Determination of developmental stages of embryo in the sea urchin, Echinometra mathaei

Item Type article

Authors Ghorani, V.; Mortazavi, M.S.; Mohammadi, E.; Sadripour, E.; Soltani, M.; Mahdavi Shahri, N.; Ghassemzadeh, F.

Download date 30/09/2021 04:46:54

Link to Item http://hdl.handle.net/1834/37280 Iranian Journal of Fisheries Sciences 11(2) 294-304 2012

Determination of developmental stages of embryo in the Sea Urchin, Echinometra mathaei

Ghorani V.1; Mortazavi M. S.2*; Mohammadi E.1; SadripourE.1; Soltani M.1; Mahdavi Shahri N.1; Ghassemzadeh F.1 Received: April 2011 Accepted: June 2011

Abstract Sea Urchin is one of the most useful tools in developmental biology studies because this organism has the simplest kind of developmental stages. We aimed to determine developmental stages and timetable of Echinometra mathaei embryo (the species of Persian Gulf). The spawning of E. mathaei was induced by 0.5M KCl injection (1ml) into the coelomic cavity. After fertilization, embryos were placed in beakers and were incubated at 29◦C and a salinity of 39 ppt until embryos reached the pluteus stage. The developmental stages of embryos and the timing of each stage including cleavage, morulae, blastula, gastrula, prism and pluteus larvae were studied under the microscope. Our results showed that after 30 hours from fertilization time, the embryos developed to pluteus larvae. E. mathaei had the shorter development time in comparison to the other Sea Urchin species. Therefore, it may be appropriate as a model organism in biological researches.

Keywords: Sea Urchin, Echinometra mathaei, Biological Model, Development, Timetable, Persian Gulf

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1- Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad-Iran. 2- Persian Gulf and Oman Sea Ecological Research Institute, P.O. Box1597, Bandar Abbas-Iran. * Corresponding author's email: [email protected]

295 Ghorani et al., Determination of developmental stages of embryo in the Sea Urchin …

Introduction Sea Urchins have an annual reproductive development, optical clarity of embryos cycle which can be affected by various have made the Sea Urchin a candidate of factors (Brooks and Wessel, 2004; Walker the suitable tool for research about the et al., 2005). Sea Urchins have separate fertility, early embryonic development and male and female sexes. Sperms and eggs also biological tests (Conway et al., 1984; are released into the water and after Semenova et al., 2006). In spite of the vast fertilization, zygotes are developed by research on the development of Sea radial holoblastic cleavage. The first and Urchins in the world, there are no reports second cleavages are both meridional and about E. mathaei from south coasts of Iran, are perpendicular to each other. The third except determination of reproductive cycle cleavage is equatorial and the fourth (Shahri et al., 2008) and recent studies divides the zygote unequally to produce about toxicity effects of metals, Hg, Cu, mesomeres, macromeres, and micromeres Pb, Cd on embryo – larval development of (Gilbert, 2003). After successive cleavage, this species. the embryo is developing to blastula stage. The objective was to determine the By this time, the cells start to occupy the developmental stage and timetable of E. periphery while leaving a central space mathaei embryo (The Persian Gulf and gradually form a central cavity called species) which may be used as a blastocoel (Briggs and Wessel, 2006). bioindicator in biological research such as Then Gastrulation is initiated by toxicity bioassays and other ecotoxicology invagination of the thickened vegetal plate researches. into the blastocoel that ultimately gives Materials and methods rise to the archenteron (Kominami and Biological materials Takata, 2000). Finally, embryo develops to About 20 samples of adult E.mathaei were the pluteus larva which starts the feeding Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 collected from Bostane costs (26˚31S, and its arms begin to develop (Yajima and 54˚39E) by snorkeling. Then samples were Kiyomoto, 2006). transferred to the laboratory of Persian In general, Sea Urchins live in a vast tidal Gulf and Oman Sea Ecological Research spectrum and have a major effect on the Institute. To induce spawning, 0.5 M structure and dynamic of their habitats potassium chloride (1 ml) was injected (Williamson et al., 2000; Williamson and into the celomic cavity of individual Sea Steinberg, 2001). The Sea Urchin, Urchins. Spawning females and males Echinometra mathaei is a member of the were inverted (oral side up) over beakers phylum Echinodermata and the class of filtered seawater (0.5 and 1 μm filters) Echinoidea (Lawrence, 1987). This species with a temperature of 29◦C so that is generally found in shallow waters of the spawned eggs and sperms were allowed to Persian Gulf and Oman Sea (Shahri et al., settle in the base of the dish and then were 2008). Some of the features like the ability collected separately. Then gametes were of spawning induction, artificial observed using a microscope to check their fertilization, coordinated and rapid Iranian Journal of Fisheries Sciences, 11(2), 2012 296

