Epizoic acoelomorph flatworms inhibit growth and expansion of the soft coral Cladiella sp. — Advanced Aquarist

1. Marine Animal Ecology, Wageningen University and Research Centre, 6708WD Wageningen, The Netherlands
2. Ouwehands Zoo, 3911AV Rhenen, The Netherlands

*Corresponding author: diedemaas@gmail.com, de Elst 1, 6708WD, Wageningen, The Netherlands
°These authors contributed equally to this work.

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Abstract

Acoel flatworms can form dense blooms on coral hosts, both in the wild and in aquaria. Adverse effects of acoels on scleractinian corals have been shown in the form of coral mucus consumption by flatworms, impairment of coral feeding and kleptoparasitism. This study investigated whether flatworms also have detrimental effects on alcyonacean corals. Growth of the soft coral Cladiella sp. was indeed affected by the presence of acoel Waminoa sp. Negative growth rates were found for flatworm–infested coral fragments throughout the experiment, in contrast to controls, possibly due to impaired photosynthesis. In addition, colony expansion was found to be negatively impacted by Waminoa. Finally, polyps of infested fragments required more time to recover after physical disturbance. These insights enhance our understanding of the coral–flatworm symbiosis, and support the view that coral–associated Waminoa are parasitic in nature.

Keywords:

Acoela; Cladiella; Waminoa; alcyonacea; symbiosis; parasitism

Highlights:

• Cladiella growth rate and colony expansion (hydrostatic skeleton maintenance) were negatively impacted by Waminoa presence.
• Polyps of Cladiella required more time to expand after physical disturbance when infected with Waminoa.
• These insights support the view that coral–associated Waminoa sp. are parasitic.

 

Introduction

Acoel flatworms (Phylum Platyhelminthes) are small (0.5–10 mm) oval–shaped, flattened organisms without a body cavity (Hyman, 1951; Bush, 1981). Certain species can form significant epizoic blooms on both wild and captive corals (Winsor, 1990; Barneah et al., 2007; Nosratpour, 2008; Haapkylä et al., 2007, 2009; Hoeksema and Farenzena, 2012). Epizoic blooms of these flatworms may have several effects on coral hosts. Firstly, it is theorized that coral photosynthesis may be hampered due to shading by dense covers of acoels, which can be up to 90–100% (Barneah et al., 2007; Haapkylä et al., 2007; Hoeksema and Farenzena, 2012). Secondly, the ability of epizoic flatworms to consume the mucus layer of scleractinian corals has been demonstrated with stable isotopes (Naumann et al., 2010). Mucus removal could affect the corals’ ability to withstand environmental stressors such as UV–radiation and sedimentation, as well as make the corals more vulnerable to microbial diseases (Shuhmacher, 1977; Krupp, 1984; Drollet et al., 1993; Ritchie, 2006; Haapkylä et al., 2007). Thirdly, a flatworm species of the genus Waminoa was found to compete with its coral host for zooplankton prey, to display kleptoparasitism, and to limit coral feeding rates (Wijgerde et al., 2011, 2013).

Thus far, all studies on interactions between corals and flatworms have been done with scleractinian corals. This study is the first to examine the potential effects of flatworms on an alcyonacean coral (Cladiella sp. Gray, 1869; family Alcyoniidae; order Alcyonacea). Fragments of Cladiella sp. were incubated with and without flatworms (Waminoa sp., Winsor, 1990; family Convolutidae, order Acoela), to test their impact on coral growth, hydrostatic skeleton maintenance (i.e. colony expansion), and polyp recovery time after physical disturbance, which may all serve as potential stress indicators (Jaap and Wheaton, 1975, Thompson and Bright, 1980). The findings of this study provide insights into the ecology of coral–associated flatworms, in particular whether Waminoa sp. are true parasites for soft corals, alike their parasitic relationship with scleractinian corals.

