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pH

7.6

8.1

7.5

7.3

7.7

7.45

7.15

7.67

7.5

8.15

7.4

8.3

Salinity (g4)

1.0

1.7

1.2

0.3

0

18

53.5

7.2

21.2

0.8

0.0

2.0

Temperature (°C)

24

23

26

25

27

21

24

23

24

26

22

25

Dissolved oxygen (mg-l)

6.1

7.3

6.4

9.4

9.4

9.0

5.9

9.9

6.9

6.5

7

7.4

Transparency (cm)

6.3

6.0

9.5

15.3

12.5

20

26

23.9

22.8

11.6

11.4

11.5

Conductivity (Scm-1)

5.3

5.8

4.4

1.4

1.0

23

72

12.5

33.9

2.15

4.9

8.5

total Nitrate (mg-l)

2.0

2.5

2.0

2.5

3.5

2.3

1.5

1.67

2.15

2.0

7.5

2.0

total Phosphate (mg-l)

0.3

0.03

0.0

0.01

0.2

0.05

0.01

0.18

0.08

0.15

0.02

0.1

Chlorophyll a (nig4)

3.2

4.6

3.5

13

7.5

2.3

1.9

1.9

5.3

24

1.2

6.3

Hydrophyte (%)

10

12

5.0

8.0

2.0

60

30

49

20

85

5.0

10

Suspended materials (mg-

l)

3.0

5.0

6.0

13

8.0

2.3

2.0

1.8

5.0

24

1.0

5.0

Results

In total five species of anostracans were identified; a parthenogenetic population of Artemia, B. orientalis, S. torvicornis, Ph. Spinosa. The collected Anostracans belong to four families; parthenogenetic Artemia (Artemidae Schlosser 1755), B. orientalis (Branchinectidae Daday 1910), S. torvicornis (Streptocephalidae Daday 1910), P. spinosa (Thamnocephalidae Packard 1883). Parthenogenetic Artemia was found coexisting with P. spinosa in 11 pools distributed in 3 sites. S. torvicornis was found only in two locations. P. spinosa was the dominating species in the studied regions identified from 15 geographic locations. A number of other invertebrates such as Leptisteria, Caenestheriella, Cayzicus, Ostracoda, Coleoptera, Cyclopoid, Harpacticioda, Cladocera, Diptera, Ephemeptera, and Odonata were found coexisting with the anostracans at study area.

Discussions

Brendonck et al. (2008) reported about 500 species of large branchiopods world-wide, 351species from Palaearctic zoogeographic region including Iran. This is the first report on existence of S. torvicornis, B. orientalis, P. spinosa from West Azerbaijan. It was observed that Artemia and P. spinosa tolerate wide variations in the environmental parameters (e. g salinity ranges 5-58 g-1). They are halophilic Anostracans that occur in the shallow brackish, saline and hypersaline waters with scarce vegetation (Alonso 1985, Thiery and Puente 2002).The pools are associated with submerged plant Carex sp. It seems that the vegetation provides a proper place for reproduction as well as a safe hide to avoid the hunters.

Conductivity reaches to higher values in the brackish and saline waters, sufficiently large enough to limit above mentioned taxa. S. torvicornis, B .orientalis, P. spinosa and parthenogenetic Artemia were found in pools with conductivity ranges between 1.4, 2- 5.8 and 12-72 S cm-1 respectively. High suspended material was recorded in the biotopes supporting S. torvicornis.

We observed lowest values of pH in biotopes supporting P. spinosa and Artemia, probably due to their higher salinity limiting the density of primary producers (see also Wetzel 2001). The results showed that B. orientalis avoids highly vegetated pools. Similar behavior was also observed in the case of S. torvicornis.

Based on our findings the anostracans do not appear until the end of February depending on the amount of flooding required to fill the biotopes. Temperature was found to be an important parameter influencing the time based appearance of branchiopod species. At the beginning of the season only P. spinosa and B. orientalis are found when the water temperature during the night is still low. But the rest of the species gradually appear when the temperature fluctuations in the night compared to the day is not too great. This may be due to the species specific adaptation of the branchiopod species to specific environmental conditions and inter species competition for occupying a common

biotope (Morin et al. 1988). This explains the succession of species in the temporary waters and the seasonal influx of aerial colonizers (Nilsson and Svensson 1994). This study presents updated information on distribution, new geographical localities and coexistence of various species of the anostracans in Iran, and thereby contributed to the knowledge of large branchiopods fauna in Iran, however still a great part of this country remains unexplored.

