The Effects of Heat Stress on the Physiology and Mortality of the Rhizostome Upside-Down Jellyfish Cassiopea xamachana—Observations Throughout the Life Cycle

Abstract

This study was designed to investigate the impact of heat stress on the physiological changes and mortality rates of different life stages of the rhizostome jellyfish species Cassiopea xamachana, including planula larvae, scyphistomae (polyps), and medusae. Both larval and scyphistoma stages of C. xamachana are relatively tolerant to high temperatures, but both experience nearly 100% mortality at 36 °C. Increasing temperatures also induced stage-specific effects. Settlement rates of artificially induced larvae were near 100% at lower temperatures but decreased at 34–36 °C; larvae were dead at 36 °C. When scyphistomae of C. xamachana were subjected to a gradual increase in temperature from 28 to 38 °C, polyp size declined steadily in starved animals, with animals showing clear signs of temperature stress between 35 and 36 °C. Small medusae of C. xamachana pulsed more than larger medusae and tended to have peak pulse rates at higher temperatures (~35 °C) compared to larger medusae (~29–33 °C), though the latter was not significant. At a temperature of 39 °C, all the medusae exhibited signs of heat stress, including pulsing erratically (generally lower) rather than steady rhythmic pulsations, releasing copious amounts of mucus, and having withdrawn oral arms. Temperature data presented here, and in the literature, show that pulsing C. xamachana medusae exhibit a bell-shaped curve, with temperatures over 38 °C being detrimental and becoming lethal at 40 °C. Based on the findings of this study, it is proposed that the medusa stage of C. xamachana has a higher tolerance for elevated temperatures compared to both the larvae and the polyps. Predictions of global climate change indicate that populations of C. xamachana will likely face longer and hotter summer periods, leading to increased population sizes. However, higher temperatures pose a greater risk to the survival of the species as they increase mortality in the polyp and larval stages compared to the medusa stage.

1. Introduction

Future climate change scenarios will make it impossible for most corals to survive on tropical reefs [1]. Reef-building corals in the Caribbean are already dying due to higher temperatures, along with associated diseases [2]. However, there are some species of animals that have wide tolerances to temperature that will, in fact, flourish in marine habitats. They will not only replace less resilient species at higher temperatures, but more invasive species will also potentially compete with native species for space in the habitat (e.g., [3]).

One such group of animals reacting positively to an increase in temperature are the jellyfish, especially scyphomedusae [4]. Scyphozoan jellyfish have a life cycle that responds positively to temperature. Under warmer temperatures and normal feeding, the polyps produce more asexual buds [5], and these polyps produce more medusae through strobilation (the production of the medusa stage from the polyp) in response to a longer period of warmer temperatures [6]. In addition, the medusae grow faster and reproduce more readily at warmer temperatures [7].

Cassiopea spp. live in mangroves and seagrass beds in relatively calm water [8,9]. These tropical jellyfish can be found globally in tropical and sub-tropical waters, alternating between the sexual medusa and the asexual polyp. They are suited for warm and sunny climates given their life history characteristics. For example, strobilation occurs when temperatures are greater than 23 °C [10]. The jellyfish are also symbiotic with dinoflagellate algae from the family Symbiodiniaceae [10,11]. The symbionts provide a source of metabolic carbon in the form of photosynthates and also provide compounds that trigger the scyphistomae (polyps) to strobilate. These host-symbiont associations withstand a wide range of temperatures, contributing to their success as an invasive species [12,13].

Populations invading new habitats often experience disruptions in their life-history stages, which can make invasion initially challenging or even impossible. For instance, in Brazil, Cassiopea andromeda typically has only male or female medusa populations that are likely clones [14,15,16]. The polyps can strobilate if temperatures are warm enough, but no planulae larvae develop, and the polyp forms buds to foster the clone. Populations frequently vanish, potentially due to the environment presenting greater variability than what the clone can endure. In the case of C. andromeda, its invasion of Brazil has been ongoing, some say, for at least 500 years [14].

