Beaker  10  is of sufficient depth to allow the individual specimens enough room so that they do not impact mesh bottom  30  with excessive force from seawater entering the container  10  from above water level  125 . For this reason 800-ml plastic beakers may be suitable, although other sizes of beakers may be used, depending on the marine life being cultivated. If such standard beakers are used, they may be modified for use with the apparatus  1  by removing the bottom of the beaker with a tool, such as a belt sander, and attaching a 340-μm size or appropriate sized nylon mesh for the organism to be cultured in its place using a bonding agent, such as commercial thermoplastic. The mesh size for mesh bottom  30  will vary depending on the organism to be cultured, but it should be smaller than the egg diameter of the organism. 
   Base  60  of agitator  1  arrays beakers  10  and provides clearance between bottom  30  of container  10  and surface  122  of table  100  so that water may flow away from the culture below container  10 , and into the surrounding sea table  100 . Base  60  can be sized to fit any sea table or accommodate any number of containers, and may be made of plywood, plexiglass, or a 6-mm polycarbonate sheet  55 , as shown in  FIG. 2 . Large (for example, 10.2-cm) diameter apertures  65  are positioned rows and columns within the sheet  55  to accommodate containers  10 . To ensure adequate spacing, apertures  65  should be spaced approximately 2.5 cm, both from each other, and from the edges of sheet  55 , as seen in  FIG. 2 . 
   Legs  70  of base  60  may be constructed from 2.5-cm (1-inch) diameter acrylic rods or another suitable material. Legs  70  should be of a length so that culture containers  10  do not touch the bottom  122  of the sea table  100  as seen in  FIG. 6 . Legs  70  are attached to sheet  55 , for example with a stainless steel bolt  75 . Preferably, legs  70  are not attached to the corners of sheet  55  as this adds stress to the base  60 , but are mounted more centrally (for example, within the first row and column on each side of sheet  55  as seen in  FIG. 2 ). Rubber caps  82  or other stoppers may be fitted onto legs  70  to provide a more secure footing on sea table  100 , or alternatively, legs  70  may be fixed to sea table  100 . Caps  82  can also be used to adjust the height and weight of base  60  by filling caps  82  with a spacer such as sand. The height of base  60  in relation to water level in sea table  100  should be such that culture containers  10  are only partly submerged (as shown in  FIG. 6 ). The water level  125  can also be adjusted by changing the length of the standpipe, rather than changing the height of base  60  as the length of the stand pipe draining water from the sea table  100  determines the height of the water surface  125  in the sea table. Therefore if a shallow depth is desired, the pipe can be shortened. 
   As seen in  FIGS. 3 and 4 , lid  80  provides a scaffold for supply tube  15  and allows access to the cultures within culture containers  10  while agitator  1  is in operation by maintaining a distance between lid  80  and base  60 . Lid  80  may be constructed from a polycarbonate sheet  85  that corresponds to base  55 , as seen in  FIG. 2 . As seen in  FIG. 6 , the edge of apertures  90  within lid  80  can be used to accommodate labels, for example 1-ml plastic pipette tips  84  with tape attached can be used to label simultaneous cultures of different species or individuals fertilized at different times. Alternatively, as seen in  FIG. 3 , lebales may be inserted in smaller apertures  86  adjacent to each aperture  90 . Spacers  200  that may be approximately 5 cm long are used to raise lid  80  above base  60  so that supply tube  15 , mounted under lid  80 , does not rest on containers  10  mounted in base  60 . Rubber leg caps  82  as seen in  FIG. 6  can be used to adjust the height of spacers  200  as seen in  FIG. 3  as well. 
   Clamps  210 , positioned between the apertures  65  that accommodate culture containers  10  and at the edges of sheet  55  as seen in  FIG. 3 , may be used to guide main supply tube  15  over the center of each culture container  10 , as seen in  FIGS. 1 and 9 . To mount clamps  210 , small apertures may be provided, for example drilled, into lid  80  to hold a nylon nut and bolt. These apertures may receive clamps  210  (which may be 2.2-cm (⅞-inch) plastic clamps) that anchor supply tube  15 , as seen in  FIGS. 7 and 8 . Clamps  210  may be fitted around the main supply tube  15  with 2.5-cm long, 1.9-cm (¾-inch) diameter PVC pipe portions before clamps  210  are attached to lid  80 . Various embodiments of clamps  210  are shown in  FIGS. 5 ,  7  and  8 . Pipe portions  240  hold main supply tube  15  more securely than clamp  210  alone. Alternatively, two small apertures may be used to hold supply tube  15  in place with a cable tie (not shown). Other means of holding supply tube  15  to lid  80  may be used including adhesives, tape or ties. Supply tube  15  is positioned at the underside of lid  80  by running it through clamps  210 . Preferably, a continuous length of clear plastic tubing is used for supply tube  15 , with both open ends joined at a Y connector  230 , as seen in  FIG. 4 . Such a design allows for maintenance of approximately equal pressure throughout the entire system. Preferred tubing may be a tube with a 1.6-cm (⅝-inch) outer diameter, and 0.95-cm (⅜-inch) inner diameter. 
   After tube  15  is aligned over each beaker  10  using clamps  210 , small apertures  220  positioned at the underside of supply tube  15  are used to direct water into containers  10 . Apertures  220  may have a 0.4-cm diameter. Preferably each aperture  220  is positioned above the center of a culture container  10 . When preparing agitator  1  for use, it is preferable to drill apertures  220  into supply tube  15 , after supply tube  15  is secured to clamps  210  so that tube  15  does not twist, upsetting the downward orientation of apertures  220 . Outlet conduits  20  may be placed through apertures  220 , to ensure that the flow of water into container  10  is gentle. Examples of such a gentle flow may include a fast drip, or a minimal continuous flow (i.e. just greater than a constant fast drip). Outlet conduits  20  may be VWR select grade plastic PVC tubes with a 0.4-cm ( 5/32-inch) outer diameter, and 0.24-cm ( 3/32-inch) inner diameter. The length of outlet conduits  20  is preferably sufficient so that the water exits outlet conduit  20  between a positioned submerged beneath water level  125 , to within about 1-cm above water level  125 , depending on the desire to break the surface tension of the water and the level of agitation. 