maturity (spherical eggs and mobile pluteus larval development was assessed in sperm). In order to fertilize a few amounts three different temperatures (27, 29 and of sperm solution were added to the 31◦C) in each of which, three different suspension of eggs and it was carefully concentrations of salinity (35, 37 and 39 stirred to allow fertilization. To ensure the ppt) with three replicates were selected. fertilization (formation of fertilization The developmental stage and timetable of membrane) has been occurred, samples E. mathaei were observed using a microscope and For determination of the developmental subsequently the fertilization eggs were timetable, after fertilization, the embryos diluted to a density of 200 eggs per ml were added to each of the test container filtered seawater (Bielmyer et al., 2005). containing filtered seawater to attain a Optimal conditions for growth of the final density of 20 embryos per ml. Then embryos samples were incubated under obtained In this study, several factors such as optimum conditions until embryos reached temperature, salinity and the type of the pluteus stage. To reduce the error rate, container required for embryos growth experiments were performed with 3 under laboratory conditions were analyzed. replicates. For the timing of each According to Fernandez and Beiras (2001) developmental stage (early cleavage, and Kurihara and Shirayama (2004), the morulae, blastula, gastrula, prism and polypropylene vials, beaker and Petri dish pluteus larvae) the samples were tested at were used to determine the suitable first in short timing intervals (~30 minute) container for embryos growth. For this and in advanced developmental stage they test, approximately equal numbers of were checked in long timing intervals (1 fertilized eggs (n=120) were placed in up to several hours) by using the beakers, vials and Petri dishes and three microscope. The timing point was replicates per container were assayed. The recorded when clear characteristics of each Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 containers containing the fertilized eggs stage were observed. were incubated at acceptable average from Data analysis temperature and salinity range for this The statistical procedure was conducted in species (temperature and salinity were two steps. In the first test, the number of 29◦C and 37 ppt, respectively). All the developed 4-arm pluteus larvae in three larvae that had developed in each containers were compared using One Way container were counted. The numbers of analysis of variance (ANOVA), followed larvae found in each container were by a Bonferroni test. In the secondary test, compared to assess any differences the percentages of pluteus larval between the containers. After the selection development were compared by two-way of suitable containers, three temperatures ANOVA and a Tukey HSD (honestly and salinities were tested within the range significant difference) multiple mean of acceptability for the species to comparison test (effects: temperature and determine optimal temperature and salinity). Also homogeneity of the salinity. However, the percentage of variance test was used prior to ANOVA. 297 Ghorani et al., Determination of developmental stages of embryo in the Sea Urchin …

Significant differences were considered at these results illustrated that significant the 95% level. differences existed between the numbers Results of developed 4-arm pluteus larvae in the Optimal conditions for growth of the aforementioned groups. Subsequently, embryos Bonferroni test indicated significant In the first step of analysis, homogeneity differences on the developmental rate of of variance test showed that all three pluteus larvae for both beaker and vial container variances were similar to each when compared to the Petri dish but the

other (F (2,6) p > 0.05). So, one- difference between beaker and vial was way ANOVA test was carried out and not significant (Table 1). Table 1: Mean number (M) and standard deviation (SD) of the developmental rate of pluteus larvae in three containers, mean comparison, one-way ANOVA, Bonferroni test, p < 0.05

Beaker Vials Petri dish F-Value p B-V B-P V-P M SD M SD M SD (df 2,6) number of developed 94.33 4.72 82.66 4.04 41.66 6.03 91.818 <0.001 * * larvae