References

Abatzopoulos TJ, N Agh, G Van Stappen, S M Razavi Rouhani, P Sorgeloos. 2006. Artemia sites in Iran. J. Mar. Biol. Ass. U.K. 86: 299-307

Agh N, F Noori. 1997. Introduction of a parthenogenetic population of Artemia from lagoons around Urmia Lake and its morphological comparison with Artemia urmiana. In: Proceedings of the First Iranian Congress of Zoology, University of Teacher Education, 17-18 September. 1997 (ed. T. Moellem), Tehran.

Alonso M. 1985. A survey of the Spanish Euphyllopoda. Miscellania Zoologica (Barcelona) 9: 179-208.

Brehm V. 1954. Filopodos de Persia recolectados por el Dr. K. Lindberg. Publ. Inst. Biol. Apl. 16: 121-125.

Brendonck L, DC Rogers, J Olsen, S Weeks, WR Hoeh. 2008. Global Diversity of large branchiopods (Crustacea: Branchiopoda) in freshwater. Hydrobiologia 595:167­176.

Brendonck L. & Williams W.D., 2000. Biodiversity in wetlands of dry regions (drylands). In: Biodiversity in wetlands: assessment, function and conservation. Gopal B., Junk W.J. & Davis J.A. (Eds.). Backhuys Publishers, Leiden, pp. 181­194.

Colburn EA. 1997. Certified: a citizen's step-by-step guide to protecting vernal pools. in R Burkholder, F Clark, E Colburn, C Feral B. Windmiller, eds. Massachusetts Audubon Society. 7th Edition. Lincoln, MA. 110 pp.

Daday DE. 1910. Monographie systematique des Phyllopodes Anostraces. Annls. Sci.nat. Zool. Paris 11: 91-489.

Eriksen CH, D Belk. 1999. Fairy shrimps of California's puddles, pools, and playas. Eureka, CA: Mad River Press.

Gunther RT. 1899. Contributions to the geography of Lake Urmia and its neighbourhood. Geogr. J. 14: 504-523.

Morin PJ, SP Lawler, EA Johnson. 1988. Competition between aquatic insects and vertebrates: interaction strength and higher order interactions. Ecology 69: 1401­1409.

Mura G, G. Azari Tatakami. 2000. A contribution to the knowledge of the anostracan fauna of Iran. Hydrobiologia, 441: 117-121

Nilsson AN, BW Svensson. 1994. Dytiscid predators and culicid prey in two boreal snowmelt pools differing intemperature and duration, Ann. Zoo. Fennici 31:

365-376.

Thiery A. L Puente. 2002. Crustacean assemblage and environmental characteristic of a man-made solar salt work in southern France, with emphasis on anostracan (Branchiopoda) population. Hydrobiologia 486: 191-200.

Wetzel RG. 2001. Limnology: lake and river ecosystems, edition. San Francisco: Academic Press. Pages: 1006.

Williams DD. 1983. The natural history of a nearctic temporary pond in Ontario with remarks on continental variation in such habitats. Int. Rev. of Hydrobiol. 68:

239-254.

The early ontogeny and embryology of a clownfish (Amphiprion ocellaris) larvae: The base study in Iran

Mahmood Azimirad*1, Naser Agh1, Rohollah Amininia2, Morteza Eijodpey2

1 Department of Fisheries, Faculty of Natural Resources, University of Urmia, Urmia, Iran

2 Center of reproduction of Clownfishes, Tehran, Iran

Abstract

The present study describes the embryonic development and early ontogeny of Amphiprion ocellaris from fertilization to post hatching. Clownfish spawned at 30°C. The newly laid eggs were orange in color and elliptical in shape (1.8x0.8 mm). We documented developmental times at 30°C to egg activation (0.5 h), cleavage (4 h), blastula (11.5 h), gastrula (20 h), neurula (24.5 h), somite (28.5 h), turnover (72 h), blood formation (113 h) and internal ear and jaw formation (144 h). Hatching occurred 168 h after fertilization. On day 3, the eye buds were pigmented and melanophores formed on the ventral surface of the embryo. Internal ear and gill formation were completed on day 5 and coincided with movement of the opercula and pectoral fins. The mouth formed on day 7 and the digestive tract appeared on day 7. By day 8, the yolk was fully absorbed and a substantial amount of food was observed in the gut. Dark and orange pigments were dispersed and aggregated through muscle contractions by days 9-14, but red pigments did not appear until the fish were three months old. This study may help improve the culture of coral reef fishes.

