research article gametogenesis, embryogenesis, and...

Research ArticleGametogenesis Embryogenesis and FertilizationEcology of Platygyra acuta in Marginal NonreefalCoral Communities in Hong Kong

Apple Pui Yi Chui Man Chung Wong Siu Hong Liu Ga Wun LeeSze Wai Chan Pui Ling Lau Sin Man Leung and Put Ang Jr

Marine Science Laboratory School of Life Sciences Chinese University of Hong Kong Sha Tin New Town Hong Kong

Correspondence should be addressed to Put Ang Jr put-angcuhkeduhk

Received 2 June 2014 Revised 31 July 2014 Accepted 16 August 2014 Published 8 September 2014

Academic Editor Nobuyuki Miyazaki

Copyright copy 2014 Apple Pui Yi Chui et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Understanding the reproductive biology of dominant coral species in subtropical nonreefal coral communities is critical inproviding important information on the processes underlying the distribution limits of coral species and communities This is thefirst study that investigates the reproduction cycle gametogenesis and fertilization ecology of Platygyra acuta Results indicatedthat P acuta is hermaphroditic and exhibits a single annual gametogenic cycle Oogenic and spermatogenic cycle occurs for 6-7months and for 2 months respectively prior to annual mass spawning event in May to June in Hong Kong It took 18 hours forP acuta to complete embryonic development develop cilia and start to rotate High (gt70) fertilization success can be achievedunder a broad range of sperm concentrations from 104 to 107 spermsmLminus1 Fertilization success remained consistently high 6 h afterspawning indicating a prolonged viability of its gametes that is much longer than that recorded for other coral species Significantlyhigher percentage of fertilization success was recorded in the first of the two consecutive nights of spawning suggesting differencesin the quality of the eggs andor sperms between days of spawningThese results serve as important baseline information for betterunderstanding of corals in marginal communities

1 Introduction

Corals show two aspects of sexual reproductive patternsThey can be hermaphroditic or gonochoric in their sexualityand they can broadcast their gametes in massmultispecificspawning (broadcaster) or brood their larvae (brooder) asa mode of larval development Hermaphroditic corals haveboth male and female gonads developed within polyp or indifferent polyps within the same colony whereas gonochoriccorals have separate sexes at the colony level Broadcastspawners carry out external fertilization in the water columnwhereas brooders carry out internal fertilization within thematernal polyp and release planula larvae [1ndash5] Overallbased on the information on sexuality and mode of devel-opment available worldwide most scleractinian corals arehermaphroditic broadcasters [3] The breeding season forbroadcast spawning species is relatively short and discrete

each colony usually spawns only once per year on a seasonalcycle with oogenic cycle typically ranging from 6 to 14months [1 3]

Since corals are sessile organisms incapable of aggregat-ing for reproduction synchronous release of gametes forexternal fertilization is crucial for reproductive success Highdegree of synchronized spawning maximizes fertilizationsuccess by increasing the chance for gametes from conspecificindividuals to meet in the water column or surface pre-venting gametes from being drifted away by current beforefertilization takes place reducing sperm dilution effect andavoiding predation [4 6 7] Therefore detailed informationon the timing of gametogenesis is needed as a baseline forbetter understanding of coral spawning pattern as well as itsreproductive strategy and implication on its sustainability ina region

Hindawi Publishing CorporationJournal of Marine BiologyVolume 2014 Article ID 953587 9 pageshttpdxdoiorg1011552014953587

2 Journal of Marine Biology

Mass spawning of scleractinian corals was first docu-mented in the Great Barrier Reef Australia in the early 1980s[8] Since then research on sexual reproduction and mass-or multispecific spawning of corals has greatly increased innumber and has expanded to different geographical regionsover the last two decades [1ndash4 9 10] Although therehave been reviews on coral reproduction and developmentdescriptions of development profile of many coral speciesare necessary [11] This is particularly true for coral speciesfound in marginal communities Marginal coral reefs andcommunities are those that exist near or beyond normalenvironmental limits of reef distribution such as low tem-perature low aragonite saturation state and light limitation[12] Studies onmarginal communities can provide importantinformation about the processes underlying the distributionlimits of coral species and communities

Hong Kong located in a subtropical region experienceslarge annual thermal variation with monthly mean SST thatranges from 14ndash16∘C in winter to 30∘C in summer [13] Thislarge temperature range makes it a marginal environment forreef building scleractinian corals Yet the coral diversity inHong Kong is relatively high with at least 84 scleractiniancoral species recorded [13] Coastal subtropical environmentlike Hong Kong may become an important refuge for coralrange expansion in the future should current pattern ofclimate change continues [14ndash17] Platygyra acuta is oneof the most dominant scleractinian corals in Hong Kongnortheastern water [18] Despite its key role in shapingthe local coral community structures its reproduction andgametogenesis have never been examined in detail neitherlocally nor elsewhere An understanding of coral reproduc-tion and development of P acuta is therefore of particularimportance if conservation of coral communities and coralreefs is considered in a global perspective

Coral assemblages in Hong Kong are usually small andisolated This is true as well in many other high latituderegions [19] The total amount of spawned gametes is muchlimited when compared to that in tropical reef areas There-fore it is important to understand the adaptive strategies ifany being employed by Hong Kong corals as exemplifiedby P acuta to ensure or promote fertilization success Thesestrategies may include the capability to fertilize under lowersperm concentration or increase gamete longevity to enhancethe chance of sperm-egg encounters [6] Manipulative exper-iments on sperm density and gamete ageing effects weretherefore conducted in this study to test if such reproductivestrategies are exhibited by P acuta In addition given thegrowing awareness of research in marginal coral areas dueto their potential importance under climate change andlimited description of coral developmental profile availablethis study also looked into detailed embryogenesis of Pacuta All these pieces of information will serve as importantbaseline for future experiments on other aspects of coralbiology in marginal environment The objectives of thisstudy are therefore to document the reproductive cycle andgametogenesis of P acuta in Hong Kong and to examine itsfertilization ecology including optimal sperm concentrationfor fertilization and gamete ageing effects that influence itsfertilization success

