A method for medusa polyp preservation and recovery

Abstract

The application discloses a method for preserving and recovering hydrozoa and medusae, and the method is characterized by the following steps: inducing the hydrozoa to degenerate and enter a dormant state by high salinity, and recovering the hydrozoa from the dormant state in 3 days at the fastest speed when the salinity is restored to 30-35 ppt, and differentiating medusae buds in one week. The method is suitable for preserving and recovering various hydrozoa such as dactylozooid hand medusae, Xiamen anthoathecate medusae and high hand medusae. The method not only solves the problems of long-term preservation and population rejuvenation of hydrozoa, but also effectively removes algae, protozoa, nematodes and other organisms, speeds up the formation and development of medusae buds, greatly saves the manpower and material resources for long-term maintenance of hydrozoa, and improves the efficiency of long-term preservation and maintenance of hydrozoa.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of germplasm resource preservation, and particularly relates to a method for preserving and recovering hydrozoa. BACKGROUND

[0002] Cnidarians have radial symmetry, simple structure, only endoderm and ectoderm, are the beginning of eumetazoan, and are an important animal group for exploring the growth and development and life evolution of animals. Among them, hydrozoa is the most diverse and has the richest life history types in cnidarians, and has polyp and medusa stages. Cladonema, Cladonema digitatum and Cladonema pacificum are selected as model organisms for research on biological development, evolution, regeneration and environmental adaptability.

[0003] Cladonema belongs to Cnidaria, Hydrozoa, Anthoathecata, Capitata and Cladonematidae, and currently has 8 valid species. Among them, Cladonema radiatum, Cladonema digitatum and Cladonema pacificum are selected as model organisms for research on biological development, evolution, regeneration and environmental adaptability. Long-term culture and preservation of model organisms will facilitate research.

[0004] Unlike medusae, polyps are small, have strong environmental tolerance, long lifespan and are easy to culture, and are the preferred stage for medusa preservation. However, the culture of polyps in vivo requires regular feeding and water changes to maintain life and water quality, which is a large workload. In addition, after a period of culture, algae, protozoa, nematodes, bacteria and other organisms are easily mixed into the culture system of polyps, affecting the growth state of polyps and even causing their death. Studies have found that the polyps of Cladonema pacificum will enter a dormant state at low temperature (5℃), and the polyp population will recover after the temperature rises to the optimum temperature, so this dormancy feature can be used for long-term preservation of germplasm. However, this method requires the use of a cold storage box, refrigerator or constant temperature incubator for preservation, increasing the cost of long-term preservation of polyps, and equipment failure will cause losses. SUMMARY

[0005] To solve the problems in the prior art, the application provides a method for preserving and recovering hydrozoa, which induces polyps to degenerate and enter a dormant state by high salt, realizes long-term preservation at room temperature, accelerates the formation and development of medusa buds, greatly saves the manpower and material resources for long-term maintenance of polyps, and improves the efficiency of long-term preservation and maintenance of polyps.

[0006] The technical scheme of the present application is as follows:

[0007] The present application provides a method for hydrula and jellyfish preservation and recovery, comprising the following steps:

[0008] S1, configuring artificial seawater: using seawater element and pure water to configure 0-70 ppt artificial seawater, after continuous aeration and filtration through 0.45 mu m filter membrane, placing at room temperature for standby;

[0009] S2, preparing bait: after mixing brine shrimp eggs with artificial seawater, pouring into the hatcher of brine shrimp, aeration culture for 24-48 h under room temperature and natural light; after completion of hatching, stopping aeration, placing after standing, taking out brine shrimp from the bottom, cleaning and removing impurities, placing in artificial seawater, and diluting according to needs for standby;

[0010] S3, daily maintenance of hydrula: hydrula can be cultured for a long time under the condition of 30-35 ppt salinity artificial seawater, 20-25 DEG C room temperature, natural light, and feeding bait once a day, and can release jellyfish body periodically;

[0011] S4, high-salt induced hydrula dormancy: under room temperature, the salinity of hydrula culture water is increased by 5 ppt per day until the hydrula completely degenerates, and finally only the creeping root enters the dormant state;

[0012] S5, recovery of hydrula dormant body: placing the hydrula dormant body under high salt into 30-35 ppt artificial seawater for culture at room temperature, so that the hydrula dormant body recovers.

[0013] Further, the salinity of the water body in which the hydrula completely degenerates in step S4 is 50-60 ppt, and the dormancy salinity of different species is different.

[0014] Further, during the process of high-salt induced dormancy in step S4, brine shrimp is fed once a day, clean high-salinity seawater is replaced after feeding for 2 hours to increase salinity; after the hydrula completely degenerates and enters dormancy, feeding is stopped; after the hydrula completely degenerates, the salinity is recorded, and thereafter the salinity is maintained, the salinity and water quality are checked once a week to ensure that the salinity increase is not more than 5 ppt and the water body is clear.

