Catostylus tagi (Class: Scyphozoa, Order: Discomedusae, Suborder: Rhizostomida, Family: Catostylidae) life cycle and first insight into its ecology

Ethics statement

The jellyfish C. tagi is not an endangered or protected species.

Fertilisation

In early October 2019, six medusae were collected from the Tagus estuary (Portugal), near the Oceanário de Lisboa, and individually transferred to the laboratory within 30 mins in 15 L buckets. Specimens’ sex and gonadal maturity were determined using a microscope. Of the six individuals, two were males while four were females. Gonads were extracted from the two most mature individuals, and gastric filaments and excess tissue removed. The female’s and the male’s bell diameters were 39.5 and 43 cm, respectively. The extracted gametes were mixed and incubated for 48 h with constant aeration in three L containers containing ultraviolet-treated artificial seawater (Red Sea’s Premium salt^®) at a salinity of 35‰ and room temperature (18 °C). After 48 h, the planulae were collected by filtering the medium on gradient mesh (200 and 55 µm). Plastic petri dishes, previously incubated for four days in natural seawater for biofilm development, were used as substrates. The petri dishes were placed at mid-height in 500 ml glass bowls, thus allowing the planulae to settle on both sides of the substrate.

Culture maintenance

Planulae and polyps were incubated in artificial seawater (salinity 35‰) at 18 °C under a natural light/dark cycle. Collected ephyrae were maintained in several 500 ml jars. Once the metaephyra stage was reached, individuals were transferred to a 60 L pseudo-Kreisel under the same conditions (i.e., temperature, salinity and feeding regime).

Polyps were fed daily with rotifers (Brachionus plicatilis) during the first week, after which newly hatched Artemia nauplii were added three times a week. Ephyrae and juveniles were fed rotifers (four times a day), Artemia (three times a day) and mashed mussel (once a day). Nauplii of AF Artemia Vietnam strain (small nauplii with high HUFA content, Inve Aquaculture NV^®, Baasrode, Belgium) and enriched EG Salt Lake Artemia fransiscana (Inve Aquaculture NV^®, Baasrode, Belgium) were used for ephyrae and juveniles, respectively. Every 2–3 days, a 50‰ water exchange was conducted.

Anatomical analysis

Two different Stereomicroscopes (Leica^® SAPO and Leica^® M165C) were used to describe the various life stages, as well as to follow the development of the gastric system, manubrium, and marginal lappets of the newly released ephyrae (stage 0) through to the metaephyra stage (stage 7).

Measurements of the scyphistoma were taken following Straehler-Pohl, Widmer & Morandini (2011): total body length (TBL), calyx length (CL), hypostome length (HL), mouth disc diameter (MDD) and stalk length (StL) (Fig. 1A). The following standard measurements were used for the young ephyrae (Straehler-Pohl & Jarms, 2010): total body diameter (TBD), central disc diameter (CDD), total marginal lappet length (TMLL), lappet stem length (LStL) and rhopalial lappet length (RLL) (Fig. 1B). Relative body dimensions (%) were calculated for scyphistomae (measurements compared with body length, e.g., CL/TBL $\times$ 100, and calyx diameter, e.g., MDD/CL $\times$ 100) and for ephyrae (measurements compared with body diameter, e.g., CDD/TBD $\times$ 100, and lappet length, e.g., RLL/TMLL $\times$ 100). A total of 11 scyphistomae and 20 ephyrae from five strobilae were measured.

