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Observation Article
Revised

Fused embryos and pre-metamorphic conjoined larvae in a broadcast spawning reef coral

[version 2; peer review: 2 approved]
PUBLISHED 05 Mar 2015
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Abstract

Fusion of embryos or larvae prior to metamorphosis is rarely known to date in colonial marine organisms. Here, we document for the first time that the embryos of the broadcast spawning coral Platygyra daedalea could fuse during blastulation and further develop into conjoined larvae, and the settlement of conjoined larvae immediately resulted in inborn juvenile colonies. Fusion of embryos might be an adaptive strategy to form pre-metamorphic chimeric larvae and larger recruits, thereby promoting early survival. However, future studies are needed to explore whether and to what extent fusion of coral embryos occurs in the field, and fully evaluate its implications.

Keywords

fusion, conjoined larvae, spawn slicks, inborn colonies, Platygyra daedalea

Revised Amendments from Version 1

According to the comments from the reviewers, we mainly made the following changes:
1) We improved the text in many instances as suggested by Prof. Rinkevich. For instance, in Materials and Methods, we used “combined” instead of “mixed”; we replaced “each type of fusion” with “chimeric larvae”. Moreover, to demonstrate the sectorial fusion, we described these conjoined larvae as multi-headed in the Discussion. 
2) We have corrected the wrong idea of the sexually produced embryos not being genetically distinct and rewritten this part in the Discussion.
3) Prof. Rinkevich suggested that the results of Mizrahi et al., 2014 would change our statement of “for the first time”. Our observation presented the embryonic chimeras as a result of fusion of embryos in a broadcast spawning coral, whereas Mizrahi et al., 2014 revealed that the larvae of brooding coral Tubastraea coccinea could metamorphose and aggregate in swimming groups. Thus, we did document for the first time the fusion between individuals at the embryonic stage in reef corals and the inborn colonies of multiple polyps upon settlement. Moreover, we followed the settlement and growth of these chimeric larvae. Overall, it is appropriate to state that we documented the embryonic chimeras and inborn colonies “for the first time” in reef corals. 
4) We point out the absence of water turbulence in this study as suggested by Prof. Baird and state that it may be a factor triggering the fusion of embryos. We added the references of Mizrahi et al., 2014 and Gauthier et al., 2008 to further discuss the potential implications of this phenomenon. 
We are really grateful for the comments from the two reviewers and the efforts of the editorial office.

See the authors' detailed response to the review by Baruch Rinkevich
See the authors' detailed response to the review by Andrew H. Baird

Introduction

In sessile colonial marine invertebrates (e.g., sponges, cnidarians, bryozoans and ascidians), fusion among conspecifics during early ontogeny could immediately lead to a marked increase in juvenile size, thereby enhancing the performance in growth, survival and competition1,2. In addition, the allogenic fusion is expected to form chimeras which possess greater genetic variability and wider ranges of physiological resistance1. Larvae of colonial marine organisms tend to settle in a gregarious manner37 and their juveniles often come into physical contact through growth and then fuse810. These life history traits increase the opportunities for fusion, and important rates of chimerism due to allogenic fusion have been detected in wild natural populations of corals and ascidians11,12. Nevertheless, fusion of embryos or larvae during planktonic and dispersive phase (i.e. prior to settlement and metamorphosis) is rarely known to date.

Modular marine invertebrates like sponges and cnidarians usually spawn their gametes in a high synchrony1315, thus also providing the chance of contact and fusion among embryos or larvae. For instance, sticky eggs released by the oviparous sponge Cliona celata were found to adhere to each other and form flattened egg mass, within which larvae fused in twos or threes. The compound larvae metamorphosed into sponges with single oscula, indicating the cytomictical fusion among embryos or larvae13. More recently, larvae of two sponges and sun coral Tubastraea coccinea have been demonstrated to fuse and generate swimming chimeras1618. Furthermore, sexually produced embryos of a non-colonial sea anemone Urticina feline were observed to fuse naturally during internal brooding, generating pre-metamorphic cytomictical and sectorial chimeras19,20. These findings suggested that the direct contact between embryos and larvae would facilitate fusion either during internal brooding or pelagic phase.

