Coral larvae on demand: a novel method for an instant acquisition of healthy brooded larvae

doi:10.21203/rs.3.rs-4616138/v1

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Coral larvae on demand: a novel method for an instant acquisition of healthy brooded larvae

https://doi.org/10.21203/rs.3.rs-4616138/v1

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Access to coral larvae is crucial for research on early life stages of corals, yet traditional methods for obtaining brooded larvae are labor-intensive and time-consuming. We present a novel method to efficiently collect viable brooded larvae within minutes from colonies of five different scleractinian coral species (Leptastrea purpurea, Leptastrea transversa, Tubastraea faulkneri, Pocillopora acuta, Favia fragum). By immersing the colonies in seawater containing 5–10 g/L potassium chloride for 5–7 minutes, we induced instant larval release. This method yielded a similar quantity of larvae compared to traditional methods. The larvae remained viable, surviving storage for several weeks and settling successfully. Our method is both faster and easier than traditional methods, suitable for application with aquarium-cultured corals or in field stations. The potassium chloride technique was effective for all tested coral species that tolerate elevated potassium ion concentrations without harm. However, it should not be applied to potassium-sensitive corals like Euphyllia.

Coral reproduction, larval release, planulation, brooder, spawning, settlement cues

Biological sciences/Physiology

Biological sciences/Zoology

Earth and environmental sciences/Ecology

Earth and environmental sciences/Environmental sciences

Earth and environmental sciences/Ocean sciences

Coral reefs are among the most threatened ecosystems worldwide (IPCC 2013, Intergovenmental Panal of Climate Change, 2013). Coral research is of immense relevance and must incorporate the entire life cycle of corals, i.e. both early and adult life stages. Critical research on early coral life stages includes the effects of stressors on larvae and juvenile corals, the settlement process, as well as post-settlement development. Acquisition of larvae needed to conduct this research is challenging and can be achieved with different approaches.

The most common reproductive strategy of scleractinian corals is broadcast spawning: during one or few nights per year, conspecific colonies simultaneously release their gametes into the water column. Approximately 80% of known species are broadcast spawners. Fertilization and embryo development commence in the water column and the emerging planulae actively search for suitable substrates to settle on and metamorphose into primary polyps. Hence, conducting larval research with broadcast spawning species comes with challenges: either be at the right location at the right time and have the necessary infrastructure available or induce spawning in an ex-situ facility. Ex-situ spawning induction is possible but comes with its own array of challenges and is thus not yet a common procedure (Craggs et al., 2017) The challenges accompanying research with broadcast spawners can be avoided by selecting species which follow the second strategy of sexual reproduction: brooding. These species usually release sperm into the water column which is taken up by conspecific colonies, followed by internal fertilization and embryogenesis into competent planulae, all within the mother polyp (Goodbody-Gringley et al., 2010; Richmond & Hunter, 1990; Szmant-Froelich, 1985). Alternatively, some species can produce clonal planulae parthenogenetically (Combosch & Vollmer, 2011; Stoddart, 1983; Yeoh & Dai, 2010). In either case, competent larvae are released into the water and are commonly ready to settle instantly (Figueiredo et al., 2013; Harii et al., 2002; Nozawa & Harrison, 2005). Compared to broadcast spawners, the number of released progenies is vastly smaller. However, due to the investment of internal development, larvae are immediately competent, often carry symbiotic microbes and even dinoflagellates and have a higher success rate in between release and settlement (Goodbody-Gringley & de Putron, 2016; Richmond, 1997). In many brooding species, reproduction happens at a higher frequency than in broadcast spawners, with some species even releasing larvae every day (Nietzer et al., 2018).

Many brooding species can be cultured in ex-situ facilities and thus allow continuous access to planulae. The challenge, however, is to obtain the larvae. Species releasing larvae on a daily basis, such as Leptastrea purpurea, allow a fairly reliable acquisition of larvae by temporarily isolating mother colonies in containers. In seasonally reproducing corals such as Pocillopora damicornis and P. acuta, the timeframe of larval release is sometimes challenging to catch. While it is known fairly precisely in some cases, e.g. in P. acuta from Guam (Richmond & Hunter, 1990) and P. damicornis from other locations (Kuanui et al., 2008; Tanner, 1996) these time frames are tied to abiotic parameters such as lunar cycle, thermal seasonality and photoperiod which would have to be simulated in an ex-situ system to maintain reproductive cycles. Exposing the corals to non-seasonal conditions will affect their reproductive periods. Pocillopora, as an example, tend to release larvae more often in lower numbers than at the concentrated high-number releases they perform in the natural environment (Jokiel et al., 1985).