Another example is in the Florida Keys, where C. xamachana is highly sensitive to the effects of cold fronts during the winter, which can plunge temperatures below 18 °C [12]. Although medusae can tolerate these lower temperatures and will continue to produce larvae, polyps disappear from the population. At temperatures below 18 °C, newly settled larvae are unable to develop functional tentacles, which hinders their ability to capture and transfer food to the mouth [12]. Consequently, the influx of new medusae into the population is constrained until the seawater warms during the seasonal transition. This warming period allows sexually produced larvae to settle, survive, develop into polyps, and eventually undergo strobilation [12].

The impact of climate change-induced higher seawater temperatures on the various stages of C. xamachana’s life cycle remains uncertain and unknown. In this study, the effect of warmer seawater temperatures was tested on each of three phases of the life cycle of C. xamachana, larvae–polyps–medusae, in an effort to determine how global climate change will influence the physiology, mortality, and life cycle of C. xamachana.

2. Materials and Methods

2.1. Temperature Stress on Settlement and Metamorphosis of Planulae Larvae

Egg masses were collected from the brooding vesicles of female medusae of C. xamachana from the Florida Keys in May 2022 and placed in a glass petri dish with 100 µg/mL antibiotics (streptomycin, neomycin, penicillin) in artificial seawater (ASW, Instant Ocean) and allowed to develop into planula larvae. Approximately 10–25 larvae were transferred (with a minimum amount of seawater containing antibiotics) into each well of a 24-well plate (Costar), then topped up with 1 mL of antibiotics-free 35 ppt ASW.

Temperatures were chosen based on previous experience [12]: 24, 30, 32, and 36 °C. There were six treatment replicates (wells) per temperature with six replicates (wells) of controls for each temperature. The temperature for the well plates was controlled with a water bath by floating the plates on the water pre-set to the treatment temperatures (i.e., no acclimation). Treatment wells had 100 µg/mL of the artificial peptide inducer (z-GPGGPA, Sigma Scientific), whereas controls had no introduced inducer. The artificial peptide inducer z-GPGGPA normally induces the larvae of C. xamachana to settle in less than 24h and metamorphose into a polyp within a week or two [8]. The larvae were monitored by counting the six replicate wells on days 0, 1, 2, 3, and 5. The number of larvae that had settled or died (disintegrated) were counted. The log-rank test was used to calculate p-values for pairwise comparison of the probabilities generated with the Kaplan–Meier estimator at the different temperatures (± 95% CI); counts were averaged across the six replicates.

2.2. Temperature Stress Assays with Scyphistomae (Polyps)

Polyps of C. xamachana were obtained from metamorphosed larvae collected as described above. Larvae were induced to settle and fed Artemia, until polyps were sufficiently large (oral disc ≈ 1.5 mm diameter) for use in experiments. Twelve polyps were individually placed in wells of a control 24-well plate (Costar) and twelve in an experimental plate, which was floated in a water bath. The temperature remained steady (28 °C) in the controls and was increased in the experimental plate at a modest rate of ≈0.5 °C per day until it reached 38 °C. The water in each well was changed about every 4 days, the plate about every 8 days, with the salinity remaining at 35 ppt. Polyps were not fed during the experiment. Tentacle lengths of the longest three tentacles were measured in relaxed polyps with an ocular micrometer in a dissecting scope, in addition to the diameter of the oral disc of the polyp, approximately every 4 days. Polyps were determined to be alive (polyp, with tentacles extended) or dead (disintegrated) by observing their morphology and response to agitation of the plate.

2.3. Temperature Stress Assays with Medusae

Experiments were conducted at the Key Largo Marine Research Laboratory in the Florida Keys in May 2022, when the seawater temperatures were about 28 °C.

Jellyfish pulse rate is a proxy for metabolism [17]. Mayer [18] found that medusae of C. xamachana exhibited an increase in pulse rate from 16 to 28 °C, followed by a broad maximum between 29 and 36 °C, before decreasing at 36–39 °C. Since the monitoring of pulse rate in this study occurred between 29 and 39 °C, there is unlikely to be an increase with temperature.

