Patent Application: US-67837908-A

Abstract:
this invention relates to a novel method of culturing coral tissues and polyps in vitro . coral tissues obtained by the method of the invention may be maintained as heterotypic spheroid tissue balls for a period of at least three months or they may be induced to undergo development into new polyps , a process termed re - morphogenesis . this method can produce genetic clones of model species from single individuals that can be propagated either as undifferentiated tissue calli or as developed polyps . the products of the invention are of value to a number of educational , scientific , and commercial endeavors . specifically , this method can be used to propagate genetic clones of a model organism for scientific research , to serve as ‘ pro - environmental conservation ’ sources of coral stock for educational specimens as well as a rapidly generated inventory for commercial aquarium industry . the method of the invention can produce sustainable test lines of corals that could be used to generate risk assessments for the impact of chemicals / activities on coral reefs , as well as being used as part of a regulatory protocol for testing waste effluent and other discharges .

Description:
the present invention provides novel methods for generating scleractinian coral tissue spheroids as well as functional coral polyps in vitro from differentiated coral tissue . the tissue spheroids and polyps are derived from a single genetic source and are therefore genetically identical , i . e . they may be defined as coral lines or clones . the coral clones or lines of the invention may be used for ecotoxicological , biomedical , and developmental studies . tissue explants obtained by the method of the invention are not only viable , but also possess the potential to undergo full re - morphogenesis to a completely developed polyp . these tissue - originating primary polyps have good survival rates (˜ 20 - 50 %). the long - term survival of the clones ( for over 3 months in tissue grade state and for over one year as polyps in culture ) provides a basis for their usefulness in short or long term coral studies . as used herein , the term “ scleractinia ” refers to “ stony ” corals which are exclusively marine animals comprising soft tissue and a hard skeleton . as used herein , the term “ ecotype ” refers to a distinct breed of organisms that is closely linked in its characteristics to the ecological surroundings it inhabits . as used herein , the term “ coral tissue ” refers to soft tissue of scleractinia corals comprising three layers : an outer epidermis ( the embryonal ectodermal layer ), a gastrodermal layer ( the embryonal endoderm layer ), and between them a mesoglea . the term “ aragonite skeleton ” refers to the rigid scleractinian skeleton , which lies external to the polyps that make it , and is composed of calcium carbonate in the crystal form aragonite . as used herein , the term “ coral tissue fragment ” refers to a fragment which includes ectoderm , endoderm , and mesoglea , but is devoid of skeletal tissue . as used herein , the terms “ spheroid ” “ callus ” and “ explant ” are used interchangeably and refer to coral a tissue fragment which is maintained viable in culture in an undeveloped form , i . e . it does not evolve into a polyp . as used herein , the term “ polyp ” refers to a coral , having a roughly cylindrical body and an oral opening usually surrounded by tentacles . as used herein , the term “ re - morphogenesis ” refers to a development process in which the spheroid reorganizes its body form into a polyp by developing mouth , septae and tentacles ( see fig2 c - f ) and thus a polyp is formed in culture . as used herein , the terms “ seawater ”, “ seawater media ” or “ seawater solutions ” are used interchangeably to denote media or solutions having seawater properties . seawater , i . e . the water of the sea , is distinguished from freshwater by its appreciable salinity . this salinity is mainly achieved due to the presence of sodium and chloride ions , however certain trace elements e . g . magnesium , calcium and potassium are also present . seawater may be obtained from a sea or produced artificially by reconstitution of the seawater content , i . e . by supplementing fresh water with ions (“ artificial seawater ”). in the context of the present invention , the concentration of the ions in the “ seawater ”, “ seawater media ” or “ seawater solutions ” may be adjusted according to the culture requirement e . g . the amount of calcium , chloride , magnesium etc may be increased or reduced . in addition , certain modifications may also be made in the seawater ph . as used herein , the term “ toxicology ” refers to the study of the adverse effects of chemical and physical agents on living organisms . excision of coral tissue fragments from polyps and cultivation of viable coral tissue explants coral tissue fragments are obtained from adult corals and excised into pieces of approximately 1 - 3 mm 3 using sterile instruments , such as fine tweezers ( no . 