For species like Pocillopora, Tubastraea or Favia, larval collection is commonly conducted by installing mesh boxes in the backflow of the broodstock tanks in order to catch the larvae being washed out of the tanks or collection containers, respectively (Jokiel et al., 1985). Alternatively, parent colonies are isolated in containers without flow-through for a certain amount of time (Kuanui et al., 2008; Stoddart, 1983). While these procedures are working well, they still come with quite some effort in both equipment and time. Larval collection in the field, e.g. on a boat, is not easily possible with these methods.

In this study, we explore a novel method that allows an instant acquisition of healthy larvae without harming the mother colonies.

For this study, we used both corals in long-term culture as well as freshly collected colonies from the field.

Coral culture in aquarium facility

The coral species used in this study - Leptastrea purpurea, Leptastrea cf. transversa, Pocillopora acuta, Tubastraea faulkneri, and Favia fragum - have been cultured in the aquarium facilities at University of Oldenburg, ICBM, in Wilhelmshaven. Leptastrea and Pocillopora colonies were originally imported from Guam, USA, Tubastraea colonies were purchased through the aquarium trade. Favia colonies were gifted by Dr. Ronald Osinga from Wageningen University. Corals were kept in a recirculating system with artificial sweater (Tropic Marin® Pro Reef). Details of the coral culture can be found online (https://uol.de/icbm/umweltbiochemie/aquarium). The corals were exposed to a 12/12 circadian rhythm and a temperature of 26°C. Illumination was provided by LED lights (EcoTech Marine Radion G4 Pro and G6 Pro), as well as T5 fluorescent bulbs (Aqua Medic Reef White and Reef blue) at an intensity of 100–150 PAR.

Larval collection with cultured corals

The initial procedure consists of preparing the potassium chloride (KCl, Carl Roth, > 99%) bath. Depending on the number of colonies that are used simultaneously, a suitable container needs to be selected. Our potassium baths were conducted in translucent plastic boxes of 20 L. The container was filled with seawater and subsequently, KCl was added to reach the desired potassium concentration. Pre-experiments were conducted in order to identify suitable ranges of both concentration and time for the potassium exposure for the respective species. Finally, KCl was used in the following concentrations: Leptastrea and Favia 5 g l^− 1 and Pocillopora and Tubastraea 7.5 g l^− 1, equaling a final K⁺ concentration of 3000 and 4300 mg l^− 1, respectively. Colonies were then placed in the container for 5 minutes except Pocillopora which was placed in the bath for 7 minutes. While colonies were in the bath, single-use Pasteur pipettes were used to agitate the colonies’ surfaces. For species such as Tubastraea faulkneri which release large and brightly colored larvae, the instant collection and transfer to untreated seawater can be carried out under white light. For species releasing larvae that contain fluorescent proteins such as GFP, the collection should be conducted in the dark; blue fluorescent light and a yellow barrier filter (for specifications see also Fiegel et al., 2023) enable the detection of released larvae. The larvae can be collected immediately after their release. After the respective exposure times, colonies can be placed into containers with untreated seawater for about 15 minutes before returning to their home tanks. In some species such as Tubastraea faulkneri, there might be some continued larval release.

When working with rather small species such as Leptastrea, we recommend keeping broodstock colonies on trays that can easily be handled and placed into treatment containers without touching or repositioning the colonies (as described in Nietzer et al., 2018).

Upon collection, the larvae should be transferred at least once to clean, untreated seawater. Subsequently, they can be stored in sterile seawater.

The number of collections varied between species and is noted in Table 1.