Image 1.  Cladiella sp.  Photo courtesy of Bob Fenner (www.wetwebmedia.com)

2. Materials and Methods

2.1. Coral husbandry and flatworm infestation

This study was carried out in the aquarium department of Ouwehands Zoo (Rhenen, The Netherlands). Live corals and flatworms were made available by the Ouwehands Aquarium directory and were taken from several display tanks in the zoo. The Cladiella colony used in this study was free of Waminoa. From this single colony, fragments for the study were obtained, hence, they were genetically identical. All fragments were approximately the same size (mean±SEM buoyant mass of 0.39±0.02 g). Cut fragments were tied to grey PVC plates of 7x7x0.5 cm using nylon thread. After a week, the nylon threads were cut as the corals had adhered to the plates, and the experiment was started. By that time, all corals appeared healthy and in a fully expanded state. In total, 32 Cladiella fragments were randomly assigned to four 200 dm³ tanks (n=8 fragments per tank). In two randomly selected tanks, 16 fragments in total were infected with Waminoa by adding a surplus (100–200 individuals) of flatworms to their tanks, while the 16 fragments in the other two tanks were left uninfected as controls (Tank numbers Z–3 and Z–6 were controls, Z–4 and Z–7 contained infected corals). This design was chosen to account for potential tank effects. On different subsets of these 32 fragments, growth rate, colony height and width (as an indicator of colony hydrostatic pressure) and polyp expansion time after physical disturbance were measured (see below for details).

All corals were fed weekly with 50 mL of freshly harvested Artemia nauplii culture (stock concentration of 800–1200 individuals mL^-1), equal to a tank concentration of 250–375 nauplii L^-1. This dosage was chosen to maintain the same feeding regimen as compared to the main display tanks at the zoo. Although it was later found the corals do not seem to capture Artemia (see results), the breakdown of organic compounds resulting from feeding Artemia could indirectly supply the corals and their symbiotic zooxanthellae with dissolved organic carbon and inorganic nutrients such as ammonia, nitrate and phosphate. Weekly, all tanks were cleaned and 10% of the seawater volume was changed with natural seawater. The seawater used to fill and change the tanks came directly from the North Sea, where it was pumped up during incoming tide at the border between the North Sea and the Eastern Scheldt estuary (Southwest Netherlands). The tanks contained a 30–50 dm³ biofilter for bacterial aerobic nitrification. Water parameters (temperature, salinity, pH, and NO₂^–) were monitored weekly to keep them within their acceptable range (Table 1).

Table 1: Seawater parameters of all four tanks during the study. Values are means±SEM (n=10 per tank).

Tank	pH	NO2–(mg/L)	Salinity (g/L)	Temperature (°C)
Z3 (control)	8.27±0.01	0.02±0.00	33.8±0.4	24.3±0.2
Z4	8.31±0.02	0.02±0.00	34.0±0.5	23.9±0.1
Z6 (control)	8.24±0.02	0.02±0.00	33.6±0.4	23.9±0.1
Z7	8.28±0.02	0.02±0.00	33.6±0.5	24.1±0.1

2.2. Growth rate

Growth rates were measured over a six–week period, during which growth was measured at the beginning of the experiment and each subsequent two weeks (t=0, t=2, t=4 and t=6 weeks). To determine growth rates (n=5 corals per tank, n=20 in total), the non–disruptive underwater buoyant weighing technique was applied (Davies, 1989; Schutter et al., 2010). To determine buoyant mass, corals on their PVC plate were hung on a hook at a fixed position in a bucket filled with seawater of a fixed volume, temperature (26°C), and salinity (35 g L^–1). The hook was connected to an under–weighing analytical scale (Osinga et al., 1999). At each weighing moment, each coral was weighed at least three times, and the average was taken from these measurements. Before the coral fragments were initially tied to their PVC plates, each individual coral fragment was weighed separately. After tying to the plates, their mass was measured again, thus generating the total mass of the coral fragment and the plate. Plate mass was calculated for each sample, and used to determine net coral mass for all subsequent measurements. Finally, specific (or relative) growth rate (µ) over three intervals were calculated using the following formula:

Here, µ is the specific growth rate expressed as gram coral gram coral^–1 day^–1 (which can be simplified as day^–1), BM is the buoyant mass expressed in grams, n–1 is the beginning of a growth interval, n is the end of that interval, and Δt is the time between measurements expressed in days.