Keywords: Embryo; Ontogeny; Clownfish; Amphiprion ocellaris, Larvae

Among all species of clownfish, Amphiprion ocellaris is the best known to aquarium traders due to its color pattern, interesting behavior and robustness. From 1997 to 2002, A. ocellaris was the most common species of marine ornamental fish and made up 15.6% of total number exported worldwide and over 25% into European countries (Wabnitz et al., 2003). Thus, clownfish are considered the 'goldfish' of marine aquaria (Hoff, 1996). Most clownfish species are symbiotic with tropical sea anemones (Dunn, 1981; Fautin, 1991; Fautin & Allen, 1997). Both clownfish and anemone benefit each other through this mutual symbiotic relationship (Porat & Chadwick-Furman, 2004). All clownfish are protandric hermaphrodite, starting life as a male and later changing to a female (Allen, 1975; Fautin, 1991; Fautin & Allen, 1997). Most clownfish species form long-term monogamous pairs. The male-female bond only breaks when one member of the pair is lost (Hattori, 1994; Buston, 2003). In most cases, the female is larger making it more vulnerable to predation. In an clownfish population, the conversion of a functional male into a female, or of an immature male into a mature male typically takes several months to a year (Hoff, 1996). Clownfish are benthic spawners (Arvedlund et al., 2000a). Their eggs are highly pigmented and contain carotenoids in the yolk sac resulting in eggs that appear whitish-orange to purple in color (Kunz, 2004). After spawning, both parents usually attend the nest until larvae hatch. The fanning behavior of the parent fish brings oxygen to the nest and removes dead eggs (Green & McCormick, 2004).

There have been extensive studies of clownfish taxonomy (Allen, 1975, 1991), their living habits associated with behavioral ecology (e.g.

Arvedlund et al., 1999; Elliott & Mariscal, 2001; Chadwick &

Arvedlund, 2005), reproductive biology (e.g. Godwin et al., 2003; Hobbs et al., 2004; Holbrook & Schmitt, 2005) and rearing methodology (e.g. Arvedlund et al., 2000c; Johnston et al., 2003). However, little attention has been paid to the embryology and early ontogeny (Green & McCormick, 2004; Yasir & Qin, 2007), except for the early development

of the olfactory system (Arvedlund et al., 2000b) and one recent preliminary study of the embryology of A. ocellaris (Liew et al., 2006). Further studies of the embryology of clownfish would improve our broader understanding of the embryology of tropical reef fish. Such studies may also improve rearing methods of coral reef fish in general. The objectives of the present study were to examine the embryonic development and ontogeny during early life from fertilization to post hatching. Descriptions were based on observations of different developmental stages using light microscopy on fertilized eggs of clownfish reared in captivity.

Materials and methods

Brood fish preparation

Six wild caught Amphiprion ocellaris from southeastern Asia waters with a mean total length of 3.5 cm were obtained from an aquarium shop in Tehran, North Iran. These fish were held communally in a 180-l glass tank with three terracotta pots on the bottom. After co-habitation for three month, spontaneous pairing occurred. Each paired couple swam together and shared one of the terracotta pots. Aggressive behavior was displayed toward other fish that came close to their pot. As soon as the pair was established, each fish was transferred into an individual glass aquarium (96-l) without substrates. One terracotta pot and an air-stone were placed inside each aquarium. The three spawning aquaria were connected to a recirculation system filtered by sand. Three submerged 300-watt heaters maintained the water temperature at 30±1°C. Salinity was maintained at 28±2 ppt by replacing evaporative loss with demineralized water. The fish were fed a mixture of flesh, mussel, squid and spirulina tablets. Frozen food was thawed at room temperature and cut into an edible size before use. The fish were fed ad libitum three times a day. Fertilized eggs used for this study were produced by these three pairs of fish. Brood fish held under the described conditions produced 100-1000 eggs every 14 days.