Mayer [19] also found that when medusae were disturbed (e.g., prodded, changes in water movement, moved between containers), they had an elevated pulse rate for a few minutes before returning to a normal rate. For this reason, counts of pulses were made at least 5 min after transferring medusae to the counting chamber. Twenty-nine medusae of C. xamachana ranging in size from 2 to 14 cm in diameter were collected and either immediately used in experiments or kept in an aquarium for less than 2 days with flow-through seawater. When medusae are removed from their natural environment and placed in an aquarium, factors such as feeding, light, and activity levels change. To minimize these effects, the medusae were kept in aquaria for no longer than two days.

Individual medusae were placed in a large glass bowl (1 L) fitted with a small aquarium heater. The temperature of the water was raised stepwise from 29 to 39 °C, taking 15 min to go to a higher temperature. The pulse rate (number of pulses per minute) at a given temperature was determined as the average from three counts per medusa. Because the pulse rate depends on the size of medusae, the medusae were assigned to seven size classes with 3–5 replicates per size class (2.0–2.9 cm n = 5, 3.0–4.9 cm n = 5, 5.0–7.9 cm n = 5, 8.0–8.9 cm n = 3, 9.0–9.9 cm n = 3, 10.0–11.9 cm n = 3, 12.0–14.9 cm n = 5, diameter in cm) and mean pulse rates within size classes were determined at six temperatures (29, 31, 33, 35, 37, 39 °C). Mean pulse rates per size class (± s.d.) were plotted at each temperature and used in statistical analyses. Medusae were not fed during experiments.

2.4. Statistical Analyses

Statistical tests and plotting were performed using R (version 4.1.0). The Shapiro test was used to decide about normal distribution of the data. To compare the means of the settlement rate, the Mann–Whitney U test was used for pairwise comparisons of settlement rates across temperature groups because the data did not meet the normality assumption required for ANOVA. For the overall comparison across all temperature groups, a Kruskal–Wallis test was conducted. Pairwise Mann–Whitney U tests were then performed as post-hoc comparisons. To account for multiple comparisons, a Bonferroni correction was applied to adjust the p-values accordingly. The log-rank test was used to calculate p-values for pairwise comparison of the probabilities generated with the Kaplan–Meier estimator at the different temperatures using the R package survival (version 3.7). Plotting of the survival curves was carried out with survminer (version 0.4.9).

Analysis of changes in pulse rates was conducted by fitting a LMER linear mixed-effects model to the data using the R package lme4 (version 1.1–35.5). To determine the effects of temperature and bell diameter on pulse rate, these factors were treated as fixed effects, while variation between individual jellyfish in each size category was accounted for as a random effect. Pairwise comparisons between size categories were computed by estimating marginal means using the R package emmeans (version 1.10.3).

3. Results

3.1. Response of Planula to High Temperature Treatment

Larvae can be induced to settle with the peptide inducer GPGGPA. Similar peptides were originally found in the bacteria Vibrio alginolyticus and likely mimic natural inducers of larval settlement [8]. Temperatures were chosen based on previous experience [5]: 24, 30, 32, and 36 °C. Larvae exposed to elevated temperatures did not show significant mortality at 24 °C and 32 °C (Log-rank test; p > 0.05) (Figure 1A). Larval mortality at 36 °C increased to an average of 97% on day 5, compared to the mortality of larvae at 24 °C, where less than 2% of larvae had died on day 5. When larvae kept at 24, 30, and 32 °C were induced to settle, over 90% of larvae settled within 1 day of exposure to the peptide (Figure 1B). At 36 °C, larval settlement decreased significantly compared to 24 °C (Figure 1B). We monitored larval mortality after settlement and found larvae that had undergone settlement showed significant mortality at 32 and 36 °C compared to 24 and 30 °C (Log-rank test; p < 0.05) (Figure 1C).

3.2. Temperature Tolerance of Scyphistomae (Polyps)

Twelve polyps were either exposed to increasing temperature (experimental, 28–38 °C) or maintained at 28 °C (control) over the course of 22 days. The diameter of the oral disc decreased steadily for both control and experimental polyps and did not differ significantly (Figure 2A). The tentacle length of experimental polyps was not significantly different from the control through 34 °C but significantly declined at 35–37 °C (Figure 2B; linear mixed-effects model, p < 0.005). Polyps and remaining tentacles did not respond to a tap of the dish at 36 °C; 100% mortality was reached by 37 °C and mean tentacle length of 0 mm (Figure 2B).