For broadcast spawning corals, synchronous spawning events usually result in billions of naked embryos floating at the sea surface in the form of spawn slicks21,22. The direct contact between naked embryos highlights the possibility of fusion of coral embryos while sticking together in slicks. Moreover, previous studies have demonstrated there is a window in ontogeny, before allorecognition system matures, when newly settled polyps can fuse23. Time for allorecognition maturation in reef corals varied from 4 months following settlement in brooding species24, to 1–3 years in spawning species9,10. This further supports the possibility of fusion at embryonic stage when allorecognition may be weak in corals. As yet, the possible occurrence of fused embryos and conjoined larvae in broadcast spawning corals has not been investigated.

Here, we happened to test this unexplored probability of fusion of embryos in broadcast spawning reef corals. We experimentally mimicked spawn slicks using gametes collected from 4 mature colonies of Platygyra daedalea, and followed the fate and development of embryos within lab-generated slicks.

Materials and methods

Ten gravid colonies of P. daedalea (20–30 cm in diameter) were collected at depth between 2–4 m from Luhuitou fringing reef in Sanya, China (18°12′N, 109°28′E). Corals were maintained in an outdoor tank with flowing sand-filtered seawater in Tropical Marine Biological Research Station in Hainan, Sanya. Four colonies spawned around 22:00 on May 18, 2014 (5 nights after full moon). Egg-sperm bundles were collected using pipettes, then combined and gently agitated to facilitate bundle disintegration and cross-fertilization. Fertilization was allowed to take place for about 2 hours, after which eggs (ca. 300, 000) were washed two times with fresh seawater and suspended in a 15 cm-diameter jar. Because of the logistical constraints, eggs were left undisturbed and they formed dense slicks on the seawater surface. The next morning around 08:30, embryos were inspected under a dissecting microscope and we accidentally discovered that some embryos fused. Embryos were washed and seawater was changed twice daily thereafter. Two days after fertilization, 500 larvae were randomly sampled to count the proportion of chimeric larvae. Seven days after fertilization, chips of crustose coralline algae Hydrolithon onkodes were used to induce the settlement of larvae and the recruits were reared in the lab at 28°C until June 26.

Results

Embryos became bowl shaped (cushion stage) 8 h after fertilization. Notably, some embryos fused (Figure 1A) and a substantial proportion even stuck together into dense aggregates (Figure 1B). It could be deduced that fusion of embryos took place some time during blastulation. Mortality of embryos within the first 2 days was extremely high (>50%) and the dense aggregates all died and decomposed. Unitary larvae became pear-shaped and began to rotate actively 20 h after fertilization, while conjoined larvae were highly variable in shape. Bi-fused larvae were dominantly peanut-shaped, and multi-fused larvae were arranged in chains or triangles, or in the form of the letter “T” or “L” (Figure 1C, D).

1a5df804-5a8a-4abd-965e-87fe972add78_figure1.gif

Figure 1. Platygyra daedalea.

(A) Fused embryos (arrows point to the fusing areas). (B) A dense aggregate comprising more than 20 embryos. (C–G) Unitary (asterisks) and conjoined larvae. (H, I) Inborn colonies. (J) Single settlers. (K) Incomplete settlement of perpendicularly bi-fused larvae, with the left partner being parallel to the substrate. (L) An inborn colony 26 days post-settlement. Roman numbers indicate visible individuals within inborn colonies. Scale bars 250 μm.