Because of the developmental rate of for comparing results of the percentage of pluteus larvae was higher in beaker than pluteus larval development in different vial and Petri dish, beaker was selected for levels of temperature and salinity. Table 2 the next step of our experiment. In the shows experimental results in different second step of analysis, beakers were used temperatures and salinities. Table 2: Mean percentage (M) and standard deviation (SD) of pluteus larval development at different temperatures and salinities Salinity (ppt) Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 Temperature 35 37 39 (◦C ) M SD M SD M SD 27 5% 1.65 15% 2.08 21% 3.11 29 54% 2.25 78% 1.93 98% 1.85 31 49% 3.16 56% 2.51 74% 2.55

The homogeneity of variance test showed temperature (F (2,18) 1659.36, p <

there is no reason to believe that the equal 0.001) and salinity (F (2,18) , 314.37, p <

variances assumption is violated (F (8,18) , 0.001). Also the Tukey HSD test indicated 0.332, p > 0.05) so two-way analysis of that significant differences existed variance (ANOVAs) test was carried out amongst all salinities and temperatures, with temperature and salinity as factors. each of which yielded significantly unique There was significance in the percentage sets of data (all p < 0.05). of pluteus larval development by Iranian Journal of Fisheries Sciences, 11(2), 2012 298

100 Salinity (ppt) 35 37 75 39

50 Mean (%) Mean

25

0 27 29 31 Temperature(C )

Figure 1: Comparison between mean percentage of pluteus larval development at different temperatures and salinities. [Error bars=SD]

As illustrated in Figure 1, with regard to formation of fertilization membrane the results of ANOVA test, temperature of around the egg (Fig. 2B). 60 minutes after 29◦C and salinity of 39 ppt were more fertilization, the first meridional cleavage suitable than the others for growth and occurred which leaded to formation of two development of embryos, in these equal size cells (Fig. 2C). The plane of the conditions 98% of embryos developed to first cell division was possessed through pluteus larvae (Table 2). the animal-vegetal axis of the egg. After a The development of E. mathaei few hours, second meridional and then an The sperm morphology in E. mathaei was equatorial cleavage generated four and Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 longer than the other echinoderms and its eight blastomere of equal size, respectively egg was completely round. When sperm (Figs. 2D and E). The fourth cleavage and egg were close together (Fig. 2A), generated a 16-cell embryo in which four sperm penetrated into the egg and vegetal cells divide unequally to form 4 fertilization occurred 2 minutes after micromeres and 4 macromeres, while four contact of sperm and egg. The obvious animal cells divided into 8 equal characteristic of fertilization was the mesomeres longitudinally (Fig. 2F).

299 Ghorani et al., Determination of developmental stages of embryo in the Sea Urchin …

Figure 2: The early development of E. mathaei occurs rapidly; (A) sperm around the egg; (B) ovum after fertilization; (C) 60 min after fertilization, 2-cell stage; (D), (E) and (F) < 4 hr after fertilization, 4-cell stage, 8-cell stage and 16-cell stage, respectively. Scale bar= 50 μm for A-F.

Cell division continued repeatedly and formed the mouth. Then continuous after 4 hours, the embryonic cell mass digestive tube was formed by connecting converted to the morulae (Fig. 3A). After 6 the mouth to the archenteron. Almost 20 hours, the seventh cleavage generated a hours after fertilization, embryos entered blastula. In this stage, the cells formed to prism stage and formed the skeletal empty spheres that surround the central elements. In this stage the embryo was Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 blastocoel. The most important prismatic and the primitive gut was morphological features in this stage were observed clearly (Fig. 3D). Finally, 30 the formation of cilium around the cell hours after fertilization, the prism mass and the mobility of embryo which developed into 4-armed pluteus larvae. At was observed microscopically (Fig. 3B). this time, a pair of frontal short arms and a Gastrula formed 14 hours after fertilization pair of dorsal long arms of larvae could be through invagination at the vegetal pole observed. Furthermore, several pigmented (Fig. 3C). The invaginated region is called cells were visible under the light the archenteron that eventually made microscope (Fig. 3E). A developmental contact with the blastocoel’s wall and timetable which was observed in the laboratory is given in Table 3. Iranian Journal of Fisheries Sciences, 11(2), 2012 300

