A method for removing filamentous algae infecting hydroids

Abstract

This invention discloses a method for removing filamentous algae infecting hydra, comprising the following steps: (1) preparing seawater containing copper ions with a concentration of 100-250 μg / L using artificial seawater as a solvent; (2) preparing food to cultivate hydra; (3) using the prepared copper-ion-containing seawater to cultivate hydra infected with filamentous algae to remove the filamentous algae, during which the copper-ion-containing seawater is replaced every 2-3 days to maintain the copper ion concentration, at which time the hydra will degenerate and form hydra dormant bodies; (4) after the filamentous algae are completely removed, the seawater is replaced with normal seawater, and the hydra population enters the recovery period; during the recovery period, the filamentous algae do not reappear, and new hydra grow from the hydra dormant bodies after 1-16 days, and the hydra subsequently return to normal. The method provided by this invention can not only effectively remove filamentous algae that infect hydra, but also allow the hydra to quickly recover into a healthy hydra colony in normal seawater after copper ion treatment, and continuously form and release healthy jellyfish.

Description

Technical Field

[0001] This invention belongs to the field of germplasm resource preservation technology, specifically relating to a method for removing filamentous algae that infect hydra bodies. Background Technology

[0002] Cnidaria are cnidarians with only endoderm and ectoderm, and their bodies are radially symmetrical. They mainly include jellyfish, corals and other groups, and occupy a unique position in the process of life evolution. Among them, jellyfish are the most biodiverse and have the most diverse life history types, making them of great research value.

[0003] Cladonema, Clytia, and Cassiopea all belong to the phylum Cnidaria and exhibit a typical two-stage life cycle: a polyp and a medusa, with alternation of generations. Among them, Cladonema pacificum and Cladonema radiatum have become model organisms in biological and physiological research fields such as development and regeneration, primarily used in studies of development, regeneration, branching of organismal structures, eyespot formation, and the cellular and molecular mechanisms of starvation. Clytia hemisphaerica has been used as a model organism in research on neural development, cell differentiation, and evolution. Cassiopea is a common species of jellyfish displayed in aquariums and has high ornamental and research value. Among them, Cassiopea andromeda and Cassiopea xamachana have been used as model organisms for research on symbiosis, regeneration, and neural development.

[0004] Species such as *Hymenopterys finata*, *Hymenopterys fusiformis*, *Hymenopterys xiananensis*, and *Hymenopterys anthocyanin* are easy to raise and have strong regenerative abilities, making them excellent experimental organisms. However, in laboratory environments, *Hymenopterys finata* polyp colonies are easily squeezed out of their living space by invasive filamentous algae, ultimately leading to death. The filamentous algae adhere to the polyps, requiring periodic manual removal of uninfected polyps to repopulate healthy, clean polyp colonies, which is quite labor-intensive. Furthermore, filamentous algae can also infect polyp colonies of other jellyfish such as *Hymenopterys xiananensis* and *Hymenopterys anthocyanin*, causing considerable damage. Therefore, a simple and effective algae control method is urgently needed to maintain the normal growth of polyps.

[0005] Copper is a cost-effective algaecide. Copper sulfate and copper-containing algaecides are broad-spectrum bactericides and algaecides, and due to their high efficiency and low cost, they have been used for decades to control phytoplankton blooms in ponds, aquaculture waters, and reservoirs. Besides phytoplankton, copper sulfate can affect the growth of filamentous algae such as Spirogyra, *Tribonemasp.*, and *Tribonemasp.* (72h-EC). 50 The concentration was 3.66 mg / L; copper-containing algaecides also showed good inhibitory effects on large algae such as *Nitellopsis obtusa*. Among many copper-containing algaecides, copper sulfate (copper ions) is low in cost and has a significant algae-removing effect. Copper is an essential trace element for living organisms, but excessive copper can be harmful to organisms. Currently, research on the effects of copper ions on cnidarians mainly focuses on corals, sea anemones, and other anthozoa, with less research on jellyfish groups such as hydrozoa and scyphozoa. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for removing filamentous algae infecting hydra, applicable to hydra that can tolerate a certain concentration of copper ion stress. It not only effectively removes filamentous algae infecting hydra, but also allows the hydra to quickly recover into a healthy hydra colony in normal seawater after copper ion treatment, and to continuously form and release healthy jellyfish.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides a method for removing filamentous algae infecting hydroids, comprising the following steps:

[0009] (1) Use seawater as a solvent to prepare seawater containing copper ions, wherein the concentration of copper ions is 100-250 μg / L;

[0010] (2) Prepare food for feeding the hydra;

[0011] (3) Use the copper-containing seawater with a concentration of 100-250 μg / L obtained in step (1) to culture the polyps infected by filamentous algae to remove the filamentous algae. During this period, the copper-containing seawater is replaced every 2-3 days to maintain the copper ion concentration.

[0012] (4) After the filamentous algae were completely removed, the seawater was replaced with normal seawater, and the hydra population entered the recovery period. During the recovery period, the filamentous algae did not reappear, and the dormant hydra grew new hydra after 1 to 16 days.

[0013] Further, in step (1), seawater with a salinity of 30-35 ppt is prepared using sea salt and pure water, filtered through a filter membrane, and then CuSO4·5H2O is added to prepare seawater containing copper ions.

[0014] Further, the preparation method of the bait in step (2) is as follows: after mixing the brine shrimp eggs with seawater, pour them into the brine shrimp incubator and aerate them for 24-48 hours under room temperature and natural light. After hatching, stop aeration, let them stand, take out the brine shrimp nauplii, clean them to remove impurities, and place them in seawater as bait to cultivate hydra.

[0015] Furthermore, during the removal of filamentous algae in step (3), the hydra are fed the feed prepared in step (2) daily, and feeding is stopped after the hydra degenerates to dormant roots.

[0016] Furthermore, after the filamentous algae are completely removed in step (4), the original copper ion concentration is maintained at 0-15 days before being replaced with normal seawater, thereby ensuring that the filamentous algae are completely removed.

[0017] Furthermore, after the filamentous algae are completely removed in step (4), the revived hydra population is fed daily with the feed prepared in step (2) to ensure that the hydra population further expands, forms and releases jellyfish.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The method of this invention uses copper-containing seawater at a concentration of 100-250 μg / L to treat filamentous algae infecting hydra. This not only effectively removes the filamentous algae infecting hydra, but also allows the hydra participating in the experiment to quickly recover into healthy hydra populations in normal seawater after treatment with copper-containing seawater. Furthermore, it continuously forms and releases healthy jellyfish, greatly saving the manpower and material resources required for long-term hydra maintenance and improving the efficiency of long-term preservation and maintenance of hydra germplasm. It also provides support for research on the toxic mechanism of copper ions to cnidarians and the tolerance of cnidarians to heavy metals.

[0020] 2. The hydra populations treated by the method of this invention are clean and free of impurities after recovery, meeting the needs of experiments and production. Furthermore, compared with hydra cultured in the laboratory for a long period, the hydra treated by this invention showed no significant differences in growth, jellyfish formation and development, and jellyfish release. This invention did not adversely affect the growth and development of any of the hydra or jellyfish species. Therefore, this method can be used for algae control in various jellyfish and hydra species. For different species, the concentration of copper ions and the treatment time need to be adjusted according to the specific species' sensitivity and tolerance to copper ions. Attached Figure Description

[0021] Figure 1-1 The effect of finger-like branched jellyfish and copper ions on the removal of filamentous algae in Example 1 of the present invention;

[0022] Figure 1-2 This illustrates the effect of copper ions on the surface area of ​​the hydroid and algae in Example 1 of the present invention.

[0023] Figure 1-3 This describes the situation in Embodiment 1 of the present invention where the finger-branched jellyfish produces jellyfish during the recovery period;

[0024] Figure 2-1 This illustrates the effect of different copper ion concentrations and treatment times on the surface area of ​​filamentous algae in Example 2 of the present invention.

[0025] Figure 2-2 The images show the filamentous algae under different copper ion treatments on days 10 and 30 in Example 2 of this invention.

[0026] Figure 2-3 The variation in the number of finger-branched hand jellyfish polyps during the copper ion treatment period and the recovery period in Example 2 of this invention;

[0027] Figure 3 The finger-branched hand jellyfish polyp in Example 3 of the present invention was restored to its original state after being treated twice with copper ions (250 μg / L);

[0028] Figure 4 This study investigates the tolerance of the white-spotted jellyfish to copper ions in Example 4 of the present invention.

