Morphological characteristics of ephyrae of Aurelia coerulea derived from planula strobilation

Ephyrae are produced through the strobilation of polyps in the general life cycle of Aurelia coerulea. However, it has been reported that planulae can also metamorphose directly into ephyrae, without passing through the polyp stage. There is a mixture of ephyrae developed from planulae (planula-strobilated ephyrae) and ephyrae developed from polyps (polyp-strobilated ephyrae) in the ephyra population. However, the effect of the planula-strobilated ephyrae on the ephyra population is yet to be determined, since their morphological characteristics have not yet been elucidated. This study aimed to determine the morphological characteristics to distinguish between planula-strobilated and polyp-strobilated ephyrae. The differences in body dimensions, such as total body diameter (TBD), central disc diameter (CDD), lappet stem length (LStL), rhopalial lappet length (RLL), and total marginal lappet length (TMLL) were compared between the two types of ephyra. Thus, we show that body proportions can be used to identify planula- and polyp-strobilated ephyrae. The ranges for identifying planula-strobilated ephyra were 35.0–38.3% for CDD/TBD, 56.7–64.9% for LStL/CDD, 84.7–99.5% for TMLL/CDD, and 31.0–37.5% for RLL/TMLL. This method could be an important basis for devising countermeasures for jellyfish blooms in areas where ephyrae deriving from planula strobilation occur.


Introduction
The mass occurrence of the scyphozoan moon jellyfish, Aurelia spp., disrupts coastal economic activities, especially electric power plants and fisheries (Purcell and Arai 2001;Purcell et al. 2007). The scyphozoan moon jellyfish Aurelia spp., alternate between asexual benthic polyp and sexual planktonic medusa stages in their life cycle. Fertilized eggs develop into planulae, which attach to a suitable substrate, and metamorphose into polyps. Polyps produce clones asexually and create colonies. The polyps subsequently then transform into strobilae, releasing planktonic ephyrae in winter and regenerate into polyps again. The released ephyrae grow into sexually mature medusae during  (Scorrano et al. 2016)] in Japan ( Fig. 1) have shown another variation in their life cycle, where the planula ostensibly metamorphoses directly into an ephyra, omitting the polyp stage after settling on substrate (Haeckel 1881;Hirai 1958;Kakinuma 1975;Miyake et al. 2019;Suzuki et al. 2019;Yasuda 1975Yasuda , 1979Lucas 2001). This was called 'Ephyra pedunculata ' by Haeckel (1881) and Hirai (1958) and is currently referred to these days as 'direct development' (Berrill 1949;Hirai 1958;Kakinuma 1975;Yasuda 1975Yasuda , 1979Arai 1997;Lucas 2001). However, Arai (1997: p 159) noted direct development of ephyrae from planulae only in Pelagia, a phenomenon also observed in Periphylla by Jarms et al. (1999), where the planula develops into an ephyra without settling on substrate. Miyake et al. (2019) suggested that the direct development of A. coerulea may be due to the strobilation of planulae because large planulae developed into ephyrae in low water temperature conditions, and into polyps in high water temperature conditions, similar to normal strobilation from polyps. Therefore, the direct development of A. coerulea planulae should be called 'planula strobilation' as it includes a benthic stage. The following details have been reported regarding this kind of development in Aurelia.
1. The planula size of the planula-strobilation type is 500-800 µm; while that of the polyp-strobilation type is 200-300 µm (Kakinuma 1975;Yasuda 1988). 2. The mature oocytes of medusae in the areas where the planula-strobilation type is distributed are large (Kon and Honma 1972;Yasuda 1988;Suzuki et al. 2019). 3. Medusae inhabiting the area where the planula-strobilation type is distributed produce large planulae from winter to spring and small planulae from summer to autumn (Yasuda 1988;Suzuki et al. 2019). 4. The planula-strobilation type Aurelia was identified as A. coerulea using DNA barcodes of the cytochrome oxidase subunit I (COI) and the internal transcribed spacer 1 (ITS-1) region (Miyake et al. 2019;Suzuki et al. 2019). Yasuda (1988) and Suzuki et al. (2019) reported that medusae with large planulae appear only during the low water temperature period (< 20 °C) from winter to spring. These reports suggested that most of the ephyrae appearing during this period are produced by direct development from planulae (planula strobilation), and that this is one of their reproductive strategies during the low water temperature period. In contrast, polyp strobilation occurred during the low water temperature period (< 20 °C) from March to May in Urazoko Bay (Yasuda 1988). It is also suggested that polyp strobilation may occur during the same period as planula strobilation in Maizuru Bay , implying that ephyrae from both planula-and polyp-strobilation coexist in the same ephyra population. It is important to study the life cycle strategies of the planula-strobilated ephyrae to determine their effect on the Aurelia medusa population. However, there are currently no methods for estimating these effects, since there is no genetic difference in DNA barcodes between planula-strobilated ephyrae and polyp-strobilated ephyrae. Moreover, the morphological differences between the two types are unclear. This study aimed to determine the morphological characteristics for distinguishing between planula-and polyp-strobilated ephyrae.