Of the 500 randomly sampled larvae, 174 (34.8%) were conjoined with 2–4 partners. Conjoined larvae clearly showed their spatial arrangement after elongation and fusion was apparently without polarity. Larvae either joined at the aboral end (Figure 1E, F), or united side by side (Figure 1G), or even fused perpendicularly (Figure 1K). Furthermore, 56 out of the 174 conjoined larvae (32.2%) united at the aboral extremity and only these larvae were potentially competent to metamorphose normally into inborn colonies (Figure 1H, I), which were prominently larger in size than the single settlers (Figure 1J). In contrast, perpendicularly bi-fused larvae settled incompletely, with one partner metamorphosing and firmly attaching while the other still being parallel to the substrate and not able to settle (Figure 1K), ultimately leading to the death of the whole entity 3 days later. Since the coralline algae provided here was not suitable for the settlement of P. daedalea larvae, only 12 inborn colonies were obtained in total and they persisted for 26 days post-settlement when the study ended (Figure 1L).

Discussion

The present study documented for the first time the fusion of embryos and inborn colonies in a broadcast spawning coral. Fusion of P. daedalea embryos was spontaneous, resulting simply from the aggregation and contact of embryos in mimicked slicks, which was analogous with that in sponge C. celata13. While unlike the cytomictical compound larvae in sponge C. celata, the chimeric P. daedalea larvae were multi-headed, suggesting sectorial fusion of coral embryos and supporting the assumption that corals typically exhibit sectorial fusion1.

Corals often spawn during seasonally calm periods and low-amplitude tides21,22,25 and spawn slicks extending up to few km in length were often observed in the field21,26. Given that slicks remained aggregated 1–2 d after spawning21,22 and embryos can fuse during embryogenesis within 8 h post-fertilization, fusion of coral embryos is highly favored in situ. On the other hand, although mass coral spawning events usually involved several species, significant temporal differences in spawning to ensure fertilization and reproductive isolation have been demonstrated for many sympatric species2729, which considerably increase the encounters between embryos of the same species in slicks. Taken together, fusion of coral embryos might be a naturally occurring phenomenon. However, the density of embryos here was 1700 cm-2 and likely to be much higher than that in the field. Moreover, water turbulence that the embryos would experience was absent in this study. Therefore, it is possible that our experimental conditions eventually led to the formation of embryonic chimeras. Likewise, larvae brooded by T. coccinea, when kept at high density in still water, could metamorphose and aggregate in clusters with extended lifespan18. Thus, whether fusion of coral embryos occurs in natural spawn slicks and the dispersal potential of these chimeric larvae remain to be determined.

At last, an important observation was that the chimeric larvae were able to settle firmly and form inborn colonies. The inborn colonies here originated from fusion of embryos and settlement of chimeric larvae, contrasting the traditional concept that the asexual budding of the primary polyp leads to the formation of a young coral colony30, and thus fusion of embryos could be an unexpected shortcut to colony formation in reef corals. Furthermore, the inborn colonies persisted for about one month and exhibited no sign of rejection, suggesting the possibility that the embryonic chimeras might contribute to recruitment in the natural environment17. These facts raise questions as to the ecological implications of inborn colonies formed as a consequence of fusion of embryos in corals. Firstly, larger coral colonies composed of multiple fused partners are known to yield remarkable gained benefits, such as enhanced survival and growth5,8. Hence, the larger initial size and the status of multi-polyp at settlement may confer these inborn colonies better capacities to compete for space and survive partial mortality.

Fusion of coral embryos also shed new light on the chimerism in scleractinian corals, which was often attributed to fusion of gregariously settling larvae5,7, or of juveniles that come into contact through growth7,9,10. However, our study documented fusion between individuals in P. daedalea occurred at the embryonic stage, earlier than any other corals studied to date. Since the embryos here were produced sexually from 4 parent colonies and they were genetically distinct, fusion of embryos could be a novel mechanism for chimerism in scleractinian corals. In that case, the increased genetic diversity within these inborn colonies may translate into versatile physiological qualities, thus enabling them to better cope with environmental changes unless negative interactions occur1,31,32.