[0029] Figure 5 This study investigates the tolerance of *Hydrocotyle maculatus* to copper ions in Example 5 of the present invention.

[0030] Figure 6 This study investigates the tolerance of the Andrographis paniculata jellyfish to copper ions in Example 6 of the present invention. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, and the following embodiments should not be regarded as limiting the scope of the present invention.

[0032] The following examples use *Dendrocalamus digitatus*, *Dendrocalamus fusiformis*, *Hydrocotyle xiaensis*, and *Dendrocalamus anthocyanin* infected with filamentous algae as research subjects;

[0033] The daily care methods for the polyps of *Jetida fingeriformis*, *Jetida salina*, *Hydrophorus maculatus*, and *Hydrophorus anthocyaninus* are as follows: They can be cultured for a long time under conditions of salinity of 30-35 ppt, temperature of 20-25℃, natural light, and feeding once a day, and can release jellyfish in stages. The culture container is a crystallizing dish with a diameter of 12.5 cm, with a 10 cm diameter polyethylene culture dish inside for the polyps to attach and grow. The culture water volume is about 300 mL, and a 15 cm diameter culture dish is placed on top as a lid to prevent the salinity from rising due to water evaporation.

[0034] Example 1

[0035] This embodiment provides a method for removing filamentous algae from the polyps of the jellyfish *Gnaphalium affine*, including the following steps:

[0036] (1) Preparation of seawater and copper-containing seawater: 30-35 ppt of seawater was prepared using sea salt and pure water and measured with an optical salinity meter. After continuous aeration, the seawater was filtered through a 0.22 μm filter membrane and placed at room temperature for later use. CuSO4·5H2O was added to the prepared artificial seawater to form copper-containing seawater. In this embodiment, six copper ion concentration gradients were also set up, namely, copper ion concentrations of 0 (control group), 25, 50, 75, 100, 250, and 500 μg / L, which were represented by the numbers Cu0 (control group), Cu25, Cu50, Cu75, Cu100, Cu250, and Cu500, respectively. Each treatment group had three replicates, and each replicate contained 70 polyps infected by filamentous algae.

[0037] (2) Mix the brine shrimp eggs with the prepared artificial seawater and pour it into the brine shrimp incubator. Aerate and culture for 24-48 hours under room temperature and natural light. After hatching, stop aeration, let stand, take out the brine shrimp nauplii, clean and remove impurities, and place them in artificial seawater as food to cultivate hydra. Dilute as needed for later use.

[0038] (3) Use the copper-containing seawater of different concentrations obtained in step (1) to culture the hydra infected by filamentous algae to remove the filamentous algae, and observe the changes of the hydra and filamentous algae regularly; during the removal of filamentous algae, replace the copper-containing artificial seawater every 3 days to maintain the copper ion concentration, and feed the hydra once a day.

[0039] in, Figure 1-1 a represents the polyp of *Jetida flavoides* that was not infected by filamentous algae; the green filamentous algae in the control group Cu0 expanded rapidly, covering the entire bottom of the culture dish within 9 days, and quickly crowding out the living space of the polyps until the polyps died. Figure 1-1 b); The Cu25 group can slow down the growth rate of filamentous algae, while the Cu50 group can effectively inhibit the growth of filamentous algae; when the copper ion concentration is less than or equal to 50 μg / L, the asexual reproduction of hydra is inhibited to some extent by copper ions, and the overall growth is slow; when the copper ion concentration is 75–250 μg / L, the hydra can degenerate into dormant roots ( Figure 1-1 c) The time it takes for the hydra to enter a dormant state decreases with the increase of copper ion treatment concentration. At this time, the filamentous algae can be effectively removed. The filamentous algae in the Cu250 group were completely removed within 2 days. When the copper ion treatment concentration is 500 μg / L, the polyps die completely within 1 day and cannot be revived after being replaced with normal seawater. During the copper treatment period, no jellyfish were produced in any of the experimental groups.

