Induction of metamorphosis in the irukandji jellyfish Carukia barnesi

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Breeding

Carukia barnesi polyps were available in culture at the James Cook University Aquarium, from a jellyfish originally collected near Double Island, North Queensland, Australia (16°43.5′S, 145°41.0′E) in 2014 and 20158. Populations increase exponentially through asexual reproduction8. Detached buds and swimming polyps were collected from the main culture and transferred to 6-well tissue culture plates in filtered natural seawater. Plates were kept in the dark to inhibit algal growth at 27°C in a constant temperature cabinet. The buds and swimming polyps were left to develop and settle to the bottom of the wells, at which point they were then fed freshly hatched Artemia nauplii and water changed 2-3 times a week. Lids remained attached to tissue culture plates to prevent water evaporation and maintain stable salinity. Polyps were maintained in this manner for at least 4 months prior to the start of experiments, with all individuals reaching maturity with the ability to asexually reproduce further buds. To preserve water quality15 the polyps were deprived of food for two days before the start of the experiment and were not fed for the duration of the tests. One day before the start of the experiment, all immature buds and polyps were removed from the wells, leaving approximately 5-10 mature polyps attached to the substrate for analysis.

Preparation of reagents

Reagents

Six chemicals were tested in the current study to induce metamorphosis in C. barnesi polyps. Four indole-containing compounds were chosen that have already been tested with other cubozoan species: 5-methoxy-2-methyl-3-indoleacetic acid, 5-methoxyindole-2-carboxylic acid, 2-methylindole16 and 5-methoxy-2-methylindole15.16. With 9-cis-retinoic acid retinoic X receptor and lugols solution.

Indole Compound Treatments

The chemical concentrations of indoles documented in the literature were used to perform preliminary concentration tests. 50 mM stock solutions were prepared with 100% ethanol, which was diluted with filtered seawater to the desired experimental concentrations: 50 μM1620μM and 5μM15. Due to the high mortality rates at all of these concentrations when used in this study on C. barnesi, all concentrations were diluted. Fifty mM stock solutions of 5-methoxy-2-methyl-3-indoleacetic acid, 5-methoxyindole-2-carboxylic acid, 2-methylindole and 5-methoxy-2-methylindole were prepared with 50% of ethanol (50% Milli-Q ® water) and stored at −20°C. 50 mM stock solutions were diluted with filtered seawater to the experimental concentrations of 5 μM, 1 μM, 0.5 μM, 0.1 μM and 0.05 μM. The 50% ethanol (50% Milli-Q® water) carrier solution was diluted to the equivalent of the experimental concentrations listed above for use as a control, and incorporated into the data as concentration 0 Seventeen ml of solution was added to the polyps to fill each well of a 6-well plate.

Treatment with iodine (lugols solution)

Aqueous iodine as a Lugols solution (0.37% iodine and 0.74% potassium iodide (sigma product information)) was prepared with equivalent concentrations in moles of iodine/ iodide: 1.5 μM, 3 μM, 6 μM, 12 μM and 24 μM. Only filtered seawater was used as a control for this treatment and incorporated into the data as concentration 0. 17 ml of solution was added to the polyps to fill each well of a 6-well plate.

Retinoid treatment

To reduce polyp mortality associated with ethanol, 0.015% ethanol in Milli-Q® water was used to prepare a 1 mM stock solution of 9-cis-Retinoic acid. The 1 mM stock solution was diluted with filtered seawater to the experimental concentrations of 5 μM, 1 μM, 0.5 μM, 0.1 μM and 0.05 μM. The 0.015% ethanol (Milli-Q® water) carrier solution was diluted to the equivalent of the experimental concentrations listed above for use as a control, and incorporated into the data as a concentration of 0. 17 mL of solution were added to the polyps to fill each well of a 6-well plate.

Metamorphosis trials

Primary trials

Experimental concentrations of reagents were added to C. barnesi polyps growing in the wells of sterile 6-well tissue culture plates. One plate was used per chemical, per concentration, in which five wells functioned as replicates containing the test chemical, while the sixth well contained only the control medium. Five concentrations were performed for each of the six chemicals; 30 plates in total.

The filtered seawater in which the polyps grew was replaced with the test chemical on day 0 and remained unchanged for the duration of the trial. The lids remained attached to the tissue culture plates to prevent water evaporation and hence salinity changes.

Polyps from each well were photographed daily using a dissecting microscope over a period of 34 days. The results were then recorded from the photographs, categorized (Fig. 6) by number of polyps that displayed:

migration of tentacles: one of the key signs of metamorphosis in this species, the polyp tentacles merge, migrating to form four distinct square-shaped wedges8.

Detached jellyfish: a jellyfish formed and detached from the polyp, recorded regardless of health status.

Movable detached jellyfish: a healthy jellyfish formed and detached from the polyp, with the ability to swim.

Polyp survival: this was then used to calculate the number of polyps that survived treatment and did not metamorphose.

Optimization test

The optimal chemical and concentration was then deduced by choosing the combination that produced the greatest percentage of healthy detached jellyfish, in this case 5-methoxy-2-methylindole at 1 μM. A final trial was then run with this to determine if the duration of chemical exposure could optimize healthy jellyfish performance. Three replicates of a minimum of five polyps were used per treatment, in which 1 μM 5-methoxy-2-methylindole (in seawater) was added to the polyps for 24, 48, 72, 96, and 120 h, before the solution was replaced with fresh seawater. A seawater-only check was also carried out. The total number of healthy detached jellyfish was recorded each day.

Data analysis

All statistical analyzes were performed in IBM SPSS Statistics Ver28. Graphs were produced in Microsoft Excel 2016 and OriginPro Graphing and Analysis 2021.

Primary trials

The effect of chemical, concentration and time was analyzed using a three-way repeated measures ANOVA for four sets of data collected during the metamorphosis process: percentage of polyps to show migration tentacles, percentage of polyps for which the jellyfish detaches, percentage of polyps for having detachment of healthy swimming jellyfish, percentage of survival of polyps that have not metamorphosed. Percentage data were transformed to arcsine square root prior to analysis. Mauchly’s sphericity test indicated that the sphericity assumption was violated on all four datasets and therefore a Greenhouse-Geisser correction was used.

Optimization test

Differences in the average percentage of healthy jellyfish produced at different exposure times were analyzed using ANOVA. Differences between means were elucidated using a Post hoc Tukey pairwise comparison test (Tukey HSD alpha 0.05).

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