[0015] Further, after the recovery of hydrula dormant body in step S5, brine shrimp is fed once a day, and the water is replaced twice a week to ensure the cleanliness of the water body.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. The application discloses a method for achieving the dormancy and recovery of hydroclones of hydromedusae by changing the salinity at room temperature, which is suitable for the preservation and recovery of various hydromedusae such as digitate hand medusae, Xiamen medusae and high hand medusae, can be applied to the short-term acquisition of the medusae of the digitate hand medusae, has great potential in the long-term preservation of the hydromedusae, and solves the problems of the long-term preservation and population recovery of the hydroclones, effectively removes algae, protozoa, nematodes and other organisms, accelerates the formation and development of medusa buds, greatly saves the manpower and material resources input for the long-term maintenance of the hydroclones, and improves the efficiency of the long-term preservation and maintenance of the hydroclones.

[0018] 2. The application uses the change of the salinity at room temperature to achieve the dormancy and recovery of the hydroclones, is simple to operate, and is free from the dependence on instruments such as refrigerators, freezers and constant-temperature incubators, and reduces the cost of the long-term maintenance of the hydroclones.

[0019] 3. Compared with the hydroclones preserved in the laboratory for a long time, the hydroclones preserved by the method have a more rapid growth after recovery and release medusae in a shorter time, and have no adverse effect on the growth and development of the medusae, the hydroclones recovered from the hydroclones have no obvious breeding of algae, protozoa, nematodes and other organisms, and the whole colony is cleaner. DETAILED DESCRIPTION

[0020] Figure 1 Fig. 1 is a schematic diagram of the hydroclones and recovery of the digitate hand medusae in Example 1 of the application;

[0021] Figure 2 Fig. 2 is a schematic diagram of the recovery of the hydroclones of the digitate hand medusae from different salinities after the salinity is restored to 35 ppt in Example 1 of the application;

[0022] Figure 3 Fig. 3 is a schematic diagram of the growth of the hydroclones from 50 ppt at 35 ppt for 100 days and the comparison with the normal population in Example 1 of the application;

[0023] Figure 4 Fig. 4 is a schematic diagram of the hydroclones and recovery of the Xiamen medusae in Example 2 of the application;

[0024] Figure 5 Fig. 5 is a schematic diagram of the hydroclones and recovery of the high hand medusae in Example 3 of the application. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges. These numerical ranges should be considered as specifically disclosed herein.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0028] Example 1

[0029] This embodiment provides a method for the preservation and resuscitation of the polyp of the finger-branched hand jellyfish, including the following steps:

[0030] S1. Prepare artificial seawater: Prepare 0-70ppt artificial seawater using sea salt and pure water, continuously aerate, filter through a 0.45μm filter membrane, and place at room temperature for later use.

[0031] S2. Prepare feed: Mix brine shrimp eggs with artificial seawater and pour the mixture into the brine shrimp incubator. Aerate and culture the brine shrimp at room temperature (20-25℃) and under natural light for 24-48 hours. After hatching, stop aeration, let the mixture stand, remove the brine shrimp larvae from the bottom, clean them to remove impurities, and place them in artificial seawater. Dilute the seawater as needed before use.

[0032] S3. Daily care of *Gnaphalium affine*: The polyps of *Gnaphalium affine* can be cultured for a long time in artificial seawater with a salinity of 30-35 ppt, at a room temperature of 20-25℃, under natural light, and fed once a day. They can also release jellyfish in stages. The culture container is a crystallizing dish with a diameter of 12.5 cm, with a 10 cm polyethylene culture dish inside for the polyps to attach and grow. The culture water volume is about 300 mL, and a 15 cm culture dish is placed on top as a lid to prevent the salinity from rising due to water evaporation.

[0033] S4, Inducing polyp dormancy of Graptolithus hydrozooides by different salinity: at room temperature, the salinity of the polyp culture water of Graptolithus hydrozooides is increased / decreased by 5 ppt per day from 35 ppt to the target salinity, and the polyps completely degenerate, finally only the creeping roots enter the dormant state; during the process of inducing dormancy by different salinity, the Artemia is fed once a day, and the clean seawater with ±5 ppt is replaced after 2 hours of feeding, and the salinity is increased / decreased until the target salinity is reached; after the polyps completely degenerate and enter dormancy, feeding is stopped, and the salinity is recorded and maintained for 1 month, and the salinity and water quality are checked once a week to ensure that the salinity is increased by no more than 5 ppt and the water is clear; the crystallization dish is covered during the culture period to prevent the salinity from rising due to water evaporation;