Tung Ping Chau Marine ParkA Ye Wan A Ma Wan

Chek Chaulowast

lowastlowast

8km

N

Figure 1 Map of Hong Kong showing the location of the study sitesChekChau andAMaWanare siteswhere samples for gametogeneticstudies were collected A Ye Wan was the site where coral spawningwas monitored and hence where sperm-egg bundles of Platygyraacuta were collected

2 Materials and Methods

21 Gametogenetic Study Samples of Platygyra acuta werecollected monthly within 5 days prior to the full moon andwithin 1ndash3m water depth range from A Ma Wan (AMW)Tung Ping Chau (22∘541015840N 114∘441015840E) for a period of 15months between May 1998 and July 1999 and again fromChek Chau (22∘501015840N 114∘361015840E) for a period of 14 monthsbetween August 2010 and September 2011 (Figure 1) Bothsites are within Tai Pang Wan (Mirs Bay) NE Hong KongOn each sampling occasion five visibly healthy colonies werehaphazardly sampled with the criteria that they were alreadyof reproductive sizegt30 cm in diameter [20] Small fragmentsof approximately 2 times 4 cm in size were removed from thecolonies using hammer and chisel Resampling of the samecolony was avoided during the study period to eliminate ifany stress or injury induced response which may affect theability of corals to reproduce properly

Coral samples were fixed in 10 formalin in seawaterimmediately after collection and kept for oneweek Followingfixation samples were decalcified in 10 hydrochloric acidfor two days with active ingredients including 100mL con-centrated hydrochloric acid (32) 07 g ethylenediaminete-traacetic acid 0008 g sodium potassium tartrate and 014 gsodium tartrate dehydrate per liter of solution Sampleswere subsequently immersed in running tap water overnightand preserved in 70 ethanol until histological processingSingle polyp was randomly selected and cut out from eachsample for dehydration processing in an ethanol series inAutomated Vacuum Tissue Processor (Leica TP 1050 LeicaInstruments GmbH) and embedded in paraffin wax Sampleswere oriented in longitudinal positions for serial cut at athickness of 7 120583m at intervals of 400 120583m and stained withhaematoxylin and eosin Slides were examined under alight microscope for oocytes or spermaries The maximumlength and perpendicular width of the six largest oocytes orspermaries were measured by the computer program Image-Pro Plus 60copyThe geometric mean diameter (GMD) for eachoocyte or spermary was obtained by calculating the squareroot of the product of the length and width measurements

Journal of Marine Biology 3

22 Embryonic Development and Fertilization Ecology ofPlatygyra acuta

221 Gametes Collection Gametes of Platygyra acuta werecollected in June 2010 and 2012 from A Ye Wan (AYW)Tung Ping Chau Marine Park (TPCMP) (22∘321015840N 114∘251015840E)(Figure 1) Ten individual colonies of Platygyra acuta weretagged at depths of minus05 to minus15m chart datum (CD)During spawning event egg-sperm bundles were collectedseparately from different colonies and transferred on shorefor further processing Bundles collected in June 2010 weretargeted for embryonic development monitoring Bundlesfrom different colonies were therefore mixed together in aculture tank and gently stirred to ensure fertilization Theculture tank was located semi-indoor and was equippedwith HOBO Temperature Pendant data loggers (Onset) torecord the water temperature every 30min The subsequentculturing procedures of the developing embryos followedthose described by Hatta et al [21] and Guest et al [22]Egg-sperm bundles collected in June 2012 were targeted forfertilization ecology studyTherefore after bundle collectioneggs and sperms were separated for subsequent experimentsusing methods adapted from Oliver and Babcock [6]

222 Embryonic Development Spawning of Platygyra acutawas observed between 2100 and 2200 h on June 6 2010 Eggbundles from 10 coral colonies were collected and mixedFertilization started at 2325 h with sperm concentration ofaround 107 sperms mLminus1 Early stages of development wereexamined by sampling the developing embryos in the culturetank at hourly intervals during the first 12 h after fertilization(119879 = 0 to 119879 = 12) every 2 h from 119879 = 12ndash24 every 4 hfrom 119879 = 24ndash72 and every 12 h thereafter Approximately50 eggs or embryos of Platygyra acuta were collected duringeach sampling and fixed in 25 glutaraldehyde (GA) for4 h at 4∘C Fixed samples were washed by 02M phosphatebuffer (PBS) and postfixed in 1 buffered osmium tetroxideat 4∘C Postfixed samples were washed by 02M phosphatebuffer (PBS) and kept in Eppendorf tubes until being readyto be processed To prepare the samples for scanning electronmicroscopy (SEM) the samples were dehydrated in increas-ing concentration of acetone-ethanol Drying was carriedout by using anhydrous acetone as intermediate liquid andtertramethylsilane (TMS) as the transition liquid After thedrying stage samples were observed under the SEM (S-3400N Hitachi Japan) with a working voltage of 20 kV

223 Optimal Sperm Concentration on Fertilization SuccessTheexperiment was conducted in June 2012 andwas repeatedon two successive nights of spawning event Concentrationof undiluted sperm stock was adjusted to about 106 to 107sperms mLminus1 These suspensions were then serially diluted10 times with 40 120583m filtered sperm-free seawater to providea standard range of sperm concentrations that ranged from102 to 107 sperms mLminus1 For each concentration 50mL ofsperm suspension was added separately to three replicateglass bottles (100mL) Approximately 100 eggs were added toeach of the bottles placed on a bench at ambient temperature

(26∘C) and left undisturbed The number of fertilized eggsthat is eggs that were at or beyond the two-cell stage wasexamined under the stereomicroscope and recorded after 4 h