Observations of the embryology

Eggs produced by the three brood pairs were collected for observations. Eggs were observed from fertilization until eight days post hatching. As soon as the eggs were fertilized, 10-20 eggs were collected with a plastic pipette from the terracotta pot and placed in a glass jar containing filtered seawater from the brood fish tank. The embryology was studied in a glass Petridish under a dissecting microscope connected to a digital camera (Canon 2004). This system was located in a room with temperature of 30°C. The newly hatched larvae were transferred to a 4-l glass aquarium containing live foods (Nannochloropsis sp. and Brachionus plicatilis). Compressed air was injected into the aquarium to produce light aeration. Images of embryonic development were made at 10 min intervals in the first 3 h, at 15 min intervals on the second day after fertilization and at 30 min intervals until the eggs hatched. The embryonic development time was described as hours post fertilization. During the post-larval stage, development events were studied daily up to eight days after hatching. The embryonic stages were defined based on the same terminology as previous studies (Green, 2004; Arezo et al., 2005).

Results

The eggs were individually attached to the inside surface of the terracotta pot. Each egg was firmly attached to the substrate by a fibrous stalk. This stalk was sticky when the egg was first released into the water, but became hardened after 5-10 min in the water. The major daily developmental events are summarized in Table 1.

Oocytes

The newly laid egg was orange in color, elliptical in shape (1.8x0.8 mm), and slightly curved around the middle part of the yolk. The chorion was transparent and the egg could be seen through the shell. The yolk was bright orange with an obvious constriction between the animal and vegetal poles, while the oil globule was clear.

Table 1. Major stages of development in embryo of Amphiprion ocellaris post fertilization (in days).

Days Major stages of development

Blastula and gastrula stages are complete. The embryo develops from a two-layer

1 blastomere to a sphere shape. The expansion of blastoderm covers 50% of the yolk

_sac_

Neurula stage: cephalization starts with a beak-like structure at the anterior part of the

2 embryo. The brain is formed at the end of the embryonic body close to the stalk. The

2 first two somites appear in the middle part of the embryonic shield. The melanophore _is covering the entire blastophore_

Body turns over and eye buds appear. Two otoliths inside the otic plate have formed as small granules on each side of the head. The embryo completes the turn-over stage

3 and tail has moved freely. The embryo size is about the same length as the eggshell with 26-30 somites. No blood cell is observed. Two lines of melanophores form at

_the ventral side of the embryonic body. Eye buds are pigmented._

4_The pale pigmented blood cells flow through the heart._

Internal ear and lower jaw form. The blood color deepens. The gills and the opercula

5 form and the body cavity is seen. The posterior otolith is slightly bigger than the anterior one. Four xanthophores appear on the ventral side of the head. The posterior

_part of the body detaches the yolksac. The opercula and pectoral fins start to move

Blood circulation is seen inside the gill. The lower and upper jaws form the mouth,

6 but the mouth was not open. The eye is fully pigmented with the lens popping out. _The posterior otolith is 3-4 times bigger that the anterior one._

The embryo hatches. The pericardium has fully developed. The digestive tract

7 appears as yellowish-silvery and the mouth frequently opens. Body cavity is enlarged

7 and becomes dark. Most melanophores at the surface of the yolksac migrate into the _body cavity_

8 The mouth frequently opens and the body movement intensifies. The yolksac shrinks _to an oil globule with <// of the eggshell length. The pericardium is fully formed.

As soon as the egg was attached to the substrate, a border between the animal pole and the vegetal pole developed. This was due to the accumulation of cytoplasm at the animal pole. Newly laid eggs had one large oil droplet and various small ones. The small oil droplets coalesced with the large ones during development. The perivitelline space was small yet transparent especially at the animal pole. The blastodisc was barely seen during this stage, but there was a large black mass at the vegetal pole surrounded by oil droplets. This black mass only appeared in

eggs within 24 h before ovulation. Eggs activation (0.5 h post fertilization)

A few minutes after spawning, water was flowing into the space between the cortex and chorion, starting from the animal pole to the vegetal pole. The incoming water suppressed the yolk and reached the vegetal pole to form the perivitelline space. This process took about 20 min to complete. The perivitelline space at the vegetal pole was larger than that at the animal pole. Immediately after the formation of the perivitelline space, the cytoplasmic area in the animal pole became thicker with an obvious blastodisc. The yolk sac in fertilized eggs shrank in size after the formation of the perivitelline space but the size of the whole egg did not change. In the activation stage, channels were formed in the cytoplasm that resulted from the spindle fibre activity during mitosis. One hour after spawning, a dome-shaped blastodisc formed, signalling the completion of the activation process.