Larval collection with freshly collected colonies

In August 2023, 100 colonies of Leptastrea spp. (L. purpurea and L. cf. transversa) were collected from Luminao reef on Guam (USA) at a depth of about 1 m (collection permit SC- 23-004A, issued by DAWR). Upon careful detachment from their substrate, colonies were transported to the University of Guam Marine Laboratory (UOGML) in cooler boxes filled with seawater. At UOGML, the colonies were placed on egg crate trays in flow-through tanks with natural light and equipped with circulation pumps (SunSun JVP-202A, SunSun JVP-102A) ensuring sufficient water circulation. Translucent boxes of 30 l volume were filled with a solution of 5 g/L KCl in seawater. Once the KCl had fully dissolved, the trays with the coral colonies were placed inside. Colonies were gently agitated with single use pasteur pipettes throughout the collection. After about 5 minutes, colonies were being gently shaken and returned to their holding tanks. The treated water containing larvae was then poured through a 30 µm mesh in order to concentrate larvae in a small volume. After being transferred to a 2 L glass bowl with natural seawater, larvae were collected by detecting them with handheld NightSea Vision emitting blue fluorescent light (460 nm). The filtered KCl solution was then used for the next tray with broodstock colonies.

Storage of larvae

The larvae for the competency affirming assays were stored in 0.2 µm filter-sterilized seawater (Chromafil® Xtra syringe filters, PES-20/25, Macherey-Nagel, 52355 Düren, Germany) in four 250 ml Schott bottles (6 larvae each) for 3 weeks and transferred to new bottles once a week. The bottles were kept in incubators at 26°C and 12/12h circadian rhythm at 80 µmol irradiance (ICBM WHV).

Competency-affirming assays

In order to affirm that the settlement capabilities of the swimming larvae were not impaired by the short-term exposure to elevated K + concentrations, larvae were settled. The novel settlement-inducing bacteria-derived compound cycloprodigiosin (CYPRO) was used to induce settlement in Leptastrea purpurea larvae (following the same protocol as described in our former publications (Fiegel et al., 2023; Petersen et al., 2021). In brief, CYPRO was dissolved in Methanol (MeOH) as a stock solution at a concentration 0.1 mg/ml. 10 µl of this stock solution were pipetted into the each well of a sterile 6 well plate. Upon evaporation of MeOH, well plates were filled up with 10 ml FSW before larvae were added. Directly after larval collection 3 wells containing each 3 larvae were setup. The rest of the larvae was stored for 1 week (4 wells with each 2 larvae), 2 weeks (4 wells with each 2 larvae) and 3 weeks (4 wells with each 2 larvae) and subsequently set up for settlement in the previous described manner. After 24 and 48 h, the larvae were checked for mortality, full settlement as well as metamorphosis without attachment. Larvae of the other species were also settled with a similar method soon to be published by Fiegel et al. (in preparation).

Control treatment

In order to assess whether simply the osmotic pressure changes of the seawater are inducing larval release, corals were treated with seawater of the respective salinity. A KCl bath with 5 g/l increases salinity from 34.5 ppt seawater to 39.5 ppt. Synthetic salt (Tropic Marin® Pro Reef) was used to reach this salinity. Colonies of Leptastrea purpurea (50 colonies), Tubastraea (5 colonies), Favia (5 colonies) and Pocillopora (5 colonies) were then exposed to this salinity the same way as in the potassium bath. Additionally, colonies were exposed to a reduced salinity of 50% (17.5 ppt) and 10% (ca. 3.5 ppt) for the same duration.

Statistic

Storage and competency experiment:

Data were entered using Microsoft Excel and statistical analysis was performed using R (R Development Core Team, 2023). The tidyverse package (Wickham et al., 2019) was used to manipulate and plot the data.

To evaluate differences in settlement behavior among age groups a Kruskal Wallis rank sum test was applied. If significant, a followed Dunn’s test (Dinno, 2017) with a Holm correction of p-values was conducted.

KCL treatment

Leptastrea purpurea

released larvae reliable with the KCl treatment at the ICBM aquarium facility as well as on Guam. We collected 27 times at the ICBM aquarium with varying amounts of corals and yielded a total of 811 larvae with a mean of 0,7 ± 0.1 SE larvae per colony. We collected 3 times on Guam with much more colonies resulting in 1260 larvae total and a mean of 6,2 ± 2 SE larvae per colony.

Leptastrea transversa

The collection method was used once with L. transversa on Guam with 24 Colonies. They released 85 larvae in total with a mean of 3.5 larvae per colony.

Tubastraea faulkneri

larvae were collected 37 times at the ICBM aquarium, yielding a total of 880 larvae with a mean of 4,7 ± 1,6 SE larvae per colony.