2.3. Colony expansion

Colony expansion (n=8 corals per tank, n=32 in total) was measured in parallel to growth. This was determined by measuring the length and width of each coral fragment in millimetres, as photographed from one fixed side of each colony. Dimensions were measured using photographs taken with an HDR–CX505VE camera (Sony Corporation, Tokyo, Japan), which were analysed via ImageJ®. Infected and uninfected corals were measured after one week and 6 weeks of infestation (t=1 and t=6 weeks) to determine the short–term potential of soft corals to adapt to Waminoa infestation. After 6 weeks, corals were photographed before being weighed. One day later, after measuring polyp recovery time (see 2.4), 14 of the infected coral fragments (n=7 corals per tank) were given a fresh water dip which lasted approximately 5 seconds, which caused the flatworms to fall off the corals and disintegrate due to osmotic pressure. The dewormed corals were then returned to their tanks and after 2 weeks (t=8) their ‘recovered’ lengths were measured and compared to 6 control fragments (n=3 corals per tank).

2.4. Polyp recovery time

After determining coral growth rates, i.e. after 6 weeks plus one day and just before the fresh water dip (see 2.3), polyp recovery time was measured for infested and uninfested corals (n=5 corals per tank, n=20 in total). Each coral was disturbed directly for a total of 5 minutes. The disturbance consisted of picking up the colony, tapping the plate against the edge of the tank for 5 times, and keeping it outside the tank for a total of 4 minutes. Directly after these 4 minutes, the coral was put in the water for 10 seconds, then taken out for 10 seconds. This in/out treatment was repeated two more times to make one minute in total. By using a stop watch, the amount of time was measured from the moment the coral was put back in its tank until the polyps were fully expanded again.

2.5. Statistical analysis

Statistical analysis was carried out using SPSS Statistics 22.0 (IBM, Somers, USA). Data series obtained were examined for conformity to the assumptions for parametric statistics. Normality of the data was tested by a Shapiro–Wilk test and Q–Q plots. Homogeneity of variances was verified by running a Levene’s test. Sphericity was verified via Mauchly’s test. A three–way mixed factorial ANOVA was used to analyse data for buoyant mass and colony size over time, with tank as a random factor and flatworms as a fixed factor. This mixed model was most appropriate since the same coral fragments were repeatedly measured over time, whereas different coral colonies were subjected to two experimental conditions (either infected or uninfected). A  two-way factorial ANOVA was used to analyse polyp recovery data, again with tank and flatworms designated as random and fixed factors, respectively. All reported data are mean±SEM. Graphs were plotted using SigmaPlot 13.0 (Systat Software, Inc, San Jose, USA).

 

3. Results

3.1. Coral health and growth rate

Infestation with Waminoa clearly changed the outer appearance of Cladiella fragments (Supplemental video 1). Infected corals were paler and polyps tended to be in a more retracted state than control colonies. Partial necrosis was observed in some of the infected fragments.

Flatworms significantly reduced Cladiella growth (Fig. 1, Table 2). Specific growth rates also varied significantly over time (Table 2), with higher overall growth rates in week 3–4 compared to week 1–2 (Bonferroni, p=0.005). Despite the absence of a significant interactive effect between flatworm presence and time (Table 2), the effect of flatworms was not consistent over time. In week 1–2, growth was not significantly different between the two treatments (simple effects contrast, F_1,18=0.712, p=0.410), in contrast to week 3–4 and 5–6, during which growth was significantly lower (i.e. negative) in the presence of flatworms (F_1,18=7.088, p=0.016, and F_1,18=12.897, p=0.002, respectively). No main or interactive tank effect on coral growth rates was found (Table 2).