5 dumont ), and aseptic techniques . immersion of corals in a modified seawater based solution ( e . g . calcium free seawater ) for up to 6 hours can also be used in several species ( faviids or pocillopora ) in order to assist in the release of tissues from the skeleton . following this the tissues are rinsed a number of times in filtered or artificial seawater . the pieces of tissue are then transferred into sterilized tissue culture solution in sterile vessels . preferably said tissue culture solution comprises seawater . the seawater may either be filtered natural seawater or artificial seawater which is commercially available . the seawater includes ca 2 + and mg 2 + . following formation of tissue spheroids ( explants ) ( between 2 - 4 days ), the spheroids are transferred to sterile culture vessels containing a culture solution , preferably seawater . spheroid growth rates can be enhanced by supplementing the culture with optimal intensities of photosynthetic photon flux densities of approximately 20 - 30 μmol / m 2 s . culturing of viable coral spheroids can be carried out in complete darkness , but this requires a specific supplemental formula to the culture solution , since in complete darkness algae survival is compromised and an external source of food is required , e . g . amino acid preparations or artemia . maintenance of coral spheroid cultures should be within the optimal temperature range for the species , with periodic changes of tissue culture solution . this may be experimentally determined for each ecotype or genotype within a specific environment . for example , the optimal temperature for maintenance of a spheroid of the red sea , e . g . f granulosa is about 19 ° c . to about 21 ° c . the temperature is preferably not higher than about 22 ° c . for this species / genotype , as in higher temperatures the spheroids will be induced to undergo re - morphogenesis . for coral species which originate from seas having a cooler lower range of water temperature e . g . the mediterranean sea , a lower temperature can be used , e . g . about 16 ° c . for oculina patagonica . coral species or ecotypes may also be obtained from cold sea environments typical for example to deep - sea waters , at which case even colder temperatures may be used for maintenance . to induce polyp development , tissue spheroids are maintained at an optimal culturing temperature for each species / ecotype ( for example for red sea f . granulosa temperature range of about 22 ° c . to about 30 ° c .) and are subjected to the following protocol : ( 1 ) one week after tissue excision from the polyp , ⅓ - ½ of the culture solution is carefully removed and new culture solution is added so that the volume remains unchanged . ( 2 ) culture solution is refreshed every 7 - 14 days as described above , while avoiding mechanical disruption of the contact between the tissue spheroid and the culture vessel surface . ( 3 ) once the tissue explants have settled on the culture vessels ( which occurs about 7 days or more after excision ), the vessels are cleaned of any algal , bacterial , or invertebrate fouling by wiping the surfaces , such as by using a sterile nylon no 2 paintbrush . these protocols , including filtration , are preferably carried out in glassware or other chemically inert material . ( 4 ) once a mouth , septae and tentacles develop ( a mouth - about two weeks after settlement , septae - after about one more week and tentacles after about another week , and in parallel skeleton deposition commences ) the culture vessels are transferred to a water table or larger culturing vessel . the polyps are fed weekly with artemia nauplii ( 1 - day following hatching ) or bryozoan recipe , or any suitable coral food known to a person skilled in the art . following the feeding the water is changed and the vessel surfaces are kept clean . the cultures are maintained under optimal temperature conditions for that coral species , for example as determined by the temperature of the sea from which the coral species is obtained . the “ red sea ” being an example of warm temperature conditions , e . g . 22 - 30 ° c . while the mediterranean sea being an example of cool temperature conditions e . g . 16 - 30 ° c . the process of culturing the biopsies creates tissue that follows polyp genesis . using this process , reorganization of the two primary tissue types occurs . this is followed by invagination and settlement of the coral tissue mass . settlement is followed by primary structure formation , including the oral invagination , septae , and tentacles . ultimately , the polyp deposits aragonite skeleton and grows . the protocol is configured for mass tissue culturing of over a hundred biopsies taken from a single coral polyp source ( i . e . single genetic source ) and can be then harvested with or without an aragonite skeleton . moreover , the re - morphognesis can repeat itself as second generation of polyp cultures can be obtained in accordance with the invention . thus suggesting line - characteristics for the coral tissues obtained in accordance with the invention . the adult polyps that the tissues were extracted from were maintained in the lab for over a year . this suggests that this method can also be successfully used in aquaculture as well as in biological studies . the formation of zooxanthellae - free polyps can be used in bleaching studies . bleaching causes great concern worldwide ( goreau and hayes 1994 , brown 1997 , hoegh - guldberg 1999 ). bleaching can be achieved by using chemical means , e . g . antimycotics or antibiotics , e . g . cycloheximide . the method of the invention is suitable for culturing scleractinian corals , including but not limited to the solitary coral fungia granulosa and , and the colonial corals favia favus and oculina patagonica . tissue from the red sea coral fungia granulosa was removed mechanically using fine tweezers . the tissues were taken from the mouth region ( m ) or from the peripheral ( p ) region of the coral polyp ( fig1 ). ten to thirty tissue explants were transferred via a number of washes in 22 μm fsw ( filtered sea water ) and then placed in 3 - 12 cm petri dishes filled with 22 μm fsw for a period of 24 hours until tissue rounding ( callus formation ) was evident . in order to minimize possible infections by mucus associated microorganisms surface mucus was removed from the corals prior to tissue excision , by placing them on a funnel and allowing the mucus to drip for 20 minutes . the corals were then returned to an aquarium with filtered seawater , and allowed to recuperate for two days prior to removal of tissue . in order to test viability of the tissue explants a neutral red assay was performed ( weeks and svendsen 1996 , stachowicz and hay 1999 ). tissues were maintained at a temperature of 24 ° c . and under 20 μmol / m 2 s of light for 10 days . the tissues were then placed in a solution of neutral red , diluted in 0 . 