The effect of temperature and salinity on planula development

Two orthogonal treatment sets were established with three temperatures (15, 20 and 25 °C) and four different salinities (20, 25, 30 and 35‰) reflecting conditions recorded in the Tagus area (Gameiro, Cartaxana & Brotas, 2007; Rodrigues et al., 2017). Water was prepared by diluting artificial seawater (35‰) with distilled water. Each treatment was tested with eighteen replicates (planulae) following the methods of Conley & Uye (2015) and Takao & Uye (2018). A total of thirty-six polycarbonate culture plates, six-wells of ten ml, were prepared (three plates per experimental condition). Culture plates were filled with natural seawater four days prior to incubation in order to allow for biofilm development. Acclimatation of the planulae to lower salinities (30, 25, and 20‰) was done in a step-wise fashion, soaking the planulae in water of each decreasing salinity for 5 min until the target salinity was reached. Over six days, planulae were surveyed daily with a stereomicroscope (Zeiss^® Stemi 305). Life stages of the planulae were recorded in the following manner: Dead; Stage 0: settled but no tentacles; Stage 1: 1–4 tentacles; Stage 2: 5–7 tentacles; Stage 3: 8–16 tentacles. None of the polyps were fed during this trial.

The effect of temperature and feeding regimes on asexual reproduction

A total of two orthogonal treatment sets were established with two temperatures (15 and 20 °C) and three feeding regimes, comprising groups being fed rotifers (Brachionus plicatilis; R_group) or Artemia sp. nauplii (A_group) or being unfed (U_group). Eighteen polyps were tested using of each treatment combination. A total of one polyp was placed per well into 6-well polycarbonate culture plates (three plates per experimental condition) filled with 10 ml of the artificial seawater (35‰). A water bath was used to maintain the designed temperatures. The photoperiod was maintained at 12 h light:12 h dark. After allowing the polyps 1 week to reattach and acclimate to the experimental temperatures, newly hatched Artemia nauplii and B. plicatilis were fed in excess every 2 days. After a feeding period of 1.5 h, the wells were cleaned with swabs and uneaten food was discarded, while the seawater was replaced with new water of the same temperature. This feeding protocol provided saturating prey briefly, resulting in equal feeding in all treatments by minimising enhanced feeding at warmer temperatures (Ma & Purcell, 2005). Specimens in the unfed treatment never received food, although the water was exchanged similar to other treatment groups. Polyps were examined daily for strobilation and ephyrae release and twice a week for podocyst production. After enumeration, new ephyrae were removed but not the new podocysts. The experiment lasted 33 days.

Several response variables were defined for analysis, comprising: the number of podocysts produced by the polyps; the time from the beginning of the experiment to strobilation onset, i.e., the “pre-strobilation” period (pre-str); the time from the beginning of strobilation to the release of the first ephyrae, i.e., the “bet-strobilation” period (bet-str); the time from the first release of ephyrae to the release of the last ephyrae, i.e., the “strobilation period” (str) the number of ephyrae produced for each strobilation event.

Community science data

This study presents data on C. tagi sightings from Tagus estuary for the year 2019 gathered in the GelAvista Project’s scope (gelavista.ipma.pt), mainly based on the GelAvista smartphone App. The project is a citizen science program that provides information on jellyfish’ presence in Portugal through volunteer contributions of jellyfish sightings via the GelAvista smartphone application, email address, and Facebook page. The collected data include GPS location, date, and hour of sighting and the approximate number of specimens spotted. Species identification is made through the examination of photographs or videos. A confidence level was assigned to all reports, taking into account the veracity and sufficiency of the information received.

Statistical analysis

Since the measurements on the planulae consisted of repeated measures of binary outcomes, data on the planktonic stage were assigned to durations from the experiment onset to settlement time (maximum of 160 h) (Takao & Uye, 2018). The combined effect of temperature and salinity on the planktonic duration was tested by two-way ANOVA followed by Tukey pair-wise comparison. Data were square-root transformed to meet the residuals homogeneity assumption.

The pre-str data were analysed with generalised linear models for counts data. Due to excessive zero in the data, a zero-inflated model was used (Zuur et al., 2009). When zero-inflated Poisson model presented overdispersion, the model was corrected with the standard errors using a quasi-GLM model and a third modelwas fitted with a negative binomial distribution. The best models was selected based on the AIC and BIC values (Zuur et al., 2009).