   Both ends of supply tube  15 , once secured to clamps  210 , are connected to Y connector  230 , which then leads to a seawater source. An inline filter may be placed between the water source and the Y adaptor  230  to remove large debris, which may otherwise collect in culture containers  10  over time. The inline filter should have a nylon mesh of a smaller size than that used for the base  30  of culture containers  10  (for example, a 200-μm mesh). A disc of such a nylon mesh can be cut to fit the inline filter. 
   Agitator  1  therefore allows the containers  10  to be cleared of debris and waste while keeping larvae intact and is therefore self-cleaning, i.e. the necrotic (dead) larvae do not need to be removed from the container using other means. Agitator can supply a very smooth stream of water that is sufficient to clear oily residue from decaying tissue but gentle enough to move the organisms within container  10  thereby mimicking movement patterns in the ocean within a small space. 
   Operation of the Agitator 
   The desired level of water flow can be controlled using a stopcock or valve near the seawater source. Preferably the water flow is gentle, for example a rate of between 2 to 2.5 liters per minute from the water source to supply tube  15 . The preferred flow from outlet conduits  20  to culture containers  10  is maintained at a gentle minimal flow. The larvae in beakers  10  do not need to be in constant motion for agitator  1  to be effective and water flow may be minimized so that the cultures are not agitated too vigorously. Outlet conduit  20  may be slightly bent to establish a cyclical stirring action within culture container  10 . Outlet conduits  20  may also be inserted deeper into main tube  15  to slow water flow. In cases where apertures  65  do not contain a container  10 , individual outlet conduits  20  may be blocked, for example using a cork fitted into a conduit  20  or aperture  220 , or valves. A plastic pipette tip inserted into a flexible outlet conduit  20 , and the submergence of outlet conduit  20  beneath the water level, slows the flow of water to minimize agitation, thereby allowing competent larvae to settle. 
   Individual specimens can be removed in small numbers from culture container  10  with a pipette as lid  80  allows access to agitator  1  via aperture  90  while it is pressurized and water is flowing through main tube  15 . Cultures can be removed from agitator  1  easily so that they can be observed, fixed, or culture containers  10  cleaned. To collect an entire culture quickly for sorting or study, lid  80  can be removed and culture containers  10  lifted out of base  60  so that only a small amount of water remains in culture container  10 . The larvae or embryos can then be easily collected with a large pipette or turkey baster. 
   It may be important to keep components of agitator  1  clean. For example, an inline filter in supply tube  15  may acquire a build up of particulate matter. Bubbles trapped over the outlet apertures  220  of main supply hose  15  during filter changes may block the flow of water. Supply hose  15  may therefore be bled by temporarily increasing the flow of water after replacing the filter. If a heavy biofilm builds up in the culture container meshes  30 , they may be rinsed if the larvae have not reached the stage at which metamorphosis is imminent. 
   Experiment 
   An agitator according to the invention was used to culture the larvae of a starfish,  Solaster stimpsoni  (Verrill). A total of 56 beakers were used as culture containers in four separate agitators (three 12-beaker arrays and one 20-beaker array using a total of two sea tables). After fertilization, the embryos were immediately transferred into the agitator. The starfish larvae were cultured at densities of up to 300 individuals per 800-ml beaker through metamorphosis and the juvenile stage (6 wks at an ambient temperature of 10° C.), although longer culture periods are possible. The agitator successfully cultured approximately 16,000 larvae during two field seasons at the Friday Harbor Laboratories (Friday Harbor, Wash.). The mortality rate was not precisely measured but no significant or dramatic reduction from the initial egg number was observable and the young starfish survived in great numbers. After nine days at 10° C., the larvae of  Solaster stimpsoni  developed adhesive disks and larval arms with which they attached either to the mesh or the walls of the beaker without the need to add settlement cues. 
   Applications and Modifications 
   The apparatus was also used to culture larvae of a holothurian,  Psolus chitonoides  (Clark), which resemble those of  S. stimpsoni  in terms of fragility and developmental mode. In addition to culturing lecithotrophic larvae, the agitator could also be used for a variety of other purposes, such as culturing small and delicate zooplankters including ctenophores, polychaete epitokes, or egg masses. Once juveniles hatch from the egg masses, they can be immediately cultured in the same container as the egg mass. It is also possible to raise planktotrophic larvae and conduct experimental manipulations of culture conditions. Such larvae could be reared within the apparatus by using a smaller mesh size and inserting a small catheter connected to an IV drip bag filled with algae into the main supply hose  15 . Alternatively, the water flow could be temporarily stopped and food added to the sea table. Treated water sources (carrying predator cues etc.) could be added to a culture via the same methods or by siphoning water from an aquarium harboring the predator into the main hose of the agitator. The construction of a sea table that is subdivided to prevent the cross-contamination of seawater along with the deployment of separate (and smaller agitators) may also be used. 
   Although the particular preferred embodiments of the invention have been disclosed in detail for illustrative purposes, it will be recognized that variations or modifications of the disclosed apparatus lie within the scope of the present invention. In particular, there are numerous ways of introducing a gentle flow of water to the containers without departing from the spirit of the invention.

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