[0040] (4) After the filamentous algae are completely removed, maintain the original copper ion concentration for 10 days and then replace it with normal seawater; if the filamentous algae are not completely removed, replace it with normal seawater after 30 days of copper ion treatment, and the hydra population will enter the recovery period; during the recovery period, feed the hydra once a day and change the water every 3 days.

[0041] The changes in hydroids and filamentous algae are as follows: Figure 1-2 As shown in the figure; where the lines indicate the average value, green represents the surface area of ​​algae (SA), and purple represents the number of polyps (P); Cu0 / 25 / 50 / 75 / 100 / 250 correspond to copper ion concentrations of 0 (control), 25, 50, 75, 100, and 250 μg / L, respectively; the red line indicates the time when the culture was replaced with normal seawater; as shown in the figure, when entering the recovery period, the filamentous algae in the Cu25 and Cu50 groups grew rapidly, covering the entire bottom of the culture dish within 3–24 days, and the number of polyps showed a trend of first increasing and then decreasing, with the number of polyps in some groups approaching zero in the later stage; the filamentous algae in the Cu75–250 groups did not reappear, while the dormant roots of the polyps grew polyps from days 1 to 16, and the polyp population recovered ( Figure 1-1 d) The recovery time increased with the increase of the concentration of copper ions in the early stage. The number of polyps continued to increase at the end of the experiment. The Cu100 group had the most polyps, which was 659.0±263.5 at the end of the experiment. During the recovery period, within 37 days from the emergence of the first jellyfish, the cumulative number of jellyfish released by the Cu75, Cu100 and Cu250 groups were 1597.7±725.1, 849.7±500.0 and 1205.7±1067.0, respectively, and there was no significant difference. After the jellyfish matured during the recovery period, the three groups were semi-ellipsoidal in shape, and there was no significant difference in morphology and size from the normal jellyfish.

[0042] In summary, based on a comprehensive analysis of the population size of the hydroids in *Jetida flavoides* during the copper ion treatment and recovery periods, the development and release of the jellyfish, and the changes in algal area, it is recommended that the copper ion concentration for removing filamentous algae infecting *Jetida flavoides* be 75–250 μg / L.

[0043] Example 2

[0044] This example studies the effects of different copper ion treatment concentrations and times on hydra and filamentous algae; the difference from Example 1 is:

[0045] In steps (1), (3), and (4), five copper ion concentration gradients were set in this embodiment: 0 (control group), 25, 50, 75, 100, and 250 μg / L, denoted by Cu0 (control group), Cu25, Cu50, Cu75, Cu100, and Cu250, respectively. Hydroids infected with filamentous algae were cultured in seawater containing different copper ion concentrations to remove the algae. Changes in the hydroids and filamentous algae were observed periodically. During the copper ion treatment, the seawater containing copper ions was replaced every two days to maintain the copper ion concentration, and the hydroids were fed once daily. Simultaneously, the time factor for maintaining the original copper ion concentration after the filamentous algae were completely removed was controlled by replacing the seawater with normal seawater. The time control for maintaining the original copper ion concentration was divided into four treatments: T0, T5, T10, and T15. T0 was the time when the filamentous algae were completely removed or the corresponding Cu... 2+ After 30 days of treatment, immediately replace with normal seawater; T5 is when filamentous algae are completely removed or in the corresponding Cu... 2+ After 30 days of treatment, continue the original treatment for 5 days, then replace with normal seawater; T10 is determined when filamentous algae are completely eliminated or at the corresponding Cu 2+ After 30 days of treatment, continue the original treatment for another 10 days, then replace with normal seawater; T15 is when filamentous algae are completely eliminated or at the corresponding Cu 2+ After 30 days of treatment, continue the original treatment for another 15 days before replacing with normal seawater.

[0046] During the copper ion treatment period, when the copper ion concentration was 50–250 μg / L, except for the T0 group of Cu50 which retained filamentous algae, the filamentous algae in the other groups were completely eliminated and did not reappear during the recovery period; while the Cu25 group only slowed down the growth rate of filamentous algae, such as Figure 2-1 , Figure 2-2 As shown, the number of hydroids increases when the copper ion concentration is less than 50 μg / L, increases slightly when the copper ion concentration is 75 μg / L, and decreases when the copper ion concentration is between 100 and 250 μg / L. Figure 2-3 As shown.