Cleavage stage (4 h post fertilization)

The cytoplasm streamed down to the blastodisc, then retracted back, leaving a furrow in the middle. The groove moved up following the retraction of the cytoplasm and divided into two cells at 1.5 h after fertilization. The cytoplasmic divisions were meroblastic. The embryo was divided into 4, 8, 16, 32 and 64 cells with 0.5 h intervals between divisions. At the 64-cell stage, the blastomeres rearranged the cells into two layers, marking the completion of the cleavage stage. The cleavage stage was completed by 4 h post-fertilization.

Blastula stage (11.5 h post fertilization)

The embryo developed from a two-layer blastomere to a sphere where the blastodisc developed into a blastoderm. This stage was completed by 11.5 h post fertilization when the yolk syncytial layer and the dorsal lip formed.

Gastrula stage (20 h post fertilization)

When the blastoderm was flattened, the dorsal lip became obvious.

The envelope layer covered the deep cells and overhung the blastoderm. The expansion of blastoderm increased the coverage over the yolk sac. The yolk syncytial layer continued to expand along the embryonic shield and joined the yolk syncytial layer to form the germ ring. Prior to the completion of the gastrula stage, the embryonic shield lengthened and the germ ring disappeared. The periblast and the envelope layer gradually migrated toward the vegetal pole to form the epiboly. At 20 h post fertilization, the blastoderm covered 50% of the yolk sac.

Neurula stage and somite development (day 2)

At 24.5 h after fertilization, a beak-like structure appeared in the anterior part of the embryo (Figure 1A), marking the process of cephalization. The brain formed at the end of the embryonic body close to the stalk (Figure 1B). Meanwhile, the eye buds appeared at 24.5 h post fertilization. Under high magnification, the black mass was embedded in oil droplets of various sizes (Figure 1C). At 28.5 h post fertilization, the first two somites appeared in the middle part of the embryonic shield and the blastopore covered 70-80% of the yolk sac. Concurrently, the Kupffer's vesicle occurred at the ventro-posterior side of the embryo. The 4th-6th somites developed in about one hour (Figure 2A). When the blastopore covered 90% of the yolk sac (27 h after fertilization), chromatophores appeared as black dots spreading all over the blastopore (Figure 2B). The structure became more obvious after the blastopore completely covered the yolk sac (Figure 2C). During this stage, 10-12 somites were formed. Turnover stage (day 3) The head orientation could be identified by the eye buds (Figure 3A). Next to the big oil globule, a black mass of melanin started to dissolve and darken the area nearby. A few melanophores appeared and migrated across the embryo. While the tip of the tail was still attached to the yolk sac, the embryonic body started to turn over (Figure 3B). The optic plate (without otolith) and the nasal vesicle were clearly visible on the head. The pigment cells spread to the ear and the ventral side of the body (Figure 3C).

The tip of the tail was freed from the yolk sac when the embryo started jerking movements (Figure 3D). By the time the embryo was half

way through the turnover stage, tail movements were clearly observed and became intense over time (Figure 3E). The embryo completed the turnover process when the head was turned from facing the attachment end to the free end of the eggshell. Intense movement of the tail freed 1/3 of the body (Figure 3F). With further tail movement, the whole body moved forward and the rest of the tail separated from the yolk sac (Figure 3G). A pair of otoliths was observed in each ear as small granules of similar size. At the end of the turnover stage (Figure 3H), 26-30 somites were observed in the embryos.