3.3. Temperature Stress on Medusae

Small medusae tended to pulse at higher rates than larger medusae. A linear mixed-effects model was fit to the data to understand the effect of temperature and size on pulse rates. The pulse rate peaked at approximately 35 °C for jellyfish < 9 cm in diameter and at 29–33 °C for those >10 cm in diameter (Figure 3A). At temperatures greater than 35 °C, the pulse rate began to decrease, making the other side of a bell-shaped curve [14]. A linear mixed effect model found significantly higher pulse rates between animals ranging between 2 and 5 cm in diameter and larger animals. All medusae showed signs of heat stress at 39 °C, consisting of withdrawn oral arms, generally pauses between pulsing (rather than rhythmic pulsing), and production of copious amounts of mucus. In the case of four medusae, a shift to double-pulsing or extremely fast and irregular beats (fibrillation), possibly proceeding death, was also observed. For these individuals, pulse rates increased at 39 °C despite showing a decreasing trend at 37 °C (Figure 3B).

4. Discussion

This study addresses the response of C. xamachana to higher temperatures, which seem unfortunately inevitable in future climate scenarios [20]. A sustained increase in temperature is expected to have several effects on the life cycle of C. xamachana. Initially, it should allow the scyphistomae (polyps) to survive through the winter period in the Florida Keys [12], whereas currently, they disappear from the population when temperatures drop below 18 °C. The additional warmer months in which polyps are present should allow longer periods where strobilation can occur, thereby allowing continuous addition of medusae into the population. Higher temperatures should also help maintain the biomass of the medusae with in situ feeding. Nevertheless, it is important to note that physiological responses to temperature typically follow a bell-shaped curve, as observed in various studies, e.g., Figure 4 in [18]. This pattern applies not only to C. xamachana, but also to many other species. It implies that while higher temperatures may initially elicit favorable physiological responses, there is inevitably a point where a lethal high temperature is reached, leading to the eventual demise of the species. The data presented here suggest that medusae of C. xamachana will survive higher temperatures for a period of time (~37–39 °C) compared to the planula larvae and the polyps, which begin to exhibit mortality around 36 °C. Larvae are particularly challenging to observe in the field due to their small size, which raises concerns about potential issues they may already be facing and remain undocumented. Unlike other marine animals, larvae are highly accessible to researchers and can be observed throughout the year in the laboratory. This unique accessibility allows researchers to closely monitor larvae and detect any signs of stress. Therefore, when researchers come across a “bad batch” of larvae during the late summer, it is possible that they are witnessing the effects of heat stress on the species or genus.

Heat stress is seen when the medusa begins pulsing erratically (generally slower) rather than steady rhythmic pulsations, releases copious amounts of mucus, and has tentacles that are misshaped and retracted (36–39 °C). The current data support those of Mayer [18], who documented increased pulsing with temperature over a 9-h period (17–36 °C). They also observed when movement had ceased (~38.5 °C) and the temperature of death (~40.0 °C).

The experiments conducted in this study were on larvae and polyps that did not possess any symbiotic algae from the Symbiodiniaceae family. Furthermore, the experiments examining the pulse rates of symbiotic medusae were conducted over a relatively short duration of less than 4 h, on a similar time scale as [18]. As a result, the role of the symbionts was not considered a factor that could influence the obtained results.

Seawater temperature increase in the environment typically occurs at a lower rate than in this experiment, but it may increase over 6 °C in a span of 3 h during the late summer. These rapid increases in temperature can even occur daily [12], which is about half the rate of temperature increase during the medusae experiments (Figure 3). This slower process might involve factors such as nutrition provided by the symbionts or the adaptation/acclimatization of the species’ response to higher temperatures. Therefore, in real-world scenarios, the presence and interactions with symbionts, as well as the potential for acclimation responses, may have a significant impact on the species’ ability to cope with and tolerate heat stress.