Overall, this is the first report of embryonic chimeras in reef corals. Fusion of coral embryos could be an adaptive strategy to form larger and chimeric recruits, thereby promoting growth and survival during the vulnerable early stages5,8. Clearly, future studies are required to explore whether fusion of embryos occurs in the field and fully evaluate its biological and ecological implications.

Ethics statement

Coral sampling was permitted by the Administration of Sanya Coral Reef National Nature Reserve, the Department of Ocean and Fisheries of Hainan Province.

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Jiang L, Lei XM, Liu S and Huang H. Fused embryos and pre-metamorphic conjoined larvae in a broadcast spawning reef coral [version 2; peer review: 2 approved]. F1000Research 2015, 4:44 (https://doi.org/10.12688/f1000research.6136.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
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PUBLISHED 05 Mar 2015
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Reviewer Report 02 Apr 2015
Baruch Rinkevich, Israel Oceanography & Limnological Research, National Institute of Oceanography, Haifa, Israel 
Approved
VIEWS 17
I confirm that I have read this submission and believe that I have an ... Continue reading
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Rinkevich B. Reviewer Report For: Fused embryos and pre-metamorphic conjoined larvae in a broadcast spawning reef coral [version 2; peer review: 2 approved]. F1000Research 2015, 4:44 (https://doi.org/10.5256/f1000research.6649.r8208)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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PUBLISHED 13 Feb 2015
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Reviewer Report 17 Feb 2015
Baruch Rinkevich, Israel Oceanography & Limnological Research, National Institute of Oceanography, Haifa, Israel 
Approved with Reservations
VIEWS 34
This short research article by Jiang et al. is timely, adds to our understanding on the magnitude of natural chimerism, and meets research standards with methodological details that made available to allow others to replicate this study. I recommend indexation ... Continue reading
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Rinkevich B. Reviewer Report For: Fused embryos and pre-metamorphic conjoined larvae in a broadcast spawning reef coral [version 2; peer review: 2 approved]. F1000Research 2015, 4:44 (https://doi.org/10.5256/f1000research.6575.r7679)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 05 Mar 2015
    Lei Jiang, Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China
    05 Mar 2015
    Author Response
    • We revised the sentence in abstract as suggested. Our observation presented the embryonic chimeras as a result of fusion of embryos in a broadcast spawning coral, whereas the results of
    ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 05 Mar 2015
    Lei Jiang, Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China
    05 Mar 2015
    Author Response
    • We revised the sentence in abstract as suggested. Our observation presented the embryonic chimeras as a result of fusion of embryos in a broadcast spawning coral, whereas the results of
    ... Continue reading
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31
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Reviewer Report 16 Feb 2015
Andrew H. Baird, School of Marine Biology and Aquaculture, James Cook University, Townsville, QLD, Australia 
Approved
VIEWS 31
This is well written article and an interesting observation although some of the literature cited is a little dated (e.g. Harrison & Wallace 1990). To my knowledge this is first time that anyone has followed these embryonic chimeras through to ... Continue reading
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HOW TO CITE THIS REPORT
Baird AH. Reviewer Report For: Fused embryos and pre-metamorphic conjoined larvae in a broadcast spawning reef coral [version 2; peer review: 2 approved]. F1000Research 2015, 4:44 (https://doi.org/10.5256/f1000research.6575.r7666)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 05 Mar 2015
    Lei Jiang, Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China
    05 Mar 2015
    Author Response
    Spawn slicks are commonly observed in the field and more importantly, they remained aggregated 1-2 days after spawning (Oliver et al., 1987; Gilmour et al., 2009). It is true that ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 05 Mar 2015
    Lei Jiang, Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China
    05 Mar 2015
    Author Response
    Spawn slicks are commonly observed in the field and more importantly, they remained aggregated 1-2 days after spawning (Oliver et al., 1987; Gilmour et al., 2009). It is true that ... Continue reading

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VERSION 2 PUBLISHED 13 Feb 2015
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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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