224 Gamete Ageing Effects on Fertilization Success Theexperiment was conducted in June 2012 and was repeated ontwo successive nights of spawning event In each case 50mLsperm suspension (106 sperms mLminus1) was added separatelyto three replicate glass bottles (250mL) Approximately 100eggs were then added to each of the bottles In the first dayof experiment each set of mixing was carried out at 30minintervals for 35 h to assess the potential longevity of gametesIn Day 2 mixing was carried out at 1 h intervals for 6 h Thischange was necessary after reviewing the results from Day1 The sperms were kept at a concentration of 106 spermsmLminus1 until the time the gametes were mixed All experimentswere conducted on a bench at ambient temperature and leftundisturbed after mixing The number of fertilized eggs wasexamined under the stereomicroscope and recorded after 4 h

225 Self-Fertilization in Platygyra acuta To assess theability for self-fertilization in P acuta egg bundles collectedfrom the same colony (119899 = 5) were stirred gently to break upthe bundles and left undisturbed for at least 4 h The numberof fertilized eggs as indicated by the presence of cleavage wascounted under the stereomicroscope This experiment wasconducted in 2012 and repeated in 2013 spawning event

23 Data Analysis All data were arcsine-transformed andtested for normality using one-sample Kolmogorov-Smirnovtest and for homogeneity of variances using Levenersquos testOne-way analysis of variance (ANOVA) was then used todetermine if there was any significant difference (119875 lt 005) inthe effect of sperm concentrations on the percent fertilizationsuccess in Experiment 1 as well as the effect of gamete ageon the percent fertilization success in Experiment 2 Posthoc comparisons of means to find significant groupings weredone using Tukeyrsquos HSD tests SPSS version 160 forWindows(SPSS Inc USA) was employed in all statistical analyses

3 Results

31 Gametogenesis of Platygyra acuta Sampled Platygyraacuta colonies covered three spawning events in 1998 1999and 2011 all had a single annual gametogenic cycle andsimilar annual patterns of gametogenesis with oogenesisoccurring for 6-7 months from November 1998 to May1999 and from November 2010 to June 2011 (Figures 2 and3) Spermatogenesis was monitored only in 2011 and wasfound to occur two months before spawning from May toJune 2011 Oocytes first appeared in P acuta tissues in bothNovember 1998 (10489 plusmn 1710 120583m) and November 2011(959 plusmn 217 120583m) (Figure 3) Their sizes increased throughthe gametogenic cycle to reach the largest mean (plusmnSD)size of 26444 plusmn 4465 120583m 3291 plusmn 257 120583m and 2571 plusmn118 120583m respectively in June 1998 May 1999 and June 2011Spermaries first appeared inMay 2011 with amean (plusmnSD) sizeof 403 plusmn 52 120583m (Figure 3) They developed rapidly within


o

nnu

o

t

Figure 2 Developing oocytes and spermaries in Platygyra acuta o oocytes n nucleus nu nucleolus and t spermaries

May Jul Sep Nov Jan Mar May0

100

200

300

400

Oocytes

1998 1999

Mea

n ge

omet

ric d

iam

eter

(120583m

)

(a)

0

50

100

150

200

250

300

OocytesSpermaries

Aug AugJunAprFebDecOct2010 2011

Mea

n ge

omet

ric d

iam

eter

(120583m

)

(b)

Figure 3 Mean plusmn SD (119899 = 5) geometric diameter of oocytes and spermaries (120583m) in Platygyra acuta colonies during the gametogenic cyclein (a) 1998-1999 and (b) 2010-2011

a month and achieved their maximum mean size of 705 plusmn249 120583m in June 2011 Oocytes and spermaries coexisted inthe polyps and all gametes disappeared in the samples of July2011 Likewise oocytes were also not detected in the samplesof July 1998 and June 1999 (Figure 3) This indicates that Pacuta in Hong Kong started gametogenesis in November andmass spawning occurred inMay or June in the following yearTwo samples collected one in November 2010 and anotherin March 2011 within the gametogenetic period were foundto contain no gamete The number of this type of samples ishowever small (5 of sampled colonies)

32 Embryogenesis of Platygyra acuta Average (plusmn SD) tem-perature of 2612 plusmn 18∘C was maintained throughout theculturing period Immediately after bundle collection sub-samples of the eggs (Figure 4(a)) released from the bundleswere measured under dissecting microscope with the mean(plusmn SD) diameter of the eggs recorded as 375 plusmn 155 120583m (119899 =20)

The first cleavage was initiated at 2 h after fertilizationCleavage furrow appeared on one side of the blastomerecreating a heart-shaped zygote (Figure 4(b)) Four equallysized blastomeres were observed at 3 h after the secondcleavage (Figure 4(c)) The third cleavage was perpendicularto the first two cleavage planes creating eight blastomeresthat further divided into 16 and 32 blastomeres in 5 hafter fertilization (Figures 4(d)-4(f)) After the 32-cell stagethe blastomeres continued to divide into highly irregularshapes (Figures 4(g)-4(h)) Embryos began to flatten andbecame bowl shaped (cushion stage) at 7 h (Figures 4(i)-4(j)) Smoothening of the embryo surface was observed at9 h (Figure 4(k)) After 10 h the irregular shaped embryosexpanded and gradually became spherical in shape (Figures4(l)-4(m)) Invagination of the blastopore began at 16 h(Figure 4(n)) followed by shrinking in diameter of the blasto-pore (Figure 4(p)) Larvae began to develop cilia and startedto rotate at 18 h Planulae were observed to elongate in theoral-aboral axis and began to be highlymobile at 36 h (Figures4(q)-4(r)) Formation of two blastopores that resulted in


(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r)

Figure 4 SEMmicrograph of embryonic developmental stages of the broadcast-spawning scleractinian coral Platygyra acuta (a) Egg wherefertilization may or may not have occurred (b) First cleavage (c) 4-cell stage (d) 8-cell stage (e) 16-cell stage (f) 32-cell stage (g)-(h)Blastomeres continued to divide into highly irregular shapes (i)-(j) Cushion stage (k) Smoothening of embryo surface (l)-(m) Embryosexpanded and became spherical shaped (o) Abnormal embryo (n)-(p) Blastula stage (q)-(r) Elongated stage Scale bar = 200120583m

separate invagination was observed but only in two embryosduring sampling at 20 and 22 hThis phenomenon is likely tobe an abnormal growth pattern (Figure 4(o))