22 μm fsw ( 0 . 57 g / l ) for 10 minutes . the tissues were washed in fsw and their viability was shown . tissues that incorporate the dye are viable tissues while those that do not are moribund ( weeks and svendsen 1996 , stachowicz and hay 1999 ). after the formation of polyps from calluses or explants , the polyps were transferred into an aquarium containing seawater and aeration , or put in a closed water flow system . polyps were then fed weekly with artemia nauplii following which water was replaced ( natural or artificial sea water ) if necessary . polyps were maintained under commercially available t5 fluorescent lights ( white and blue spectra ) or natural sunlight . tissue was excised from two 10 - month old cultured polyps that had been maintained in an aquarium ( see tissue origin experimental conditions ). after forming calluses the f2 were placed in glass petri dishes filled with 0 . 45 μm fsw , in 32 μmol / m 2 s of light and a daily temperature cycle of 23 - 30 ° c . in addition , in order to activate swift release of fungiid polyps from their substrate and from their stalks , the explants were maintained in two light regimes , high ( 130 μmmol / m 2 s ) and low light ( 20 mol / m 2 s ). tissue from the red sea colonial coral favia favus was removed mechanically using fine tweezers . tissues were rinsed in filtered natural seawater ( 0 . 22 μm fsw ) placed in glass petri dishes 24 hours after removal . the tissues were maintained under the same conditions as the fungiid corals . tissue from the mediterranean coral oculina patagonica was removed mechanically using fine tweezers . tissues were rinsed in filtered natural seawater ( 0 . 22 μm fsw ) placed in glass petri dishes 24 hours after removal . the tissues were maintained under the same conditions as the fungiid corals . polyps were transferred into different concentrations of the fungicide cycloheximide ( sigma cat no : 01811 ) ( 10 mg / 1 , 20 mg / l and 28 mg / l ) for a period of one month . polyps were placed under 20 μmol / m 2 s of light and a daily temperature of 25 ° c . using fine tweezers tissue fragments were explanted from an adult coral fungia granulosa that had been fragmented using a hammer and clean chisel . approximately 24 hours after explanting , the tissues rounded up into a planula - like morphology ( see fig2 a ) and became very motile . in order to determine viability of the tissues a neutral red viable staining test was performed . as shown in fig2 a the live tissues were dyed red , whereas the dead or disintegrated ones did not take in the dye . the tissue explants , which can also be referred to as calluses , were maintained in this form for up to three months when the water temperature was low (˜ 19 ° c .). the explants were not only viable , but also showed the potential of becoming a fully - grown polyp . when maintained in the proper conditions , the callus or explant settles and develops a mouth , septae and tentacles ( see fig2 c - f ). this process is referred to as re - morphogenesis in which a tissue from an adult polyp reorganizes its body form into a new polyp . this process in the optimal conditions occurs within a month : settling after a week , forming a mouth after two weeks , forming septae after three weeks and tentacles after four weeks . the optimal protocol for maintaining this polyp culture was determined after a series of experiments . the main parameters that were examined are survivorship of the explants ( or polyps in the later stages ) or mouth development — a stage which represents the turning point in which an explant or callus becomes a polyp . experiments were performed to determine the optimal conditions for survivorship and development ( formation of mouth as a characteristic of polyp formation ) of the tissues . the survivorship parameters refer to tissue survivorship without taking into account if the tissues developed into polyps or remained at tissue grade stage . this parameter was used to establish optimal conditions for primary stages of tissue or polyp culture . on the other hand , mouth formation is a characteristic of re - morphogenesis and therefore the establishment of polyp culture . i . general : in order to examine the effect of the substrate , excised tissues of approximately the same size were placed in petri dishes with 7 different substrata . in each experiment the fragments were maintained at 23 ° c . under constant light and examined daily for settlement . this experiment was concluded after three months . 6 ) coral skeleton fragments . skeleton fragments were crushed using a hammer and sterilized in an autoclave . they were then glued to a plastic petri dish using super glue and rinsed three times in ddw and once in fsw . 