Since only two combinations produced ephyrae, bet-str, str and ephyra released by strobilation event were analysed with a T or Wilcox ranking test depending on the variance homogeneity.

Data were analysed with the free R platform (version 3.0.2; R Development Core Team 2011) using car (Fox & Weisberg, 2019) and glmmTMB (Brooks et al., 2017).

Life cycle

Catostylus tagi displayed a typical metagenetic life cycle including scyphistoma and ephyra phases (Fig. 2). The first polyps were observed approximately 96 h after the planulae had been added to the culture plates. The young polyps were translucent-white, cone-shaped, and typically had four tentacles. Mature scyphistoma (Fig. 2A) had 16 tentacles in a single whorl around a slightly sunken mouth disc. The four-lipped hypostome was short, 325 ± 64 µm (≈22% TBL) and club-shaped. The calyx had an elongated cup shape. Scyphistoma colour varied from white to pale orange depending on the feeding. Measurements of C. tagi mature scyphistoma are summarized in Table 1.

The scyphistoma proliferated asexually via podocysts (Fig. 2A); this proliferation was observed starting as a periderm-enclosed podocyst. The podocysts were typically yellow or brown. A finger-shaped stolon developed from the lower part of the stalk and attached to the substrate allowing the scyphistoma to shift over. No other reproduction modalities, such as lateral budding by stolon, lateral scyphistoma budding or pedalocysts were observed.

Both monodisc (producing one ephyra) and polydisc (producing multiple ephyrae) strobilation were observed; monodisc strobilation was observed only once. At the first stage of strobilation, the calyx elongated and the first marginal lobe formed via constriction of the upper part of the calyx (Fig. 2B); additional, marginal lobes progressively formed beneath this initial one (Fig. 2C). The lappets and rhopalii appeared (Fig. 2D) and the scyphistoma tentacle progressively achieve complete resorption. After release of the final ephyra, the residuum developed new tentacles (n = 16) and hypostome (Fig. 2E).

Newly released ephyrae (stage 0) typically had eight lappets with a pair of antler palm-like rhopalial lappets with two to seven finger-like appendages (Fig. 2F). There was one rhopalium per lappet. The tips of the rhopalial canals ended at the red-coloured rhopalium base; however, the eight velar canals were either not developed or undistinguishable. The eight rhopalial canals were slightly forked and with rounded points. There were one or two gastric filaments per quadrant. The manubrium presented a four-lipped shape (Fig. 3A). Ephyrae exhibited colours from dark pink to dark red. Measurements of newly released C. tagi ephyrae are summarized in Table 1.

As the timing of the developmental stages of ephyrae are affected by environmental conditions (e.g., temperature, feeding), the stages are listed below in chronological order without designated time period:

Stage 1 (Fig. 2G): The TBD doubled to 4.8 ± 0.4 mm. Eight rhombical velar canals appeared. The velar canal thickened and the tips rounded-up. The first oral tentacles developed on the distal ends of the manubrium (Fig. 3B).

Stage 2 (Fig. 2H): Lappet bulbs grew between the marginal lappets. The velar canals formed a pair of branches midway. The manubrium started to split into four oral arms (Fig. 3C).

Stage 3 (Fig. 2I): The lappet bulbs developed into serrated velar lappets. The velar canals lengthened centrifugally and the rhopalial canals formed a pair of side branches that grew centrifugally toward the velar canals. The four oral arms divided to form eight arms (Fig. 3D).

Stage 4 (Fig. 2J): The side branches of the velar canals fused with the pair of side branches of the rhopalial canals to form a primary ring canal. The velar lappets extended outward while their serrations retracted.

Stage 5 (Fig. 2K): The velar lappets continued their extension outwards. The serrations on the antler palm-like rhopalial lappets retracted. Two new canals grew on the velar canal, developing centripetally (tertiary canals) parallel to the radial canals, while two additional canals developed horizontally toward the rhopalial canal.