Blood formation, internal ear and lower jaw formation (day 4,5,6)

Transparent and spherical blood cells were first observed entering the heart chamber 113 h after fertilization. The number of blood cells quickly increased and circulated within the body and in blood vessels on the surface of the yolk sac. At 96 h after fertilization, elliptical blood cells showed pinkish. When the eye buds were pigmented the embryo reached 1.25 times the size of the eggshell. Two lines of melanophores ran along the ventral side of the embryonic body. Four orange-yellow casts on the ventral side of the head next to each eye marked the first appearance of xanthophores. By the end of day 4, the peritoneal cavity became dark on the dorsal surface as a result of melanophore migration (Figure 4A).

The pink color of the blood cells was intensified by 103 h post fertilization. The length of the embryo was now 1.3 times the length of the eggshell and half of the body length was already separated from the yolk sac. On day 5 after fertilization, the embryo reached 1.5 times the eggshell length. The posterior otolith was slightly larger than the anterior one. The eyes with the retina surrounding the lens were highly pigmented while the choroid fissure was still obvious. On the 5th day, three lines of xanthophores were observed along the middle through to the posterior part of the body. Three to five xanthophores also appeared on the dorsal part of the body. The gill and the opercula were distinct with blood circulation in the gill and pectoral fins. The opercula and the pectoral fins started moving frequently.

Hatching (day 7)

On day 7, embryos were bent inside the eggshell and the tail reached the posterior of the eyes. The lower jaw formed when the embryo reached 1.7 times the eggshell length, but the mouth was not yet open. The digestive tract was distinct on the left side of the body. More iridophores created iridescent color on the digestive tract and the lining of the peritoneal cavity. The eyes were fully pigmented with iridophores scattered on the retina. A pair of otoliths became obvious with the posterior otolith 3-4 times larger than the anterior one. At 152 h after fertilization, body movement was intensified and the tail ruptured the area next to the base of the eggshell (Figure 4A). With a few jerking motions, the fish freed its whole body. The yolk sac was less than half of the eggshell length, with only a big oil droplet remaining (Figure 4B).

On day 7, the digestive tract was yellowish-silvery and the mouth opened frequently (Figure 4C). In some cases where the embryo failed to complete the turnover stage, the hatching process was delayed. Melanophores concentrated on the dorsal side and xanthophores concentrated on the lateral side of the body. The well-developed sensory and olfactory systems enabled the larva to detect and ingest exogenous food on the first day after hatching.

Post hatching (days 8-14)

On day 8, the yolk size had reduced to only a half of its original size. The dorsal fin fold at hatching connected the caudal and anal fin folds (Figure 5A). On day 9, the larvae still contained some yolk and one large oil droplet (Figure 5B). The fin rays under the urostyle became obvious while the dorsal and the anal fin rays started to develop on the fin folds (Figure 5C). The yolk was fully absorbed by day 10 after hatching but the oil droplet still remained. The dorsal and anal fins formed with obvious fin folds. The urostyle developed more rays and started to bend. The digestive tract became green with a substantial amount of food in it. Yellow-orange pigments were spread all over the body (Figure 5D) and the pelvic fin buds became obvious. On day 11, the urostyle was clearly bent, and the pelvic fins formed (Figure 5E). On day 12, the fin fold gradually disappeared and

the anal and dorsal fins separated (Figure 5F). On day 13, pelvic fin rays were clearly observed and melanophores formed within the fin folds. The fish body looked paler when the melanophores retracted (Figure 5G) or darker when they dispersed (Figure 5H). The pigment cells were scattered all over the body except on the fins and the body parts close to the fin base (Figure 5I). On day 14, both dark (melanophores) and orange (xanthophores) pigments were seen to disperse and aggregate through muscle contractions. By three months after hatching, red pigment cells (erythrophores) were seen on the caudal fin.

Discussion

Mated pairs of Amphiprion ocellaris spontaneously spawned in captivity at 30°C. The eggs were elliptical and possessed a black mass at the vegetal pole, which is rarely seen in eggs of other fish species. This black mass is believed to have a close association with the formation of melanophores. The pattern of embryonic development of this species is similar to other clownfish, but the hatching time is shorter than other species in this genus. During embryonic development, the head turned from the attachment end to the free end upon completing turnover. This seems to be a critical stage for survival since failure to turn the body around would lead to death.