Beziat and Kunzmann [21] found that warmer temperatures have the potential to drive invasions of Cassiopea spp. into new habitats, depending on the thermal tolerance of their symbionts. The type of Symbiodineaceae in the tissues was not monitored in their study; however, the C. andromeda were maintained at a constant 24 °C at the Leibniz Centre for Tropical Marine Research in Germany, and all medusae died at 34 °C. This is a lower threshold than medusae of C. xamachana in the current study, possibly because of interspecies variation, a difference in symbionts, or different maintenance-experimental conditions.

4.1. Larval Settlement and Metamorphosis

The sea surface temperatures in Florida Bay generally range from 17 to 38 °C (NOAA). Larvae of C. xamachana appear to be resilient to increased temperatures, tolerating temperatures of 32 °C but exhibiting mortality at 36 °C. Interestingly, nearly 100% of larvae triggered to settle with the peptide inducer z-GPGGPA underwent the transition at all temperatures tested within 24 h. The only exception was larvae kept at 36 °C, which began dying within 24 h of induction. Larvae untreated with the peptide inducer generally did not settle within 5 days.

4.2. Scyphistomae (Polyps) and Medusae

The scyphistomae and medusae utilized in these experiments were collected in the Florida Keys and identified as C. xamachana. However, morphological and genetic similarities between medusae of C. xamachana and C. andromeda, have led to researchers suggesting the species found in Florida may be due to past introductions of C. andromeda [22]. Muffet et al. [23] have further proposed that 90% of the medusae found in the Florida Keys are C. xamachana, while the remaining 10% are C. andromeda. Muffet et al. [23] analyzed a limited sample size of 55 individuals. Further investigation into potential genetic differences between the two species in Florida would provide valuable insights.

Scyphistomae of C. xamachana exhibited similar temperature tolerance as the larval stage, exhibiting mortality at 36 °C. For the duration of the experiment, polyps were treated to increasing temperatures without food. As a result of combined stress, polyp size decreased gradually. However, scyphistomae are capable of long durations without food (>2 months, WKF, unpublished). Polyp deaths during the experiment are, therefore, likely the result of acute temperature stress.

The pulse rate of medusae is thought to be a function of metabolic rate [24,25]. Mayer [19] found rhythmic pulsation of small C. xamachana faster than that of larger medusae, and [17] documented medusae of C. xamachana having a higher pulse rate (implying metabolic rate) than C. frondosa. Mayer [18] was apparently the first to notice that the pulse rate increases with temperature of the seawater: “Cassiopea xamachana with motor centers intact pulsates at its maximum rate at about 33 °C, ceasing to pulsate if cooled to 16.6 °C or if heated to 38.5 °C”.

The results presented here support that small medusae pulse faster and tend to have higher maximum pulse rates at higher temperatures compared to larger medusae (Figure 3A). This suggests that younger medusae exhibit a higher metabolic rate per gram of biomass or unit of surface area compared to larger medusae. Thus, smaller jellyfish may be more susceptible to an increase in temperatures. This observation aligns with the concept of allometric scaling, which explores the relationship between an organism’s size and its metabolic characteristics [26].

5. Conclusions

The response of the notoriously invasive jellyfish C. xamachana to higher temperatures is predictable. While drawing conclusions about global climate change based on short-term experiments has limitations, further research, including long-term observations, is needed. Initially, as temperatures rise, both polyps and larvae are expected to increase in numbers alongside the medusae, particularly in populations of C. xamachana at the edges of the tropics. However, as global climate change continues to raise temperatures, it is hypothesized on the basis of our results that the larvae and polyps will encounter lethal temperatures (~36 °C) earlier than the medusae. This would leave the medusae to endure and survive at higher temperatures, ranging from 36 to 39 °C, before eventually succumbing to temperatures exceeding 39 °C.

Note: Seawater temperatures during the El Niño of 2023 exceeded 38 °C in Florida Bay (“A world record for coral reef areas?” New York Times, 26 July 2023), with 36 °C recorded in Buttonwood Sound where Key Largo Marine research Laboratory is located and the experiments detailed above occurred. Medusae of C. xamachana were observed bleached white and starving (getting smaller). No developing larvae were observed on the tentacles of the female medusae, and no polyps were observed (personal observation). It appears that the lethal temperatures of this species have arrived.