Table 2: Three–way mixed factorial ANOVA, demonstrating effects of flatworms, tank and time on specific growth rate and colony expansion, and two–way factorial ANOVA for polyp recovery time of the soft coral Cladiella sp.

Source	df	F	P
	specific growth rate
Flatworms	1	11.478	0.004*
Tank	1	0.909	0.355
Time	2	9.409	0.001*
Flatworms * Tank	1	0.076	0.786
Flatworms * Time	2	1.125	0.33
Tank * Time	2	0.783	0.466
Flatworms * Tank * Time	2	0.424	0.658
	colony height
Flatworms	1	1.721	0.208
Tank	1	0.244	0.628
Time	2	3.229	0.053
Flatworms * Tank	1	0.314	0.583
Flatworms * Time	2	13.621	0.000*
Tank * Time	2	0.532	0.592
Flatworms * Tank * Time	2	0.189	0.828
	colony width
Flatworms	1	4.193	0.057
Tank	1	3.244	0.091
Time	2	1.111	0.342
Flatworms * Tank	1	0.184	0.674
Flatworms * Time	2	7.818	0.002*
Tank * Time	2	0.993	0.382
Flatworms * Tank * Time	2	0.076	0.927
	polyp recovery time
Flatworms	1	1443.991	0.017*
Tank	1	5.429	0.258
Flatworms * Tank	1	0.033	0.858
*Indicates significant effect (n=5–8).

3.2.  Colony expansion

Next to inhibiting coral growth rates, flatworms significantly reduced colony expansion after 1 and 6 weeks, as indicated by reduced height and width of infested colonies compared to worm–free corals (Fig. 2). The interactive effect of flatworm presence and time on colony height and width (Table 2) was reflected by the absence of a significant difference between worm–free and dewormed corals at the end of week 8 (Fig. 2). Vice versa, the interaction was reflected by a positive trend in time for colony height and width for the (initially) flatworm–hosting corals only, due to increased expansion after flatworm removal. No main or interactive tank effect on colony expansion was found (Table 2).

3.3. Polyp recovery time

Flatworms also significantly affected polyp recovery time (Table 2), with infested polyps exhibiting longer recovery times compared to worm–free polyps (Fig. 3). No main or interactive tank effect on polyp recovery time was found (Table 2).

4. Discussion

This study has yielded new insights into the effects of flatworms on soft corals, which support the emerging view that acoelomorph flatworms should be considered coral parasites, rather than commensals (Wijgerde et al., 2013). First of all, it was found that Cladiella growth is inhibited by the presence of Waminoa flatworms, which has not been shown before. This suggests that flatworms can significantly affect the ability of corals to invest energy in growth. Some tissue loss was also observed in infected corals, in line with field observations by Hoeksema and Farenzena (2012). Since Cladiella is a particularly sturdy genus (Wabnitz et al., 2003), the negative effects of epizoic flatworms could be even more devastating in less resilient corals.

Although this study represents the first experimental evidence for flatworm–induced growth retardation in corals, earlier studies already proposed several potential mechanisms by which flatworms may affect coral growth. One mechanism relates to kleptoparasitism and feeding impairment, where flatworms steal planktonic prey from corals and reduce their capacity to capture and retain prey items, thus impacting their ability to feed heterotrophically (Wijgerde et al., 2013). In the current study, it was found that Waminoa was indeed able to efficiently capture Artemia nauplii when present on their soft coral host (Supplemental video 1). However, Cladiella fragments themselves were not observed to capture Artemia, in line with the view that soft corals are inefficient zooplanktivores (Fabricius et al., 1995a,b). Hence, for this particular species of soft coral, it is unlikely that Waminoa infestations affect its ability to capture zooplankton. It is possible that uninfected Cladiella may be able to retain Artemia more effectively, as flatworms have been found to remove the sticky mucus layer from coral polyps (Naumann et al., 2010), resulting in less effective prey capture (Wijgerde et al., 2013). However, prey capture by flatworm–free Cladiella was also not observed. Another study suggested that autotrophic feeding of corals is hampered by flatworm presence due to shading (Barneah et al., 2007), which reduces illumination of coral tissue and the symbiotic zooxanthellae, thus decreasing the corals’ photosynthetic ability. Since photosynthesis is thought to be the primary energy source for Cladiella, and given the fact that flatworms can cover corals to a considerable extent, such a shading effect could explain the observed growth retardation.