In this study, A. ocellaris hatched in slightly over 7 days (168 h) after fertilization, but Liew et al. (2006) reported that this species did not hatch until 7.5 days (180 h) under 27-28°C. In comparison, Green (2004) found that A. melanopus did not hatch until 7.5 days. Cell cleavage in A. ocellaris started at 1.20 h (Liew et al., 2006), but we did not observe cleavage until 4 h after fertilization. In the first 2 days, A. ocellaris embryos completed blastula and gastrula. This developmental rate was similar to A. melanopus (Green, 2004).

Body movement is a critical part of development between major stages of organogenesis (Patterson & Martin- Robichaud, 1983). Body turnover of A. ocellaris embryos occurred on day 3 after fertilization. Similarly, tail movement of A. melanopus occurred by day 3 (Green,

2004). Tail-flicking could increase the oxygen circulation within the embryonic capsule especially for benthic eggs (Cronin & Seymour, 2000). Similar to A. melanopus (Green, 2004), A. ocellaris developed a rudimentary heart on day 3 and haemoglobin on day 4. Amphiprion ocellaris formed gills and opercula on day 5, but gills did not occur until day 6 in A. melanopus (Green, 2004). The development rates of the eye and the digestive system in A. ocellaris were similar to those reported by Green (2004), Liew et al (2006) and Yasir and Qin (2007).

In this study, immediately after fertilization, the perivitelline space in clownfish eggs started to develop but the space at the vegetal pole was larger than that at the animal pole. Unlike demersal eggs in Lepistoseus osseus (Long & Ballard, 2001), where the perivitelline space was formed by the expansion of the chorion, the egg yolk of A. ocellaris actually shrank in size without any change in the egg size during perivitelline formation.

The black mass in the embryo has not been reported by other authors who studied the early life of clownfish (Allen, 1991; Hoff, 1996; Green, 2004; Liew et al., 2006). We found that this black mass on the yolk surface was surrounded by numerous small oil droplets. Hoff (1996) described the A. ocellaris egg as a 'clear yellow-orange with a small white dot on the tip of the egg'. This 'small white dot on the egg' may be the black pigment as we observed in this study because the dark pigment could be seen as a white dot under a dissecting microscope with the light source from the top. This black mass in A. ocellaris seems to be the melanin mass, but this has not been reported in other fish eggs (Ahlstrom & Moser, 1980; Matarese & Sandknop, 1984; Kunz, 2004).

Once ovulated, fish eggs quickly absorb water. Other materials including vitamins and metals required for enzyme activity in the egg should be obtained from the female (Brooks et al., 1997). Melanin synthesis is carried out by the tyrosinase family protein and is auto-regulated and activated by its own substrate and cofactor tyrosine (Stearns & Wang, 1987). The process of melanin synthesis usually occurs inside melanophores or melanosomes in the dermis, or in melano-macrophage cells situated in the spleen, kidney and liver (Agius & Roberts, 2003). Therefore, the appearance of melanin inside the eggs of

clownfish is unusual. This mass of melanin was only observed within 24 h before spawning, suggesting that the steroid that triggers oocyte maturation and ovulation (i.e. maturation inducing hormone) may also trigger the chain reaction of the melanin precursors in the egg yolk.

According to Ahlstrom & Moser (1980), the melanophores usually first appear along the dorso-lateral surfaces of the embryo and on the yolk sac. Melanophores in teleost fish arise from the neural crest, apart from the cell layer adjoining the retina. During neurula and somite stages, the melanoblasts in other species begin to migrate from the neural crest through the dermis and form the melanophores (Kunz, 2004). When the first melanophores reach the dorsal part of the embryonic head, the stellate melanophores become prominent and start to produce melanin (Bagnara & Hadley, 1973). The present research showed that during the early blastula stage, some black mass dispersed into the yolk. During the late gastrula stage, however, the boundary of the distinctive black mass in the yolk sac gradually dissolved into the yolk. The dissolving process continued to the somite stage and then became stagnant as the melanophores appeared all over the yolk sac. The dermal melanophores on the embryo had a typical stellate form with dark color, while melanophores on the ventral side of the yolk sac were large brownish and round. During migration, most of the melanoblasts would not synthesize melanin until they arrived at their final sites. This suggests that the melanin mass in the yolk of clownfish eggs might contribute to the early formation of the melanophores (Yasir and Qin, 2007)

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