33 Optimal Sperm Concentration on Fertilization SuccessIn both experimental days (10th and 11th of June 2012)

spawning of Platygyra acuta was observed between 2050and 2210 h Fertilization success varied significantly withdifferent sperm concentrations (Day 1 119865

(512)= 18257 119875 lt

0001 Day 2 119865(614)

= 13248 119875 lt 0001) High percentfertilization successes (gt95 in Day 1 and gt73 in Day 2)were obtained at a broad spermdensity range between 104 and


Fert

iliza

tion

()

0

20

40

60

80

100

Sperm concentration (per mL)

A

B

CCD CD D

Control 102 103 104 105 106

(a)

0

20

40

60

80

100

Fert

iliza

tion

()


A

B

C

A

C CC

Control 102 103 104 105 106 107

(b)

Figure 5 Mean plusmn SD (119899 = 3) percent () fertilization success of the coral Platygyra acuta under different sperm concentration treatmentsExperiment was conducted on (a) Day 1 June 10 2012 and (b) Day 2 June 11 2012 Data indicated with the same letter showed no significantdifference in fertilization as determined by Tukeyrsquos HSD post hoc test Percent fertilization of eggs in sperm-free filtered sea water (FSW)served as the control to evaluate the effectiveness of sperm wash in separating eggs from the sperms before mixing

106 spermsmLminus1 (Figures 5(a) and 5(b))Mean (plusmnSD) percentfertilization success was lowest at sperm concentration of 102sperms mLminus1 on Day 1 (126 plusmn 93) and Day 2 (105 plusmn 50)Very high sperm concentration of 107 sperms mLminus1 couldonly be obtained on Day 2 of the experiment and showedno sign of significant inhibition of fertilization (Tukeyrsquos HSDtest 119875 = 0066) (Figure 5(b)) However percent fertilizationsuccess varied at sperm concentration of 103 sperms mLminus1 inthe two experimental days with 881 plusmn 18 observed in Day1 but only 313 plusmn 67 in Day 2 Highest fertilization successwas achieved at 106 sperms mLminus1 in both days with 989 plusmn 1in Day 1 and 850 plusmn 28 in Day 2

34 Gamete Ageing Effect on Fertilization Success In bothexperimental days egg bundles were collected from taggedcolonies that spawned between 2050 and 2210 h The fer-tilization experiment started a bit later after sperm washto separate sperms from the eggs Therefore time = 0indicated here referred to 2330 h around 25 h after spawningFertilization success varied with different ages of gametes(Day 1 119865

(818)= 11239 119875 lt 0001 Day 2 119865

(716)= 18787

119875 lt 0001) Mean percent fertilization success was relativelyhigh for the first 3 h (119879 = 0 to 119879 = 3 h) of the experimentsranging from 96 to 94 in Day 1 and from 75 to 73in Day 2 (Figures 6(a) and 6(b)) Significant reduction infertilization success was observed after 3 h (Tukeyrsquos HSD test119875 lt 005) with only 380 plusmn 31 fertilization success at 6 hafter the commencement of the experiment (Figure 6(b))

35 Self-Fertilization in Platygyra acuta No fertilized eggs orvery low fertilization lt06 was observed in this experimenteither in 2012 or in 2013 indicating that eggs and sperms fromthe same colony of P acuta cannot self-fertilize

4 Discussion

In the present study oocytes appeared from November toMay or June suggesting that the duration of the oogenic cycleis between six and seven months The spermatogenic cyclewas much shorter Spermaries started to appear only approx-imately two months before spawning Harrison and Wallace[3] reported that broadcast spawning coral species releasetheir gametes subsequent to the full moon after gametematuration The disappearance of oocytes and spermariesafter May (1999) or June (1998 2011) therefore indicated thatspawning of P acuta in Hong Kong occurs after the full moonin May or June each year Spawning of P acuta has also beenconfirmed by field observations Our results also supportedthose from previous studies from other localities suggestingthat most Platygyra species are hermaphroditic broadcastspawners with oocytes and spermaries present within thesame polyp [7 8 23 24]

Broadcast spawning of Platygyra species has been doc-umented in different regions [4] However there are onlytwo studies describing in detail the length and pattern ofgametogenic cycles ofPlatygyra species includingP daedaleain Kenya [25] and P pini in Singapore [24] Single annualgametogenic cycles were found in majority of P daedaleacolonies in Kenya whereas a low proportion of the popu-lation spawned biannually Duration of oogenic cycle lastedbetween six and sevenmonths and spermatogenic cycle lastedfive months [25] Two spawning seasons were observed inP pini colonies in Singapore but whether the two spawningseasons were caused by multiple gametogenic cycles orpopulation split spawning is not known Duration of oogeniccycle of P pini lasted between five and eight months andspermatogenic cycle approximately two to threemonths [24]In the present study P acuta has a single annual gametogeniccycle with comparable length of oogenic cycle as that of both

Journal of Marine Biology 7Fe

rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)

Figure 6 Mean plusmn SD (119899 = 3) percent () fertilization successof the coral Platygyra acuta gametes mixed at different times afterspawning to examine the effect of ageing Eggs and sperm were ofthe same age at the time ofmixing Experimentwas conducted on (a)Day 1 June 10 2012 and (b) Day 2 June 11 2012 Data indicated withthe same letter showed no significant difference in fertilization asdetermined by Tukeyrsquos HSD post hoc test The background controlsin Day 1 and Day 2 were 170 plusmn 11 and 98 plusmn 39 respectively

species of Platygyra described above Its spermatogenic cycleis however more comparable to that of P pini Biannualspawning is not restricted to equatorial reefs [24] Futureresearch is needed to investigate whether single annualgametogenic cycle is the more common reproductive modeat marginal reef communities Having annual gametogeniccycle may be one of the adaptive reproductive strategies toensure high sperm density for fertilization success [6]