[0047] The polyps of *Tetranychus digitata* and filamentous algae entered the recovery period. Normal seawater was replaced with different amounts of raw copper ion treatment time as described above. During this period, the plants were fed once daily, and the water was changed every two days to maintain water cleanliness. In the early stages, when the copper ion concentration was less than 50 μg / L, the number of polyps decreased during the recovery period, with the Cu0 and Cu25 treatment groups approaching zero by the end of the recovery period. In the early stages, when the copper ion concentration was 75 μg / L, the number of polyps first increased and then decreased, resulting in overall population decline by the end of the recovery period. In the early stages, when the copper treatment concentration was 100–250 μg / L, the number of polyps consistently increased during the recovery period. The Cu100 T5 treatment group had the highest number of polyps at the end of the recovery period, with an average of 149.7 ± 41.3. During the recovery period, the treatment time that maintained the original copper ion concentration had no significant impact on the removal of filamentous algae, the recovery of hydroids, or the yield of jellyfish. The treatment concentration and time in the early stage had no interaction effect on the cumulative number of jellyfish released during the recovery period.

[0048] In summary, based on a comprehensive analysis of factors such as the population size of hydroids, jellyfish development and release, and changes in algal surface area during the treatment and recovery periods at different copper ion concentrations, it is recommended that the copper ion concentration for removing filamentous algae be 100–250 μg / L. After the filamentous algae are completely removed, the seawater should be replaced with normal seawater. The original copper ion concentration can be maintained for 0–15 days after the filamentous algae are completely removed.

[0049] Example 3

[0050] In this embodiment, seawater containing 250 μg / L copper ions was used to treat the filamentous algae infecting the hydroid of the finger-branched jellyfish multiple times;

[0051] The difference between Example 3 and Example 1 is as follows:

[0052] In step (3), when treating the filamentous algae infecting the polyps of the finger-branched jellyfish with seawater containing copper ions, seawater containing 250 μg / L copper ions was used twice. After the first use successfully removed the filamentous algae, the lush polyp population was re-infected by the filamentous algae on the 66th day due to water pollution. Therefore, the seawater containing 250 μg / L copper ions was used again to successfully remove the filamentous algae. The polyps recovered and entered a rapid growth period within a week of the recovery period.

[0053] The filamentous algae were removed within a week, and the cells were then soaked in seawater containing 250 μg / L copper ions for another week before being replaced with normal seawater. The recovered polyps treated with 250 μg / L copper ions showed no significant differences in morphology, size, population density, medullary formation and development, or medullary size compared to the polyps not treated with copper ions. Figure 3As shown, in this embodiment, there were 20 independent samples of hydroids that underwent two copper ion treatments (250 μg / L). The recovery rate during the recovery period was as high as 95%. One sample did not recover in the later stage because the hydroid was in poor condition in the early stage.

[0054] Example 4

[0055] The difference between Example 4 and Example 1 is that: in this example, the white-spotted jellyfish is used as the research object, and the white-spotted jellyfish is treated with seawater containing 100 μg / L copper ions. The remaining steps are the same as in Example 1.

[0056] like Figure 4 As shown, where, Figure 4 a represents a healthy polyp of the jellyfish *A. elegans*. Figure 4 b. When treated with 100 μg / L copper ions, the hydroid roots of *Tetranychus fusiforme* degenerate into dormant root state; Figure 4 c represents the revival of the hydra's dormant roots after the seawater was replaced with normal seawater; Figure 4 Figure d shows the regrowth of polyps from dormant roots during the recovery period. As shown in the figure, under 100 μg / L copper ion treatment, filamentous algae were completely eliminated, and the polyps degenerated into dormant roots. After 5 days in seawater with the original copper ion concentration, the seawater was replaced with normal seawater. During the recovery period, the dormant polyps of *Tetranychus fusiformis* regrowed on the second day, showing no morphological difference from normal polyps.

