Coral reef restoration approaches have often utilized adult colonies as sources for fragments (i.e. clones) to be transplanted. Although restoration through this method is fast and cheap, it has been pointed out that it may reduce genetic diversity of the restored population. Low genetic diversity is a concern for reef restoration when seed fragments are raised asexually from only a few donor colonies. This can lead to lower fertilization rates among seed fragments, and reducing the longterm benefits of reef restoration in particular areas. Additionally, low genetic diversity can compound the effects of increased ocean temperature and other environmental stressors, further jeopardizing the health of a reef. An alternative approach through sexually propagated coral cultures and out-plantings can alleviate this problem. Sexually produced offsprings are more genetically diverse. They can be produced in far greater numbers than coral fragments and do not imply destructive methods. Ongoing research at the Akajima Marine Science Laboratory in Okinawa, Japan has helped to improve the production and maintenance of sexually propagated larval cultures. Our results show that crosses between gametes from 6 or more colonies will provide the highest fertilization rate (>95%). Based on the results, we suggest the use of 6 or more donor colonies for practical gamete fertilization in sexually derived coral culture.
Currently, the most common approach to coral reef restoration is through the transplantation of fragments (i.e. clones). However, restoration practices that use a limited number of donor colonies can reduce gene flow [
An alternative approach through the farming of coral offspring from larval cultures can alleviate these problems. The method involves the collection and cross-fertilization of gametes from gravid coral colonies during the spawning period, and the subsequent maintenance of the larval culture. Despite the potential cost, in addition to time that is required for this approach, it is a key solution that it could produce far greater numbers of offsprings than coral fragments and do not imply destructive methods which can impair donor coral population. Sexually produced offsprings are more genetically diverse, and provide long-term benefits than asexually produced coral fragments. As a result, higher fertilization rates can be expected from out-planted populations produced from sexually propagated offsprings than those from asexually raised fragments.
During past production of recruits using sexual propagation of acroporid corals, low fertilization rates were occasionally experienced when only a few donor colonies were used for the crosses. Although the reason for this is unknown, it was suspected that this occurred due to: 1) higher genetic similarity between donor colonies, 2) presence of morphologically indistinguishable cryptic species, or 3) the release of unhealthy gametes. Therefore, based on our experience, mixing the gametes from more than 3 donor colonies [
In the present study, fragments of gravid donor colonies from two locations in Kerama Islands, Okinawa, were collected and fertilization rates of crosses were compared by changing the number of colonies from l to 6 in order to corroborate our previous (above stated) recommendation experimentally.
In 2011, coral fragments (ca. 16 - 22 cm in length) from 13 gravid, donor colonies of Acropora tenuis were collected from the northern side of Yakabi Island (26˚13.070'N, 127˚14.274'E) on June 5 and the northeastern side of Kuba Island (26˚10.711'N, 127˚14.582'E) on June 11 (
The gametes were then mixed in a pair-wise combination within glass vials, containing 50 mL seawater each. Sperm adjusted to a concentration of 105/mL along with 300 - 470 eggs were added to the vial for fertilization. The fertilization success can be influenced by competition between spermatozoids of distinct parents. Therefore, the concentration-adjusted sperm from 2 or more donor colonies was pooled together prior to mixing with eggs.
All combinations of crosses were conducted once within 3 hours after spawning. The gametes were allowed to fertilize for approximately 20 minutes in a room kept at approximately 25˚C, and percent fertilization was obtained by examining at least 100 eggs per cross under the dissecting microscope. Counts were obtained within
Map of Kerama Islands. Sampling locations of wild donor colonies (Kuba Island and Yakabi Island: red spots) and cultured colonies (Majanohama in Aka Island: blue spot)
5.5 hours of fertilization. In addition to the fertilization rate, the developmental stages of the embryos were scored. The presence of abnormally developing embryos, if any, was also recorded.
Cross-fertilization between 2 colonies were arranged as follows: A × b, A × c, A × d, B × c, B × d, B × e, C × d, C × e, C × f (
Because of shortage of time, the experiment in 2012 was carried out using 6 colonies of A. tenuis from only Kuba Island (the same sampling location as 2011). The fragments were collected on June 1, and spawning occurred on June 11 (19:35 hr). The eggs (G, H, I, J, K, L) and sperm (g, h, i, j, k, l) from the 6 colonies were maintained in separate vials, and then mixed in a pair-wise combination following the procedure from the previous experiment. In addition to the six self-fertilization crosses, fertilization of eighteen 2-colony cross combinations, two 3-colony crosses (GHI × ghi and JKL × jkl), and three 6-colony crosses (GHI × jkl, JKL × ghi and GHIJKL × ghijkl) were conducted (
Results of the cross-fertilization using 6 donor colonies in 2011 are shown in
Comparison of fertilization ratio (%) between different combinations of 2-, 3-, 6-colony crosses. (a) Experiment in 2011; (b) In 2012. Number in each frame provides rates for 2-colony crosses. The rates for 3- and 6-colony crosses are given in the margin
mixed, which yielded high fertilization rate.
Instead of mixing gametes from donor colonies from 2 distinct sites in 2011, gametes from only Kuba Island were mixed in 2012. However, the fertilization rates between gametes from 2-, 3-, and 6-colony crosses were higher than those from the initial experiment (
In 2011 most of the embryos developed to or beyond the 128-cell stage between 5.5 and 7.0 hours after fertilization (
Genotypes of donor colonies used for the cross-fertilization were not determined in the present study. Use of colonies of unknown genotypes may reduce the potential for true crosses between distinct genotypes and may result in lower fertilization success. This may be the explanation for higher success in the 2012 experiment vs. the 2011 experiment. However, measurement of genetic similarity before fertilization is impractical for nursery farming of sexually propagated corals. The present cross-fertilizations were only conducted once for each combination, and therefore, were unable to confirm the results statistically. Lack of replicates may be criticized, but for the scope of this study, grasping general tendencies of the variation in fertilization rates was more meaningful than detailed analysis of the variation. It is important that the following similarities in outcome were obtained between the two years. 1) With 2-colony crosses, the fertilization rate varied widely; 2) with 3-colony crosses the fertilization rate increased but some showed relatively lower rates; and 3) with 6-colony crosses the rate was consistently higher than 95%.
Composition of developmental stage of embryo originated from cross-fertilization between different donor colonies
A rarefaction analysis of population genetic datasets conducted by Shearer et al. [
In the present experiment, the number of donor colonies was limited to 6. It lacks replication and statistics, but shows evidence that fertilization rate increases with the number of donor colonies, as if there exists a threshold where the relationship reaches an asymptote. The purpose of the present study was not to assess how genotypic differences between the donor colonies affected fertilization rate, but aimed to investigate how many donor colonies are necessary to consistently achieve high fertilization rate. If the importance of allelic diversity of a population is taken into consideration, cross-fertilization with 10 colonies or more may be needed, but for practical nursery farming of sexually propagated corals, cross-fertilization with 6 or more colonies could be recommended in the laboratory.
We are grateful to H. Taniguchi who kindly collected coral colonies from Kuba and Yakabi Islands. Advices concerning laboratory experiments offered by M. Hatta and H. Fukami are also highly appreciated.