Our study is the first detailed examination of the embry-onic development of P acuta In general the developmentalstages are similar to those described for P sinensis [7] and Pcontorta [11] The embryogenetic pattern of P acuta also fitsthat of robust corals described by Okubo et al [11] HoweverOkubo et al [11] documented the formation of blastopores

with separate invagination in P contorta with the two poresthat subsequently merged In our current study this stagewith two pores was only observed in two but not in the rest ofgt100 embryos We therefore suspect this to be an abnormalgrowth stage for P acuta It is still possible that this stage wasvery short and hence could have been missed between oursampling intervals of one h or such characteristic of doubleinvagination is not shared among Platygyra species

While the general embryogenetic patterns and stages of Pacuta are similar to those of many of the other coral speciesexamined thus far they differ from those of the branchingAcropora spp [11 26] Instead of forming a very thin singlelayer prawn-chip stage which is typical for Acropora speciesP acuta develops a cushion stage (Figures 4(i) and 4(j))which subsequently expands and develops a depression onits side referred to as ldquopseudoblastoporerdquo by Okubo et al[11] This is then followed by a return to spherical shapebefore commencing a second invagination (Figure 4(m)) Itis reported that the timing required for coral embryo tocomplete embryonic development through to larval motilitystage varies among families and species Acropora hyacinthusand Pectinia lactuca embryos were motile at 48 h and 18 hrespectively [27] Favites pentagona and F abdita embryosstarted swimming approximately 15 and 22 h after first cleav-age [11] In this study P acuta developed cilia and started torotate 18 h after fertilizationThis is comparable to P contortawhose embryos were reported to start swimming 19 h afterfertilization [11] In both the present and P contorta studiesthe culturing temperature for embryonic development wasmaintained around 26∘C

Fertilization successes of P acuta were strongly influ-enced by sperm concentrations The pronounced decrease inpercent fertilization success at low sperm concentrations isbelieved to be due to the decreased probability of successfulegg-sperm encounters [6] Our results were comparableto those from Oliver and Babcock [6] for P sinensis butwith even broader optimal sperm concentrations recordedfrom 104 to 107 sperms mLminus1 Inhibition of fertilization wasreported when sperm concentration was greater than 106

sperms mLminus1 [6] but such inhibition was not observed inthe current study However it should be noted that thelevel of statistical significance to evaluate the difference infertilization success between the highest sperm concentrationof 107 sperms mLminus1 and the lower sperm concentrations wasmarginal (ie 119875 = 0066) Significant inhibitory effect mightbe detected with more replicates or in repeated experimentsNonetheless we were unable to test sperm concentrationhigher than 107 spermsmLminus1The overall fertilization successin the second day of spawning was also lower with thehighest only around 80 This suggests that the quality ofthe eggs and sperms may differ between spawning days andmay ultimately affect the optimal concentration of sperm forsuccessful fertilization In the present study themost strikingdifference on optimal sperm concentration for fertilizationsuccess between the two days of spawning was observed at103 sperms mLminus1 concentrationThe reason for a much lowerpercent fertilization success at this sperm concentration inthe second day of spawning is not known but may be related


to the lower quality of the sperms or their activity fromsubsequent days of spawning Additional experiments shouldbe carried out to evaluate this hypothesis

Gamete ageing effect experiment indicated that viabilityof the gametes for successful fertilization is relatively shortThe capability for fertilization remained high for up to 35 hafter the experiment started Given that the start of theexperiment (119879 = 0) was about 25 h after spawning thisindicates that highest gamete viability could only last for 6 hNonetheless the duration of this viability is already muchlonger than that reported for other corals Oliver andBabcock[6] showed thatfertilization for P sinensis remained highfor only up to 2 h after spawning The mechanisms behindsuch prolonged viability of P acuta gametes in the presentstudy are not clear Apparently given that the supply ofgametes is usually limited in a marginal environment suchprolonged viabilitymay be a reproductive strategy to enhancefertilization success by increasing the chances of egg-spermencounter over time It is also not clear whether the loss ofviability was largely due to the eggs or the sperms or both Ithas been suggested that the loss of viability may be related toa drop in spermmotility due to depletion of its limited energyreserves [6] Prolonged gamete viability of more than 5 h hasbeen demonstrated in other marine invertebrates such asthe abalone Haliotis laevigata [28] and mussel Mytilus edulis[29] Even longer sperm longevities of more than 24 h werereported in the ascidians Ascidia mentula [30] Althougheggs and sperm of the coral P acuta were still fertilizable forup to 6 h after release the age of the gametes for optimalfertilization is comparatively short

Plasticity in coral gametes released over the spawningperiod of several days has been documented before HedouinandGates [31] reported a significant difference in the fertiliza-tion success of Montipora capitata between two consecutivespawning nights within a month and between spawningmonths under copper exposure It has also been shown thatthe number size and lipid and protein concentrations ofgametes released at different spawning days were differentthus contributing to differences in their quality [30 31] Thegenerally much lower fertilization success observed in thesecond day of spawning in P acuta observed in the presentstudy may be part of an adaptive reproductive strategy forthis coral The best and most mature egg bundles couldbe released immediately upon receiving the first cue ofa favorable condition for fertilization to ensure a higherprobability of fertilization success Those released in thesecond or subsequent days may thus be of a lesser qualityFurther investigations should be conducted to assess thishypothesis

Results of self-fertilization experiment indicated that eggsand sperms from the same colony of P acuta were not ableto self-fertilize Scleractinian corals have been reported todemonstrate a self-recognition response [32] However thedegree of specificity may vary with species [33] One thingto be noted was that although self-fertilization experimentin this study was ended at least 4 h after gamete mixingit has been reported that the effectiveness of the barrierthat inhibits self-fertilization may decrease several hoursafter spawning [34] There remains a possibility that the

level of self-fertilization success might increase beyond 4 hafter spawning in P acuta Nevertheless the ability to avoidself-fertilization together with successful fertilization underlower sperm concentration and prolonged gamete longevitywill all provide time for gametes from different coloniesto mix hence enhancing the chance of successful cross-fertilization in P acuta under the natural condition [6]