[0034] In this embodiment, the target salinity is set to 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70 ppt, and each salinity group has three replicates. The polyps of Graptolithus hydrozooides finally degenerate in water bodies with salinity of 15, 50, 55, 60 and 65 ppt and retain the organization of the polyp root, and completely degenerate in water bodies with salinity of 0-10, 70 ppt, leaving only the empty shell wrapped around the polyp root Figure 1 A); and degenerate into dormant roots Figure 1 B) at 15, 50-65 ppt;

[0035] S5, Resuscitation of Graptolithus hydrozooides polyp dormancy: Graptolithus hydrozooides polyps that have been dormant at high salinity are placed back in 35 ppt artificial seawater. After three days of salinity recovery to 35 ppt, the polyp dormancy begins to germinate, as shown by the white arrows in Figure 1 C; after two weeks of salinity recovery to 35 ppt, the polyps differentiate into medusae buds, as shown by the white arrows in Figure 1 D; during the resuscitation of the polyp dormancy in 35 ppt, the Artemia is fed once a day, and the water is changed twice a week, and the culture is observed at room temperature, so that the Graptolithus hydrozooides polyp dormancy grows well after resuscitation.

[0036] In this embodiment, the resuscitation of Graptolithus hydrozooides polyp dormancy is carried out, as shown in Figure 2 A-2D, Graptolithus hydrozooides polyp dormancy from different salinities is resuscitated in 35 ppt, wherein the 15→35 ppt, 50→35 ppt, 55→35 ppt, 60→35 ppt groups correspond to Figure 2 A, Figure 2 B, Figure 2 C, Figure 2D, while the hydrocauli of 65 ppt could not recover in 35 ppt; in the 15→35 ppt, 55→35 ppt, and 60→35 ppt groups, the hydrocauli of the tentaculate medusae recovered into hydrocauli within 1 week; the growth of the hydrocauli in the four groups of recovery from different salinities showed a J-shaped growth curve, and the medusae buds differentiated from the hydrocauli within 20 days, and the medusae buds developed into medusae within 6-12 days; among them, the recovery of the hydrocauli from 50 ppt in 35 ppt was the best, the hydrocauli recovered into hydrocauli within 3 days, and the number of the hydrocauli increased to 2793.7±411.7 within 100 days; and the medusae buds differentiated from the hydrocauli after 7 days, and a total of 3193.3±1181.7 medusae were released within 100 days of culture;

[0037] The hydrocauli in this example were compared with the hydrocauli living in 35 ppt for a long time (e.g. Figure 3 ), wherein Figure 3 A is the 35 ppt group, i.e., the hydrocauli living in 35 ppt for a long time, Figure 3 B is the 50→35 ppt group, i.e., the recovery and growth of the hydrocauli from 50 ppt in 35 ppt; it can be seen from the figure that the recovery of the hydrocauli from 50 ppt in 35 ppt is good, and there is no significant difference in the number of hydrocauli and the number of medusae released within 100 days between the 50→35 ppt group and the 35 ppt group, and the medusae are released 38 days earlier. The medusae released by the hydrocauli after recovery can develop normally to maturity. In addition, the hydrocauli in 50 ppt that have been dormant for 6 months are placed back in 35 ppt water, and the hydrocauli can recover within 10 days; therefore, 50 ppt is the final target salinity for high-salt induction.

[0038] Example 2

[0039] The present example provides a method for preserving and recovering the hydrocauli of Xiamen Muggiaea medusae, comprising the following steps:

[0040] Steps S1-S3 are the same as the operation in Example 1;

[0041] S4, inducing the hydrocauli of Xiamen Muggiaea medusae into dormancy by high salinity: at room temperature, the salinity of the culture water is increased by 5 ppt per day until the hydrocauli completely degenerate, and only the creeping roots enter a dormant state, Figure 4 A is the degeneration of the hydrocauli of Xiamen Muggiaea medusae into dormant roots in 50 ppt; during this process, Artemia is fed once a day, and clean high-salinity seawater is changed after 2 hours of feeding to complete the operation of increasing salinity; after the hydrocauli completely degenerate and enter dormancy, feeding is stopped; the salinity is recorded after the hydrocauli completely degenerate, at which time the salinity is 50 ppt; thereafter, the salinity and water quality are detected once a week to ensure that the salinity is increased by no more than 5 ppt, and the water is clear; during this period, the covers of the crystallization dishes are covered to prevent the salinity from rising due to water evaporation;

[0042] S5. Revival of dormant polyps of *Hylocereus macrantha*: Dormant polyps of *Hylocereus macrantha*, after one week of dormancy under 50 ppt, were directly placed in 30 ppt of water and cultured at room temperature. The revival of the polyps was observed daily. The polyps regrowed within 3 days. Figure 4 B; During the revival period, the hydra hibernating bodies were fed with artichokes once a day and the water was changed twice a week, and they grew well.