7 ) mesoglea strips . mesoglea strips ( excised from the bell of rhopilema nomadica , class : scyphzoa ) were rinsed three times in ddw and were placed in a plastic petri dish with fsw . ii . substrate and antibiotics : in order to determine whether antibiotics have an effect on the survival of the tissues , tissue explants or spheroids were placed on 4 different substrates ( sterile glass petri dishes , sterile scratched glass , plastic and scratched plastic ) in fsw or fsw + antibiotics ( 0 . 5 mg / ml kanamycin and penicillin g ) sigma cat no . n2889 . the tissues were maintained at a constant temperature of 23 ° c . for two months under constant light . iii . transparencies : polyester transparency films were used in order to assess if tissue would settle on substrate that could be easily cut and manipulated . for sterility the transparencies were soaked in 70 % ethanol for 24 hours , washed in fsw before being placed inside plastic petri dishes . growth on transparencies was compared with growth on other substrates i . e . plastic and glass . glass petri dishes containing tissue explants were placed under four different light regimes : high light ( 106 μmmol / m 2 s ), medium light ( 85 μmol / m 2 s ), low light ( 22 μmol / m 2 s ), and dark ( 2 . 5 μmol / m 2 s ). in the first three weeks of the experiment the tissues were maintained under 12 : 12 light / dark regime , which was then changed to 9 hours light : 15 hours dark ( due to polyps bleaching at high light intensities ). in all experiments the temperature regime was 26 ° c . during the day and 23 ° c . during the night . two different temperature regimes were used — constant temperature ( 25 ° c .) and a cycling of daily temperature ( 23 - 30 ° c .). both regimes used white and blue light ; however the constant temperature was under 20 μmol / m 2 s of light and the cycling under 32 μmmol / m 2 s of light . tissues were separated to mouth region ( 1 cm away from parent polyp mouth ) and peripheral region . the resulting tissue explants were placed in glass petri dishes filled with fsw under ambient light conditions and under a diurnal temperature cycle of 20 - 28 ° c . some explants were maintained at low temperatures ( 19 ° c .) and monitored for morphological changes . tissues were separated to mouth region and peripheral region as above . tissue explants from each tissue type were placed in glass petri dishes filled with fsw filtered with 0 . 22 μm pore filter or 0 . 45 μm pore filter , under ambient light and an average diurnal temperature cycle of 20 - 28 ° c ., or in artificial seawater ( produced from commercially available sea salt ). the percent of explants with the characteristic in question ( survivorship or mouth development ) was counted in each dish within a treatment . it is noted that most of the explants that developed mouths survived and developed into polyps . the scoring was calculated by averaging the measured percentages . a meier - kaplan survivorship curve ( kaplan and meier , 1958 ) was developed and a cox — mantel log rank test was carried out ( see http :// www . medcalc . be / index . php ). according to the kaplan meier survival test the longest survival times in the first experiment were in the scratched plastic and scratched glass ( see table 2 ). the cox mental tests shown in table 1 , indicate that explants on the scratched substrates showed significantly higher survival rates than those on the non - scratched substrates ( p & lt ; 0 . 05 ). according to fig3 , the scratched glass shows the highest average percent survival compared to all other substrates . the lowest survivorship was demonstrated in the mesoglea and skeleton fragments substrates compared to all the other substrates in this experiment ( table 1 p & lt ; 0 . 05 ), and therefore were not used again . a second experiment was performed using four of the substrates included in the first experiment , with a supplement of antibiotics in order to examine if antibiotics may have an effect on the survival of the tissues . according to the kaplan meier overall comparison test , a significant difference was found ( p & lt ; 0 . 05 ) in survivorship between explants in antibiotics and those without antibiotics , suggesting that antibiotics has a positive effect on the survival of the tissues . interestingly in this experiment , the scratched substrates did not appear to be the best substrates for survival . according to fig4 and table 4 the substrate that shows the highest survival rates and survival time is plastic ( see table 4 ), there is a significant difference between plastic and all the other substrates ( see table 3 ). a third experiment was performed using glass and plastic substrates . in addition plastic transparencies were added as a substrate to assess the usefulness in providing a substrate which is easy to manipulate . the high survival percentage was found in explants cultured in the glass plates compared to the other substrates . there is a significant difference between glass and the other substrates ( see table 5 table 6 , fig5 ). transperancies proved to be ineffective and none of the explants survived by the end of the experiment . in the first experiment only the explants cultured on glass or scratched glass plates developed mouths ( see fig6 ). in addition in the glass substrate there were significantly more polyps that developed mouths than in the scratched glass ( table 7 , p & lt ; 0 . 