5 Conclusions

Reproductive ecology and strategy of corals from marginalenvironment are understudied Yet marginal environmentcould be refuge for future coral expansion under the threatof global climate change Our study provided the firstdetailed documentation on gametogenic cycles embryonicdevelopment and fertilization ecology of P acuta one ofthe most dominant massive coral species in subtropicalmarginal nonreefal coral communities in Hong Kong Ourstudy also contributed new insights to the understanding ofthe reproductive strategy of corals These baseline pieces ofinformation and insights may shed light on further under-standing coral responses to and adaptations in a changingocean environment and could contribute to the developmentof strategies for their conservation and protection

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The study was partly supported by the Research Grant Coun-cil of Hong Kong General Research Fund no 460013 andthe Chinese University of Hong Kong Research CommitteeGroup Research Scheme The authors thank K H ChanC H Kwok W W Wah H B Yeung and Y H Yungfor technical support M H Chau K W Chu Y L HuiY L Kam C K Kwok K Y Lam H N Leung Y HLeung T Y Ng H L Tsang C W Yeung L C Yeungand final year project students of 2011 2012 and 2013 forfield and lab assistance Permission to work in Tung PingChau Marine Park was kindly granted by the AgricultureFisheries and Conservation Department of the Hong KongSAR Government

References

[1] AH Baird J R Guest and B LWillis ldquoSystematic and biogeo-graphical patterns in the reproductive biology of scleractiniancoralsrdquo Annual Review of Ecology Evolution and Systematicsvol 40 pp 551ndash571 2009

[2] P L Harrison ldquoSexual reproduction of scleractinian coralsrdquo inCoral Reefs An Ecosystem in Transition Z Dubinsky and NStambler Eds pp 59ndash85 Springer New York NY USA 2011

[3] P Harrison and C Wallace ldquoReproduction dispersal andrecruitment of scleractinian coralsrdquo in Ecosystems of the WorldCoral Reefs Z Dubinsky Ed pp 133ndash207 Elsevier New YorkNY USA 1990


[4] R H Richmond and C L Hunter ldquoReproduction and recruit-ment of corals comparisons among the Caribbean the TropicalPacific and the Red SeardquoMarine Ecology Progress Series vol 60pp 185ndash203 1990

[5] Y H Fadlallah ldquoSexual reproduction development and larvalbiology in scleractinian coralsmdasha reviewrdquoCoral Reefs vol 2 no3 pp 129ndash150 1983

[6] J Oliver and R Babcock ldquoAspects of the fertilization ecologyof broadcast spawning corals sperm dilution effects and in situmeasurements of fertilizationrdquo Biological Bulletin vol 183 no3 pp 409ndash417 1992

[7] R C Babcock and A J Heyward ldquoLarval development ofcertain gamete-spawning scleractinian coralsrdquo Coral Reefs vol5 no 3 pp 111ndash116 1986

[8] R L Harrison R C Babcock G D Bull J K Oliver CC Wallace and B L Willis ldquoMass spawning in tropical reefcoralsrdquo Science vol 223 no 4641 pp 1186ndash1189 1984

[9] R H Richmond ldquoReproduction and recruitment in coralscritical links in the persistence of reefsrdquo in Life and Death ofCoral Reefs C Birkeland Ed pp 175ndash197 Chapman amp HallNew York NY USA 1997

[10] J R Guest A H Baird B P L Goh and L M ChouldquoSeasonal reproduction in equatorial reef coralsrdquo InvertebrateReproduction and Development vol 48 no 1ndash3 pp 207ndash2182005

[11] N Okubo T Mezaki Y Nozawa et al ldquoComparative embryol-ogy of eleven species of stony corals (Scleractinia)rdquo PLoS ONEvol 8 no 12 Article ID e84115 2013

[12] J A Kleypas JWMcManu and L A BMene ldquoEnvironmentallimits to coral reef development where do we draw the linerdquoThe American Zoologist vol 39 no 1 pp 146ndash159 1999

[13] P O Ang L S Choi M M Choi et al ldquoHong Kongrdquo in Statusof Coral Reefs of the East Asian Seas Region 2004 pp 121ndash152Japan Wildlife Research Centre Ministry of the EnvironmentGovernment of Japan 2005

[14] W F Precht and R B Aronson ldquoClimate flickers and rangeshifts of reef coralsrdquo Frontiers in Ecology and the Environmentvol 2 no 6 pp 307ndash314 2004

[15] B J Greenstein and J M Pandolfi ldquoEscaping the heat rangeshifts of reef coral taxa in coastal Western Australiardquo GlobalChange Biology vol 14 no 3 pp 513ndash528 2008

[16] H Yamano K Sugihara and K Nomura ldquoRapid polewardrange expansion of tropical reef corals in response to rising seasurface temperaturesrdquo Geophysical Research Letters vol 38 no4 Article ID L04601 2011

[17] E Couce A Ridgwell and E J Hendy ldquoFuture habitat suitabil-ity for coral reef ecosystems under global warming and oceanacidificationrdquo Global Change Biology vol 19 no 12 pp 3592ndash3606 2013

[18] T-W Tam and P O Ang Jr ldquoRepeated physical disturbancesand the stability of sub-tropical coral communities in HongKong Chinardquo Aquatic Conservation Marine and FreshwaterEcosystems vol 18 no 6 pp 1005ndash1024 2008

[19] L C Wicks J P A Gardner and S K Davy ldquoSpatial patternsand regional affinities of coral communities at the KermadecIslands Marine Reserve New Zealand-a marginal high-latitudesiterdquoMarine Ecology Progress Series vol 400 pp 101ndash113 2010

[20] C C Wallace ldquoReproduction recruitment and fragmentationin nine sympatric species of the coral genus Acroporardquo MarineBiology vol 88 no 3 pp 217ndash233 1985