Materials and methods
The planula-strobilation type of A. coerulea is found in Nanao Bay (Miyake et al. 2019). Two hundred and fortyone medusae of A. coerulea were collected near the Notojima Aquarium (Nanao, Ishikawa Prefecture, Japan), which is located in Nanao Bay, in April 2020 (Fig. 1). The medusae were kept in an exhibition tank at the Notojima Aquarium. Water temperature was maintained at approximately 15 °C. On 23 June 2020 planulae were collected from three medusae (approximately 300 individuals per medusa) in the exhibition tank, and each set of planulae from the three medusae was transferred into separate Petri-dishes (63 mm in diameter, 34.6 mm in height). The planulae were transferred to the laboratory in a cooler box to maintain a low temperature and were kept in an incubator at 15 °C to collect ephyrae strobilated from large planulae. The ephyrae strobilated from large planulae were defined as E1 (Fig. 2).
On 19 March 2020, large planulae (n = 66) were collected from a medusa in Miyazu Bay (Kyoto Prefecture) next to Maizuru Bay (Fig. 1), where the planula-strobilation type is distributed . Individual planulae were placed in each well of 12-well plates and incubated at 15 °C in the laboratory to distinguish polyps developed from residua after planula strobilation (PRP: planula-residuum polyp in Fig. 2) and polyps developed directly from planulae (lPP: large-planula polyp in Fig. 2). Polyps (nPP: normalsize planula polyp in Fig. 2) collected from Aburatsubo (Kanagawa Prefecture) were used as a control strain of A. coerulea, common along the Pacific coast of Japan (Fig. 1). The polyps from Aburatsubo were identified as A. coerulea by rearing the medusa generation as well as DNA barcoding using cytochrome oxidase subunit I (COI). The polyps were fed Artemia nauplii every 2 days and kept at a temperature of 23 °C. Three to four hours after feeding, the rearing water was replaced with filtered seawater (pore size: 1 µm). Polyp strobilation was induced in an incubator by decreasing the temperature to 15 °C to obtain ephyrae. The ephyrae were divided into two categories and four types of ephyra in this study, as follows in Fig. 2. • E1: Ephyra strobilated from a large planula. • E2: Ephyra derived from polyp strobilation. • E2.1: Ephyra liberated through strobilation of the residual polyp after the strobilation of a large planula. • E2.2: Ephyra liberated through strobilation of the polyp developed directly from a large planula. • E2.3: Ephyra liberated from a normal-sized planula.
Ephyrae were photographed using a digital camera (EOS Kiss X6i; Canon, Tokyo, Japan) mounted on a stereomicroscope (SZX12; Olympus, Tokyo, Japan) within 24 h after liberation. Body dimensions like the total body diameter (TBD), central disc diameter (CDD), lappet stem length (LStL), rhopalial lappet length (RLL), and total marginal lappet length (TMLL) of ephyrae were measured according to Straehler-Pohl and Jarms (2010), using the image analysis software, ImageJ (Schneider et al. 2012) (Fig. 3). Statistical analysis was performed in R version 4.0.3 (Team 2020). An ANCOVA was used to compare the differences in the regression coefficients between each measurement part and Tukey's multiple comparison test was used to compare the difference in the ratios of each body proportion between the four types of ephyrae.

Results
Planulae in one of three sets of Petri-dishes, which were collected at the Notojima Aquarium, strobilated and released single ephyrae 4 days after collection. Ephyrae with a perfect marginal lappet were selected for measurement of the body dimensions (n = 105, E1 in Figs. 2, 4). The size range of these large planulae was 389-558 µm, and the average size was 444.0 ± 35.8 µm (n = 50).