[0057] Example 5

[0058] The difference between Example 5 and Example 4 is that: in this example, *Hydrocotyle maculatus* is used as the research object, and the remaining treatment steps are the same as in Example 4;

[0059] like Figure 5 As shown, where, Figure 5 a represents a healthy polyp of the Xiamen Hydra jellyfish; Figure 5 b. When treated with 100 μg / L copper ions, the hydroid roots of *Hydrophorus macrantha* in Xiamen degenerate into a dormant root state. Figure 5 c represents the revival of the hydra's dormant roots after the seawater was replaced with normal seawater; Figure 5 d represents the regrowth of hydra from the dormant roots during the recovery period. As shown in the figure, after treatment with 100 μg / L copper ions, the hydra of *Hydrocotyle xiaensis* degenerates into hydra roots, and the filamentous algae are completely removed. After being kept in seawater with the original copper ion concentration for 5 days and then replaced with normal seawater, the dormant hydra revives on the second day of the recovery period.

[0060] Example 6

[0061] The difference between Example 6 and Example 4 is that: in this example, Andrographis paniculata is used as the research object, and the other processing steps are the same as in Example 4;

[0062] like Figure 6 As shown, where, Figure 6 a represents a healthy Andorium jellyfish polyp; Figure 6 b and 6c, when treated with 100 μg / L copper ions, the tentacles of the Andorian jellyfish polyp retracted; Figure 6 Figure d shows the re-extension of the tentacles of the Andorian jellyfish polyp after being replaced with normal seawater. As shown in the figure, when the Andorian jellyfish polyp was treated with 100 μg / L copper ions, the tentacles contracted and the filamentous algae were completely removed. After being kept in seawater with the original copper ion concentration for 5 days and then replaced with normal seawater, the tentacles of the Andorian jellyfish polyp re-extensioned within 5 hours during the recovery period.

[0063] In addition to the above-mentioned examples of *Gnaphalium affine*, *Gnaphalium affine*, *Gnaphalium anthopterum*, and *Gnaphalium spp.*, this invention can also be used for other hydra that can tolerate a certain concentration of copper ion stress. Therefore, the method provided by this invention can be used for algae removal applications that remove infestations from various jellyfish hydra.

[0064] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for removing filamentous algae infecting hydroids, characterized in that, Includes the following steps: (1) Prepare copper-containing seawater using seawater as a solvent, wherein the concentration of copper ions is 100-250 μg / L; (2) Prepare food for feeding the hydra; (3) Use the copper-containing seawater with a concentration of 100-250 μg / L obtained in step (1) to culture the polyps infected by filamentous algae to remove the filamentous algae. During this period, the copper-containing seawater is replaced every 2-3 days to maintain the copper ion concentration. (4) After the filamentous algae were completely removed, the seawater was replaced with normal seawater, and the hydra population entered the recovery period. During the recovery period, the filamentous algae did not reappear, and the dormant hydra bodies grew new hydra bodies after 1 to 16 days.

2. The method for removing filamentous algae infecting hydroids according to claim 1, characterized in that, In step (1), seawater with a salinity of 30-35 ppt is prepared using sea salt and pure water. After filtration through a filter membrane, CuSO4·5H2O is added to prepare seawater containing copper ions with a copper ion concentration of 100-250 μg / L.

3. The method for removing filamentous algae infecting hydroids according to claim 1, characterized in that, The preparation method of the bait in step (2) is as follows: after mixing the brine shrimp eggs with seawater, pour them into the brine shrimp incubator and aerate them for 24-48 hours under room temperature and natural light. After hatching, stop aeration, let them stand, take out the brine shrimp nauplii, clean them to remove impurities, and place them in seawater as bait to cultivate hydra.

4. The method for removing filamentous algae infecting hydroids according to claim 1, characterized in that, During the removal of filamentous algae in step (3), the hydra are fed the feed prepared in step (2) daily. Feeding is stopped after the hydra degenerates to dormant roots.

5. The method for removing filamentous algae infecting hydroids according to claim 1, characterized in that, After the filamentous algae are completely removed in step (4), the original copper ion concentration is maintained at 0-15 days before being replaced with normal seawater to ensure that the filamentous algae are completely removed.

6. The method for removing filamentous algae infecting hydroids according to claim 5, characterized in that, After the filamentous algae are completely removed in step (4), the revived hydra population is fed the feed prepared in step (2) daily to ensure that the hydra population further expands, forms and releases jellyfish.

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