Close–ups of several Cladiella colonies without (first sequence) and with Waminoa flatworms (second and third sequence). Note the retracted polyps of infested colonies in the second and third sequence.  Also note that Waminoa efficiently capture Artemia nauplii, in contrast to the coral.

In the current study, it was observed that corals infected with Waminoa had more retracted polyps compared to the fully extended polyps of uninfected corals. This can be regarded as a sign of stress, as normally polyps would be continuously expanded to aid photosynthesis by efficiently exposing the symbiotic zooxanthellae to light (Levy et al., 2001). Polyp retraction may therefore further impair the photosynthetic capacity of the corals, beyond the shading effects mentioned above, and thus lead to reduced growth rates. Discolouration of the corals was also observed, suggesting a loss of zooxanthellae and/or photosynthetic pigments (Rogers, 1979). Loss of zooxanthellae may further reduce the photosynthetic capacity of the coral.

Waminoa did not only impair Cladiella growth, but also affected colony expansion, or hydrostatic skeleton maintenance. Infested Cladiella exhibited reduced colony expansion as compared to flatworm–free corals, which in addition to growth retardation, necrosis, polyp retraction and discolouration suggests that Waminoa impose physiological stress on Cladiella. Soft coral polyps normally maintain their hydrostatic skeleton through water intake, an energy–demanding process, and Waminoa clearly affect this ability. This negative effect did not seem to increase over time. Apparently, flatworms rapidly (within one week) reduce colony expansion, an effect which remains constant over a time period of at least several weeks. In addition to reduced polyp retraction, impaired colony expansion could result in denser packing and thus self–shading of zooxanthellae, with less photosynthetically active surface available for infected corals (Stambler and Dubinsky, 2004). This further supports the earlier proposition that the observed flatworm–induced growth retardation in Cladiella is primarily caused by impairment of photosynthesis. As removal of the flatworms using a freshwater dip increased colony expansion, this indicates that negative effects caused by Waminoa are (at least partially) reversible. Whether this also holds true for growth rates is yet unclear, but likely.

Another sign of impaired coral health caused by Waminoa was a marked increase in polyp recovery time for infested corals after physical disturbance. After a disturbance, polyps of flatworm–infested corals took almost twice as long to expand to their original state when compared to controls. This indicates that infested corals are less resilient to physical disturbances as they require more time to recover, possibly due to reduced energy reserves caused by impaired photosynthesis.

Finally, a secondary effect of Waminoa infestation on soft corals could be decreased competitiveness, as smaller and less healthy corals are more likely to be overgrown by algae and other competitors for space (Lirman, 2001). Hence, Waminoa blooms are likely to render coral reefs more susceptible to overgrowth, although it is currently unknown what factors control flatworm densities. In this era of rapid climate change and coral reef degradation, further research on the role flatworms play in coral reef ecosystem functioning is warranted.

It is noteworthy that during this study, the control corals showed a decreasing trend in terms of growth rate and colony expansion. This may have been due to inherent quality fluctuations of the seawater used. Nevertheless, this should have similarly affected the flatworm–infested corals, maintaining the validity of our study.

In conclusion, this study shows that Waminoa flatworms negatively affect the soft coral Cladiella, in terms of reducing growth and colony expansion, inducing necrosis and colony discolouration, and increasing polyp recovery time after disturbance. Thus, this study supports the emerging view that Waminoa are parasites which may significantly affect (soft) coral growth and health.

 

Acknowledgements

We would like to thank Ulf Jondelius of Zoologiska Institutionen for identifying the Acoel species, and Leen van Ofwegen of Naturalis for identifying the soft coral species.

 

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