[0043] Example 3

[0044] This embodiment provides a method for the preservation and revival of the polyp of the jellyfish *Echinochloa crus-galli*, including the following steps:

[0045] Steps S1-S3 are the same as those in Example 1;

[0046] S4. High salt induction of expert jellyfish into dormancy: At room temperature, the salinity of the culture water is increased by 5 ppt per day until the polyps completely degenerate, and finally only the creeping roots remain, entering a dormant state; Figure 5 A. The jellyfish, known as A, degenerated into a dormant root at 50 ppt. During this process, it was fed with brine shrimp once a day, and clean, high-salinity seawater was introduced two hours after feeding to increase salinity. Feeding was stopped after the polyps had completely degenerated and entered dormancy. The salinity was recorded at 50 ppt after the polyps had completely degenerated. Salinity and water quality were then tested weekly to ensure that the increase in salinity did not exceed 5 ppt and that the water remained clear. The crystallization dish was covered during this period to prevent salinity from rising due to water evaporation.

[0047] S5. Revival of dormant *Echinochloa crus-galli* hydromes: Dormant *Echinochloa crus-galli* hydromes that had been dormant for one week under 50 ppt were directly placed in 33 ppt of water and cultured at room temperature. The revival of the dormant *Echinochloa crus-galli* hydromes was observed daily. *Echinochloa crus-galli* regrowth occurred within 3 days. Figure 5 B; During the recovery period, the dormant jellyfish of the expert jellyfish were fed with brine shrimp once a day and the water was changed twice a week, and they grew well.

[0048] In summary, this invention utilizes the characteristic of hydroids in a high-salt environment (50-60 ppt) to induce dormancy in hydroids, thus achieving long-term preservation and population rejuvenation. Furthermore, unlike low-salt environments, high-salt environments can kill some algae, protozoa, nematodes, bacteria, and other organisms, resulting in a cleaner post-recovery hydroid population. This invention is applicable to hydroids of genera such as *Hymenoptera*, *Hymenoptera*, and *Hymenoptera* that can remain dormant in high-salt environments. For hydroids of other species, the method provided by this invention can be adjusted to suit the specific biological characteristics of the species.

[0049] The above merely illustrates the embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent flow transformation or direct or indirect application in other related technical fields based on the content of the present application should be included in the patent protection scope of the present application.

Claims

1. A method for preservation and recovery of a hydrozoan medusa, characterized by, The method comprises the following steps: S1, configuring artificial seawater: using seawater and pure water to configure 0-70 ppt artificial seawater, continuously aerating, filtering through a 0.45 μm filter membrane, and placing at room temperature for use; S2, preparing bait: after mixing brine shrimp eggs with artificial seawater, pouring into the hatching device of brine shrimp, and culturing under room temperature and natural light for 24-48 h, stopping aeration after hatching, taking out brine shrimp from the bottom after standing, cleaning and removing impurities, and placing in artificial seawater, and diluting according to needs for use; S3, daily maintenance of hydrocladium: the hydrocladium can be cultured for a long time under the conditions of 30-35 ppt salinity artificial seawater, 20-25℃ room temperature, natural light, and feeding once a day, and can release medusas periodically; S4, high-salt-induced hydrocladium dormancy: the salinity of the hydrocladium culture water is increased by 5 ppt per day until the hydrocladium completely degrades at room temperature, the salinity of the water body when the hydrocladium completely degrades is 50-60 ppt, and finally only the creeping root enters the dormant state; S5, resuscitation of hydrocladium dormant body: the hydrocladium dormant body under high salt is placed in 30-35 ppt artificial seawater at room temperature for culture, so that the hydrocladium dormant body recovers.

2. A method of medusa preservation and recovery as claimed in claim 1, wherein: During the process of high-salt-induced dormancy in step S4, the brine shrimp is fed once a day, and clean high-salinity seawater is replaced after 2 hours of feeding to increase the salinity; after the hydrocladium completely degrades and enters dormancy, feeding is stopped; after the hydrocladium completely degrades, the salinity is recorded, and the salinity is maintained thereafter; the salinity and water quality are checked once a week to ensure that the salinity increase does not exceed 5 ppt and the water body is clear.

3. A method of medusa preservation and recovery as claimed in claim 1, wherein: After the resuscitation of the hydrocladium dormant body in step S5, the brine shrimp is fed once a day, and the water is replaced twice a week to ensure the cleanliness of the water body.

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