Pocillopora acuta

colonies from the ICBM aquarium were bathed 9 times, collecting a total of 97 larvae with 1.6 ± 0.3 SE larvae per colony. Along with the planulae, microscopic organelles, likely acrospheres, were often released in large numbers.

Favia fragum

where exposed to the KCl treatment 25 times releasing a total of 205 larvae with a mean of 0.5 ± 0.1 SE larvae per colony.

Collection data is presented in Table 1. Figure 1 shows photos of adult colonies, larvae, and settled recruits, while Fig. 2 illustrates the larval output per colony.

Control treatment:

While there were 2 larvae collected in the 50% salinity exposure with Leptastrea purpurea, the other control treatments did not yield any larvae. The same 50 Leptastrea purpurea colonies released more than 200 larvae when exposed to the potassium treatment.

Table 1

summary of larval release methods and results
species	location	exposure time	KCL	No. collections	total larvae	mean per colony	SD	SE
Favia fragum	Aquarium	5 min	5 g/L	25	205	0,5	1	0
Leptastrea purpurea	Aquarium	5 min	5 g/L	27	811	0,7	1	0
Leptastrea purpurea	Guam	5 min	5 g/L	3	1260	6,2	3	2
Leptastrea transversa	Guam	5 min	5 g/L	1	85	3,5
Pocillopora acuta	Aquarium	7 min	7.5 g/L	9	97	1,6	1	0
Tubastraea faulkneri	Aquarium	5 min	7.5 g/L	37	880	4,7	10	2

Larval survival: Leptastrea purpurea

All larvae survived the long-term storage over 3 weeks. None settled or died.

Larval settlement: Leptastrea purpurea

The stored larvae were competent to settle over the total storage duration of 3 weeks. With a settlement response over 60% for all time points (direct after collection: 77.8% ± 22.2 SE; week 1: 75% ± 14.4 SE; week 2: 62.5% ± 23.9 SE; week 3: 75% ± 14 SE). There was no statistically significant difference in the settlement behavior among the age groups (Kruskal Wallis rank sum test, χ² (df = 3) = 0.189; p = 0.98).

Additional effects: Pocillopora acuta

In Pocillopora acuta, the immersion in the potassium bath led to an immediately release of large quantities of very small (ca. 100 µm) organelles containing green fluorescent protein (Fig. 3) in addition to the above described larval release. The release usually happened within seconds after exposure. The organelles contained zooxanthellae. A colony of around 20 cm in diameter could release several thousand of these organelles. This phenomenon was common in P. acuta but was not observed in every collection. While they seemed to be alive for some time, decomposition would start after one to two days at 26°C.

The procedure described in this study is the first of its kind to allow an instant acquisition of viable planula larvae from brooding corals. The method can be applied without any elaborate preparation and does not require intricate infrastructure. An application in field stations both with or without flow-through systems, as well as in ex-situ aquaria or on a boat is possible. Even an in-situ application could be conducted by injecting a higher concentrated solution into a bag placed over a colony on the reef. Introducing KCl into the reef environment is harmless since both potassium and chlorine occur in high concentrations in the seawater.

The mechanism enabling the instant expulsion of larvae has yet to be investigated. Potassium ions are involved in many cellular pathways, such as cell potential regulation. A sudden stimulation of excitable cells in the corals by increased potassium concentrations seems to initiate a cascade leading to the expulsion of developed larvae. It is generally known that potassium concentration changes play important roles in many invertebrate cellular mechanisms. Behavioral changes by increasing potassium Ions have been shown for larvae of several invertebrates where it induces settlement (Pearce & Scheibling, 1994). A widely used method to obtain gametes from sea urchins is to inject KCl into the coelom of mature adults (Strathmann, 1987).

However, very little is known about cellular mechanisms regarding the development and propagation of planulae in brooding scleractinians. Our study proposes that a sudden increase in potassium concentration of the surrounding medium to about 7–11 x of the natural concentration (around 400 mg/L) is triggering the instant release of fully developed larvae in brooding species across the scleractinian taxonomic tree. With species of the genera Pocillopora, Leptastrea, Favia and Tubastraea, our study covers a wide range of the scleractinian clades. In order to rule out that the reaction is related to changes in salinity and thus creating an increase in osmotic potential, we tested both increased and decreased salinity with no larval release effect. Our assumption is that the mechanism is closely tied to the potassium concentration.