Stage 6 (Fig. 2L): The midway-side branches of the velar canal fused with the radial velar canals to form a secondary ring canal. The centripetal canal fused with the second ring and continued growing centripetally. Below the second ring, the radial canal developed another set of side branches to form the final ring canal. The velar lappet extremities extended until reaching the rhopalial lappets yo complete the umbrella.

Stage 7 (Fig. 2M): The tips of the centripetally growing canals avoid the fusion, instead protruding into the space between the last ring canal and the stomach. The floor and the roof of the canals fused, forming woven “Inseln”, constituting a mesh network of anastomosing canals.

A total of 5 months after ephyrae are released from the strobilae, juvenile C. tagi are fully developed (Fig. 4).

Temperature and salinity effect on planulae development

Planulae of C. tagi showed relatively low mortality (≤20 %), especially at 15 °C. No mortality was observed at 30‰ salinity (Fig. 5). The first planulae settlement occurred within four to six hours in all treatment combinations. The final settlement proportion varied from 53% (15 °C and 25‰) to 100% (25 °C and 25–35‰). The planktonic duration (Fig. 6, Table 2) was significantly influenced by temperature (p < 0.001) and salinity (p < 0.01), however, no interaction was detected (p = 0.31). Tukey post hoc testing revealed planula settlement to be faster at higher temperatures (25 °C), while the planktonic stage duration was significantly prolonged at 30‰ salinity when compared to 35‰.

No polyps with tentacles were observed at 15 °C, all salinities considered. Polyp development was enhanced at higher temperatures, being optimal at 25 °C for all salinities. Up to 54.2% of the planulae developed into polyps under conditions of 25 °C and 20‰. During the experiment, polyps developed a maximum of eight tentacles, seen at the highest temperature (25 °C) with lower salinities (20 and 25‰). Morphological deformities were not detected.

Temperature and food effect on asexual reproduction

Podocyst production, which varied between 0.8 ± 0.9 and 1.7 ± 1.5 podocysts per scyphisotma, was neither influenced by temperature (p = 0.21) nor by feeding regime (p = 0.3), with no significant interaction detected (p = 0.17) (Fig. 7, Table 3).

At 20 and 15 °C, strobilation occurred only in three groups: 20 °C-R_group (61.1%), 20 °C-A_group (61.1%) and 15 °C-A_group (28%). Pre-str, which was significantly influenced by temperature and diet, was shorter in the 20 °C-R_group (11 ± 4 days) and 20 °C-A_group (15 ± 7 days) than in the 15 °C-A_group (32 ± 3 days) (Tables 2, 3).

Ephyrae release was only observed at 20 °C. The duration of bet-str (R_group: 5.7 ± 1.4 days; A_group: 5.6 ± 2.6 days) and the number of ephyrae produced by strobilation (R_group: 6.3 ± 3.5; A_group: 6.9 ± 3.9) were not significantly affected by the diet (Tables 2, 3). The strobilation period was significantly shorter in the R_group (2.5 ± 2.3 days) than in the A_group (26.4 ± 3.9 days) (Tables 2, 3). Twenty-seven per cent of the scyphistomae in the 20 °C-R_group were able to perform new strobilation 10 to 16 days after ending the first strobilation. The number of ephyrae produced by those scyphistomae did not change between the first (4 ± 1.7) and the second strobilation (3.7 ± 0.6).

Seasonal distribution of Catostylus tagi adults in the Tagus estuary

During 2019, sightings of adult C. tagi were reported via the GelAvista smartphone App along both margins of the Tagus estuary, from the inner bay and Cala do Norte to the estuary’s opening to the Atlantic Ocean (Fig. 8A). Sightings were recorded in all months of the year except April (Fig. 8B). Many sightings (>5) were recorded from September to February, a proxy for a higher abundance of this species.