05 ). furthermore , in the glass substrate the mouth development time was shorter than in all other substrates ( see table 8 ). in the second experiment , tissues in all substrates developed mouths , however in low percentages and only following a long period of time ( see fig7 and table 10 ). a supplement of antibiotics was used in order to examine if it had an effect on the mouth development , thus affecting the rates of transformation into polyps . according to the cox - mantel comparison test , no significant difference was shown ( p & gt ; 0 . 05 ) between treatments , suggesting that antibiotics have no effect on the rates of mouth development . fig7 shows , however , that the highest mouth development percentage is in the scratched plastic + antibiotics substrate . the scratched glass showed a significant difference compared to all substrates and took the longest to develop mouths ( see table 9 , 10 ). in the third experiment , there was no mouth development at all on the transparencies since none of the explants survived ( fig8 ). the highest rates of mouth development and the shortest amount of time until development was observed in explants grown on the glass substrate , with a significant difference compared to plastic ( see table 11 , 12 ). overall , it appears that glass is the most effective substrate in terms of mouth development . in this experiment , the most effective light regime for tissue survival was tested . fig9 and table 13 show that there is a significantly higher survival percentage under the dark and low light regimes . the highest average survival time ( see table 14 ) was under the low light regime . interestingly it is evident that high light showed significantly better results than medium light ( see table 13 , 14 ). in this experiment , the most effective light regime for mouth development was tested . as can be seen in table 16 , there was a significant difference between all light regimes except between low and medium light . mouths developed fastest in the high light regime ( see table 15 ). however there was some mortality in the high light regime , resulting in a lower percentage of explants with mouth at the end of the experiment ( fig1 ). a more successful light regime therefore is the low light regime that shows high percentage of mouth development , which remains persistent throughout the experiment . according to fig1 and table 17 , constant temperature showed higher rates of survivorship and higher survival time , with significant differences between the temperature regimes ( p & lt ; 0 . 05 ). the cycling showed higher rates of mouth development and shorter development time ( see fig1 and table 18 ), with a significant difference between the temperature regimes ( p & lt ; 0 . 05 ). two different seawater filtration protocols were examined in order to test their influence on the survivorship of the tissues . 0 . 45 μm filtered fsw showed higher rates of survivorship with a significant difference from 0 . 22 μm - filtered fsw ( p & lt ; 0 . 01 , see fig1 ). the effect of tissue origin from the adult polyp was examined in terms of survivorship . no significant difference between the two tissue origins ( mouth tissue and peripheral tissue ) was shown ( see fig1 ), and they had very similar survival time ( see table 21 ). the effect of tissue origin from the adult polyp was examined in terms of mouth development . no significant difference between the two origins was shown ( see fig1 ), and the time until mouth development was very similar ( see table 22 ). the effect of tissue origin was also examined in terms of tentacle development . no significant difference between the two origins was shown ( see fig1 ), and they had very similar survival time ( see table 23 ). cultured fungiid polyps develop on a short stalk attached to the glass petri dish in the aquarium . following release from the substrate , the polyp detaches from the stalk and the stalk develops into an additional polyp . in order to activate swift release of fungiid polyps from their substrate and from their stalks , the high surface light regime ( 130 μmmol / m 2 s ) was used and resulted in faster release than the low regime . to test the possibility of cultivating a second generation of tissues or polyps in culture , tissues were explanted from 10 month old adult polyps that had been previously cultivated in the lab ( see fig1 and fig2 ). mouth development reached 19 % and septal and tentacle development reached 18 % by week 8 . developmental parameters show that mouth , septae and tentacles start to develop in the third week . in order to examine the ability of other species to undergo re - morphogenesis , a similar protocol was used on the coral favia favus . the tissues roundedup 24 hours after the removal from the adult colony and became explants ( formed calluses ). four weeks later complete polyps had developed with a mouth , septae and tentacles . in order to examine the ability of other species to undergo re - morphogenesis , a similar protocol was used on the coral oculina patagonica . the complete development of the polyp is shown in fig2 . the tissues rounded up 24 hours after the removal from the adult colony and became the explants ( formed calluses ). two weeks later complete polyps had developed with a mouth , septae and tentacles . by the third week 7 / 105 fragments developed into polyps . polyps bleached one week after adding cycloheximide in all the concentrations that were used . in fig2 a healthy coral vs . a bleached coral is shown . arvedlund , m ., j . craggs , and pecorelli j . 2003 . coral culture — possible future trends and directions . in marine ornamental species : collection , culture & amp ; conservation , ed . j . c . cato and c . l . brown , 233 - 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