Bl

Ar

2nd 1st

Figure 3: The developmental stages of morulae to 4-armed pluteus larvae

Ar, primary gut or archenteron; Bl, blastocoel. (A) 4 hr after fertilization, morulae stage, (B) 6 hr after fertilization, blastula stage, showing blastocoel (C) 14 hr after fertilization, gastrula stage, archenteron not visible clearly in this image, (D) 20 hr after fertilization, prism stage, showing primitive gut, (E) 30 hr after fertilization, 4- armed pluteus larvae, showing first (1st) and second (2nd) pairs of arms. Scale bar= 50 μm for A-D. Scale bar= 100µm for E. Table 3: Developmental timetable of E. mathaei. Time after Developmental stages fertilization Formation of fertilization 2 min membrane First cleavage 60 min Morulae 4 hours

Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 Blastula 6 hours Gastrula 14 hours Prism 20 hours 4-armed pluteus larvae 30 hours

Discussion For determining the optimum conditions, on developmental rate of pluteus larvae the experiment was conducted in different was significant and the mean of larval containers. Because the type of the development in beakers was higher than container can affects the embryos growth vials and Petri dish so beaker was selected in laboratory conditions (Lera et al., 2006), as a suitable container for experiment. The we used different containers including results showed that our optimal polypropylene vials, Petri dish and beaker. temperature and salinity were higher than Our results showed the effect of container the other similar researches. Fernandez 301 Ghorani et al., Determination of developmental stages of embryo in the Sea Urchin …

and Beiras (2001) reported the optimal in E. mathaei whereas Shimek (2003) temperature for embryos growth of the Sea reported that embryos develop into prism Urchin Paracentrotus lividus at 20ºC. after 24 hours. Finally, the researches Cesar et al. (2004) also reported the reported that Paracentrotus lividus and optimal temperature of 22ºC for this Sterechinus neumayeri spend 48 hours and species. Bielmyer et al. (2005) showed that 20 days after fertilization to reach the optimal salinity for Diadema antillarum pluteus larvae, respectively (Russo et al., was 33 ppt and King and Riddle (2001) 2003; King and Riddle, 2001) while in E. reported an optimal salinity of 34 ppt for mathaei that occurred after 30 hours. Our Sterechinus neumayeri embryos. Probably results showed E. mathaei has a shorter difference of optimal temperature and developmental timing and the pluteus salinity for embryos growth is related to larvae developed in a shorter time the difference in the habitat of studied compared to the other species of Sea species. The optimal temperature for Urchins. Probably the environmental growing E. mathaei embryos that live in conditions that lead to the different the warm area of Iran (Bandar Abbas) was adaptation of the species to the 29 ºC and optimal salinity for this species environment resulted in different was 39 ppt. developmental timing. Whereas this According to table 3, 60 minutes after experiment was carried out in the warm fertilization, the first cleavage occurred area of Iran, thus, one of the important similar to Arbacia punctulata Sea Urchin environmental factors was temperature. (Shimek, 2003). Also, King and Riddle King and Riddle (2001) indicated that Sea (2001) showed that in Sterechinus Urchin embryos in cool regions reach to Downloaded from jifro.ir at 13:58 +0330 on Tuesday February 13th 2018 neumayeri the embryos develop into the pluteus larvae sooner than similar species blastula 2-3 days after fertilization, while from warmer regions. that occurs after 12 and 6 hours in In conclusion, we demonstrated Paracentrotus lividus (Russo et al., 2003) that E. mathaei, as other species of warm and E. mathaei (in the present study), regions, has a shorter developmental respectively. In the present study, timing compared to other studied species. developing into the gastrula was 14 hours Researches showed that Sea Urchins are a after fertilization while King and Riddle suitable model for biological studies due to (2001) showed that Sterechinus neumayeri some of features such as ability of embryos developed into the gastrula after spawning induction and artificial 9-10 days and that occurred in Arbacia fertilization, rapid development and also punctulata after 24 hours (Shimek, 2003). optical clarity of embryos (Conway et al., Prism occurred 20 hours after fertilization 1984; Semenova et al., 2006). As a result, Iranian Journal of Fisheries Sciences, 11(2), 2012 302

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