Settlement competency of the larvae was not affected by potassium exposure, as demonstrated in our assays with L. purpurea. We further excluded any delayed detrimental effects on the planulae by storing them for four weeks and testing settlement weekly. Although only the CYPRO-induced settlement with L. purpurea was statistically evaluated for this publication, similar settlement assays were conducted with other species (Fiegel et al., in preparation). Additionally, all species were settled on crustose coralline algae (CCA)-covered tiles (see Fig. 1).

In Leptastrea purpurea, our novel collection method yields at least the same number of larvae as the classical collection approach as described in Nietzer et al., 2018. We found the method just as effective with L. purpurea colonies in our aquarium facility as the overnight collection method and it simultaneously helped to preemptively control parasite populations such as ciliates and amphipods. The effort of both material and time required to obtain the planulae, however, is reduced to a minimum and allows spontaneous and instant collections. Our working group has entirely switched to the KCl method for larval collection.

We tried the method with both laboratory grade KCl (Carl Roth, > 99%) as well as the much less costly food grade KCL (KaliSel with 0.5% aerosile) and both works well. Food grade KCl usually contains aerosile (silicate) as an anti-caking agent. This kind of KCl is safe to use as the silicate nano particles are inert and do not affect the corals.

Additional effects

In Pocillopora acuta, we regularly observed the release of very small, round organelles of about 100 to 150 µm in size (cf. Figure X). In comparison, planulae of P. acuta are about 1.5- 3 mm in length (cf. Figure 1B2). The organelles were released immediately under potassium exposure. They don’t seem to be underdeveloped larvae, particularly due to their size and quick release, often within seconds. These structures may originate from the corals’ exterior tissue. Their precise origin and function are yet to be investigated. Our current assumption is that these structures are tentacle tips, the acrospheres. Despite the assumed loss of acrospheres, colonies always displayed full polyp extension shortly after being exposed to potassium treatments and appeared to be very healthy.

Treatments with potassium have long been applied for corals to remove parasitic organisms, such as ciliates, flatworms or crustaceans in the aquarium hobby. There are commercially available products based on potassium chloride (such as Reef Primer by Polyp Lab®). The effectiveness of increased potassium concentrations in order to remove parasites while not harming corals in combination with an instantaneous release of viable larvae make the KCl treatment an ideal tool for the long-term culture of brooding species in ex-situ systems as well as a fast method to obtain planulae in general. However, there are some species which react adversely to an increase in potassium concentration: albeit not a brooding species, Galaxea spp. react to an immediate increase in potassium with the loss of intercorallite tissue. While the colonies can recover from the damage without problems, it temporarily causes severe damage. The same goes for Euphyllia spp.. An exposure to high K + concentrations usually leads to the immediate loss of intercorallite tissue. We have only found adverse effects for members of the Euphyllidae family however, we want to stress a careful approach using this technique with new species.

Due to its easy application, low cost and time-saving design, the here described method could be a useful tool to drastically increase efficiency in coral research. It is an easily applicable tool to determine whether brooding colonies are carrying planulae and could be used in field studies to assess when species are reproducing. Making coral research more efficient is a key achievement in the ongoing endeavor to preserve coral reef ecosystems.

Contributions

SN discovered the method and designed assays. SN and MM wrote the manuscript. LJF conducted most of the collection assays, and prepared statistics and figures. MM took part in the collection assays. PJS supplied lab space, resources, and took part in the field campaign on Guam. All authors worked on the manuscript.

Author Contribution

S.N. discovered the method and designed assays. S.N. and M.M. wrote the manuscript. L.F. conducted most of the collection assays, and prepared statistics and figures. M.M. took part in the collection assays. P.S. supplied lab space, resources, and took part in the field campaign on Guam. All authors worked on the manuscript.

Acknowledgement

We would like to thank staff and students of both UOL and UOG for their support and assistance. We also want to tank Dr. Ronald Osinga and his group at Wageningen University for our collaboration.

Data Availability

Data is provided within the manuscript

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No competing interests reported.

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Coral larvae on demand: a novel method for an instant acquisition of healthy brooded larvae

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Abstract

Figures

Introduction

Methods

Coral culture in aquarium facility

Larval collection with cultured corals

Larval collection with freshly collected colonies

Storage of larvae

Competency-affirming assays

Control treatment

Statistic

Results

KCL treatment

Control treatment:

Discussion

Additional effects

Declarations

Contributions

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1