[21] M Hatta K Iwao H Taniguchi and M Omori ldquoRestorationtechnology using sexual reproductionrdquo in Manual for Restora-tion and Remediation of Coral Reefs M Omori and S FujiwaraEds pp 14ndash33 Nature Conservation Bureau Ministry of theEnvironment Tokyo Japan 2004

[22] J R Guest A Heyward M Omori K Iwao A Morse andC Boch ldquoRearing coral larvae for reef rehabilitationrdquo in ReefRehabilitation Manual A J Edwards Ed pp 74ndash98 CoralReef Targeted Research amp Capacity Building for ManagementProgram 2010

[23] Y Shlesinger T L Goulet and Y Loya ldquoReproductive patternsof scleractinian corals in theNorthernRed SeardquoMarine Biologyvol 132 no 4 pp 691ndash701 1998

[24] J R Guest L M Chou and B Goh ldquoReproductive seasonalityof the reef building coral Platygyra Pini on Singaporersquos reefsrdquoRaffles Bulletin of Zoology vol 25 pp 123ndash131 2012

[25] S Mangubhai and P L Harrison ldquoGametogenesis spawningand fecundity of Platygyra daedalea (Scleractinia) on equatorialreefs in Kenyardquo Coral Reefs vol 27 no 1 pp 117ndash122 2008

[26] N Okubo and T Motokawa ldquoEmbryogenesis in the reef-building coral Acropora spprdquo Zoological Science vol 24 no 12pp 1169ndash1177 2007

[27] T C Toh J Guest and L M Chou ldquoCoral larval rearing in Sin-gapore observations on spawning timing larval developmentand settlement of two common scleractinian coral speciesrdquo inContributions to Marine Science pp 81ndash87 National Universityof Singapore 2012

[28] R Babcock and J Keesing ldquoFertilization biology of the abaloneHaliotis laevigata laboratory and field studiesrdquo Canadian Jour-nal of Fisheries and Aquatic Sciences vol 56 no 9 pp 1668ndash1678 1999

[29] L A Levy and C A Couturier ldquo Effects of sperm longevityand gamete concentrations on fertilization success in the bluemusselrdquo Bulletin of the Aquaculture Association of Canada vol96 pp 71ndash73 1996

[30] J N Havenhand ldquoFertilization and the potential for dispersalof gametes and larvae in the solitary ascidian Ascidia mentulaMullerrdquo Ophelia vol 33 no 1 pp 1ndash15 1991

[31] L Hedouin and R D Gates ldquoAssessing fertilization success ofthe coral Montipora capitata under copper exposure does thenight of spawning matterrdquo Marine Pollution Bulletin vol 66no 1-2 pp 221ndash224 2013

[32] W H Hildemann R L Raison G Cheung C J Hull L Akakaand J Okamoto ldquoImmunological specificity and memory in ascleractinian coralrdquoNature vol 270 no 5634 pp 219ndash223 1977

[33] J A Stoddart D J Ayre B Willis and A J Heyward ldquoSelfrecognition in sponges and coralsrdquo Evolution vol 39 no 2 pp461ndash463 1985

[34] A J Heyward and R C Babcock ldquoSelf- and cross-fertilizationin scleractinian coralsrdquo Marine Biology vol 90 no 2 pp 191ndash195 1986

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Mass spawning of scleractinian corals was first docu-mented in the Great Barrier Reef Australia in the early 1980s[8] Since then research on sexual reproduction and mass-or multispecific spawning of corals has greatly increased innumber and has expanded to different geographical regionsover the last two decades [1ndash4 9 10] Although therehave been reviews on coral reproduction and developmentdescriptions of development profile of many coral speciesare necessary [11] This is particularly true for coral speciesfound in marginal communities Marginal coral reefs andcommunities are those that exist near or beyond normalenvironmental limits of reef distribution such as low tem-perature low aragonite saturation state and light limitation[12] Studies onmarginal communities can provide importantinformation about the processes underlying the distributionlimits of coral species and communities

Hong Kong located in a subtropical region experienceslarge annual thermal variation with monthly mean SST thatranges from 14ndash16∘C in winter to 30∘C in summer [13] Thislarge temperature range makes it a marginal environment forreef building scleractinian corals Yet the coral diversity inHong Kong is relatively high with at least 84 scleractiniancoral species recorded [13] Coastal subtropical environmentlike Hong Kong may become an important refuge for coralrange expansion in the future should current pattern ofclimate change continues [14ndash17] Platygyra acuta is oneof the most dominant scleractinian corals in Hong Kongnortheastern water [18] Despite its key role in shapingthe local coral community structures its reproduction andgametogenesis have never been examined in detail neitherlocally nor elsewhere An understanding of coral reproduc-tion and development of P acuta is therefore of particularimportance if conservation of coral communities and coralreefs is considered in a global perspective

Coral assemblages in Hong Kong are usually small andisolated This is true as well in many other high latituderegions [19] The total amount of spawned gametes is muchlimited when compared to that in tropical reef areas There-fore it is important to understand the adaptive strategies ifany being employed by Hong Kong corals as exemplifiedby P acuta to ensure or promote fertilization success Thesestrategies may include the capability to fertilize under lowersperm concentration or increase gamete longevity to enhancethe chance of sperm-egg encounters [6] Manipulative exper-iments on sperm density and gamete ageing effects weretherefore conducted in this study to test if such reproductivestrategies are exhibited by P acuta In addition given thegrowing awareness of research in marginal coral areas dueto their potential importance under climate change andlimited description of coral developmental profile availablethis study also looked into detailed embryogenesis of Pacuta All these pieces of information will serve as importantbaseline for future experiments on other aspects of coralbiology in marginal environment The objectives of thisstudy are therefore to document the reproductive cycle andgametogenesis of P acuta in Hong Kong and to examine itsfertilization ecology including optimal sperm concentrationfor fertilization and gamete ageing effects that influence itsfertilization success