Discussion
The total body diameter (TBD) of the planula-strobilated ephyrae (1.63 ± 0.24 mm) was approximately half the size of the TBD of the polyp-strobilated ephyrae (3.02 ± 0.31 mm) ( Table 1 and Fig. 7). Almost all planula tissue was used to produce a small ephyra by planula strobilation. As a result, only a small number of tiny residua was produced (27 out of 44). Only 27 residua developed into tiny polyps while 17 disappeared, suggesting that strobilating planulae use their energy to release an ephyra that can swim away rather than producing polyps or regenerative tissue residua for further polyp development. Fu et al. (2014) studied the point of no return from starvation in the ephyrae of A. aurita sensu lato (later A. coerulea). They determined that the PNR 50 (the starvation period at which 50% of ephyrae recovered from starvation and grew to the advanced ephyrae stage) was the point at which the disc diameter decreased by 30% compared with ephyra size just after liberation from strobila, and was longer under lower water temperature conditions. The average TBD at PNR 50 was 1.36 mm at 15 °C, 1.69 mm at 12 °C, and 1.65 mm at 9 °C, similar to those of the planula-strobilated ephyrae. The starvation tolerance of ephyrae is an adaptive strategy to survive in the primary season of ephyrae liberation when zooplankton biomass is low (Fu et al. 2014). This suggests Fig. 6. The difference of the ratios of each body proportion between the four types of ephyrae. Asterisk shows significant differences: *: p < 0.05, **: p < 0.01, ***: p < 0.001 that the size of the planula-strobilation ephyrae is the biological minimum size of ephyrae for survival in nature. Thus, planula strobilation is an adaptive strategy to increase fitness by releasing a minimum-sized ephyra, which can survive on minimal energy, during the low water temperature season.
The ephyra types can be distinguished by body dimensions (Table 1). However, Suzuki et al. (2019) reported no differences in size between ephyrae strobilated by planulae and polyps. Any difference may depend on the difference in planula sizes (679 ± 142 μm in Suzuki et al. (2019) and 444.0 ± 35.8 μm in the current study). The ratios of these planula sizes are also similar to the ratios of ephyra sizes (2.5 mm in Suzuki et al. (2019) and 1.6 mm in the current study). However, the mean TBD and CDD were 2.5 mm and 0.9 mm, respectively, and the CDD/TBD ratio was 36% for Suzuki et al. (2019); and the CDD/TBD value was within 35.0-38.3% of the interquartile range of the CDD/TBD ratio in our study. In addition, among the planula-strobilated ephyrae observed in this study, an ephyra with a TBD of 2.5 mm had a CDD/TBD value of 31.5%, which does not fall within the range but indicates that the ratio of CDD/TBD is remarkably small. Moreover, this specimen had an LStL/ CDD value of 60.1%, a TMLL/CDD value of 99.2%, and an RLL/TMLL value of 39.5%. All these values fall within the range to identify planula-strobilated ephyrae, except for RLL/TMLL. Even if TBD is similar to that of polyp-strobilated ephyrae, it is possible to identify the strobilation type of ephyrae based on body dimensions. Therefore, CDD/TBD, LStL/CDD, TMLL/CDD, and RLL/TMLL ratios can be used to distinguish between planula-strobilated and polyp-strobilated ephyrae. In addition, the interquartile ranges of the ratios can be used for identifying the strobilation type. The ranges for identifying planula-strobilated ephyrae were 35.0-38.3% for CDD/TBD, 56.7-64.9% for LStL/CDD, 84.7-99.5% for TMLL/CDD, and 37.5-31.0% for RLL/TMLL. These results indicate that the planula-strobilated ephyrae have long marginal lappets compared with central disk diameter. The lack of difference in the RLL/CDD ratio indicates no difference in the lappet's crack depth between the ephyra types, suggesting that planula-strobilated ephyrae have long marginal lappet stems. The body size of planula-strobilated ephyrae was smaller than that of polyp-strobilated ephyrae, and the swimming ability of planula-strobilated ephyrae was low, regardless of the long marginal lappet of planula-strobilated ephyrae acting as a paddle (Higgins et al. 2008;Feitl et al. 2009), and generating propulsion to allow the ephyrae to swim.
The appearance of planula-strobilating moon jellyfish has been observed only in a few places in Japan (Mutsu Bay, Aomori Prefecture; Urazoko Bay, Fukui Prefecture; Maizuru Bay, Kyoto Prefecture; and Nanao Bay, Ishikawa Prefecture) (Hirai 1958;Kakinuma 1975;Miyake et al. 2019;Suzuki et al. 2019;Yasuda 1975Yasuda , 1979 and Germany (Haeckel 1881). However, planula-strobilated ephyrae have been observed in areas with thermal and nuclear plants in the Wakasa and Maizuru Bays (Yasuda 1988;Suzuki et al. 2019). To prevent power plant accidents due to invasion of the water intake by a large number of A. coerulea, a series of ecological studies of A. aurita were conducted by Dr. Toru Yasuda (1988). The bell-size of mature medusae, oocyte, and planula sizes were the only parameters used to distinguish between planula strobilation and polyp strobilation in previous studies (Kon and Honma 1972;Kakinuma 1975;Yasuda 1975;Suzuki et al. 2019). This study provides a method to discriminate between the strobilation types based on the morphology of ephyrae. Yasuda (1975) and Suzuki et al. (2019) speculated that the Aurelia population was mixed, with both planula-strobilated and polyp-strobilated ephyrae occurring in their sampling field. The methodology of this study could clarify the proportion and population dynamics of planula-strobilated ephyrae in the field. Elucidation of the population dynamics of planula strobilation ephyrae using this method could be an important basis for preparing countermeasures for jellyfish blooms in areas where planula strobilation occurs.