Tung Ping Chau Marine ParkA Ye Wan A Ma Wan

Chek Chaulowast

lowastlowast

8km

N

Figure 1 Map of Hong Kong showing the location of the study sitesChekChau andAMaWanare siteswhere samples for gametogeneticstudies were collected A Ye Wan was the site where coral spawningwas monitored and hence where sperm-egg bundles of Platygyraacuta were collected

2 Materials and Methods

21 Gametogenetic Study Samples of Platygyra acuta werecollected monthly within 5 days prior to the full moon andwithin 1ndash3m water depth range from A Ma Wan (AMW)Tung Ping Chau (22∘541015840N 114∘441015840E) for a period of 15months between May 1998 and July 1999 and again fromChek Chau (22∘501015840N 114∘361015840E) for a period of 14 monthsbetween August 2010 and September 2011 (Figure 1) Bothsites are within Tai Pang Wan (Mirs Bay) NE Hong KongOn each sampling occasion five visibly healthy colonies werehaphazardly sampled with the criteria that they were alreadyof reproductive sizegt30 cm in diameter [20] Small fragmentsof approximately 2 times 4 cm in size were removed from thecolonies using hammer and chisel Resampling of the samecolony was avoided during the study period to eliminate ifany stress or injury induced response which may affect theability of corals to reproduce properly

Coral samples were fixed in 10 formalin in seawaterimmediately after collection and kept for oneweek Followingfixation samples were decalcified in 10 hydrochloric acidfor two days with active ingredients including 100mL con-centrated hydrochloric acid (32) 07 g ethylenediaminete-traacetic acid 0008 g sodium potassium tartrate and 014 gsodium tartrate dehydrate per liter of solution Sampleswere subsequently immersed in running tap water overnightand preserved in 70 ethanol until histological processingSingle polyp was randomly selected and cut out from eachsample for dehydration processing in an ethanol series inAutomated Vacuum Tissue Processor (Leica TP 1050 LeicaInstruments GmbH) and embedded in paraffin wax Sampleswere oriented in longitudinal positions for serial cut at athickness of 7 120583m at intervals of 400 120583m and stained withhaematoxylin and eosin Slides were examined under alight microscope for oocytes or spermaries The maximumlength and perpendicular width of the six largest oocytes orspermaries were measured by the computer program Image-Pro Plus 60copyThe geometric mean diameter (GMD) for eachoocyte or spermary was obtained by calculating the squareroot of the product of the length and width measurements










3 Results



o

nnu

o

t



100

200

300

400

Oocytes

1998 1999

Mea

n ge

omet

ric d

iam

eter

(120583m

)

(a)

0

50

100

150

200

250

300

OocytesSpermaries


Mea

n ge

omet

ric d

iam

eter

(120583m

)

(b)






(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r)





(512)= 18257 119875 lt

0001 Day 2 119865(614)



Fert

iliza

tion

()

0

20

40

60

80

100


A

B

CCD CD D

Control 102 103 104 105 106

(a)

0

20

40

60

80

100

Fert

iliza

tion

()


A

B

C

A

C CC

Control 102 103 104 105 106 107

(b)




(818)= 11239 119875 lt 0001 Day 2 119865

(716)= 18787



4 Discussion




rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)














5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










3 Results



o

nnu

o

t



100

200

300

400

Oocytes

1998 1999

Mea

n ge

omet

ric d

iam

eter

(120583m

)

(a)

0

50

100

150

200

250

300

OocytesSpermaries


Mea

n ge

omet

ric d

iam

eter

(120583m

)

(b)






(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r)





(512)= 18257 119875 lt

0001 Day 2 119865(614)



Fert

iliza

tion

()

0

20

40

60

80

100


A

B

CCD CD D

Control 102 103 104 105 106

(a)

0

20

40

60

80

100

Fert

iliza

tion

()


A

B

C

A

C CC

Control 102 103 104 105 106 107

(b)




(818)= 11239 119875 lt 0001 Day 2 119865

(716)= 18787



4 Discussion




rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)














5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


o

nnu

o

t



100

200

300

400

Oocytes

1998 1999

Mea

n ge

omet

ric d

iam

eter

(120583m

)

(a)

0

50

100

150

200

250

300

OocytesSpermaries


Mea

n ge

omet

ric d

iam

eter

(120583m

)

(b)






(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r)





(512)= 18257 119875 lt

0001 Day 2 119865(614)



Fert

iliza

tion

()

0

20

40

60

80

100


A

B

CCD CD D

Control 102 103 104 105 106

(a)

0

20

40

60

80

100

Fert

iliza

tion

()


A

B

C

A

C CC

Control 102 103 104 105 106 107

(b)




(818)= 11239 119875 lt 0001 Day 2 119865

(716)= 18787



4 Discussion




rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)














5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r)





(512)= 18257 119875 lt

0001 Day 2 119865(614)



Fert

iliza

tion

()

0

20

40

60

80

100


A

B

CCD CD D

Control 102 103 104 105 106

(a)

0

20

40

60

80

100

Fert

iliza

tion

()


A

B

C

A

C CC

Control 102 103 104 105 106 107

(b)




(818)= 11239 119875 lt 0001 Day 2 119865

(716)= 18787



4 Discussion




rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)














5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Fert

iliza

tion

()

0

20

40

60

80

100


A

B

CCD CD D

Control 102 103 104 105 106

(a)

0

20

40

60

80

100

Fert

iliza

tion

()


A

B

C

A

C CC

Control 102 103 104 105 106 107

(b)




(818)= 11239 119875 lt 0001 Day 2 119865

(716)= 18787



4 Discussion




rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)














5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


rtili

zatio

n (

)

0

20

40

60

80

100

Time (hours)

Control 353252151050

A

B BBCC

BC BC BCBC

(a)

Fert

iliza

tion

()

Time (hours)

0

20

40

60

80

100

Control 6543210

A

B B B B

C

DD

(b)














5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusions




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article gametogenesis, embryogenesis, and...

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