Embryogenesis of cherry barb Puntius titteya

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EMBRYOGENESIS OF CHERRY BARB PUNTIUS TITTEYA

Agata Korzelecka-Orkisz

Adam Taсski

Magdalena Kaczmarek

Adam Brysiewicz

Andrzej Sobociсski

Krzysztof Formicki

Department of Fish Anatomy and Embryology,

The Westpomeranian University

of Technology in Szczecin, Poland

ABSTRACT

The study of morfogenezis and tmbrions function Puntius titteya Deraniyagala 1929 has shown that small sizes(amounts) (v = 0,97mm3) egg(ovum) covers the sticky shell fastenning egg to itself mutually, as well as to(towards) plants, amongst which(who) most often proceeds the incubation, who at the temperature 26 C longs ebaut 550 H . Typical eggs of fish is a side location disk and division of the yolk on two parts.

Key words: cherry barb, embryogenesis, egg, embryonic morphometry

INTRODUCTION

Cherry barb (Puntius titteya) is one of the most beautiful ornamental fish people like to keep in their aquaria. It belongs to the cyprinid family.

The origin of this endemic fish is the waters of Sri Lanka in south Asia. It inhabits benthic dark areas of the sandy bottoms in the streams (Sundarabarathy et al. 2004).

Cherry barb is a species recorded in the World Red Book of Endangered Species as highly threatened species (IUCN 2000).

The possible cause of their population decline is their commercial overfishing and the contamination of their natural environment (Pethiyagoda 1994, 1999; Sundarabarathy et al. 2004).

Males which are slightly darker than females have red body. In the mating period of their life the red color of males becomes even more intensive. Females are on the other hand grey-brown and along their body length they have a dark streak. Males also have this streak and even more pronounced than in females (Jakubowski and Ring 1983). The fins of the male are pink-organge and in females they are orange-grey. Their sexual dimorphism is visible. These fish grow up to 5cm in length (Kornobis 1989). Cherry barb is an active and quite friendly fish. It spends most of its time near the bottom or in the middle of the water column. Barb cherries swim in schools.

They are oviparous and mature after first 4-6 months of their life. They attempt the spawning multiple times during the year. Culturing of the cherry barbs is quite easy if one can provide an adequate aeration, proper temperature, lighting and the right bottom substrate. Their spawning act lasts several hours. Sticky eggs are laid onto the aquatic plants. In order to protect the eggs from the cannibalism by their parents, one can separate the bottom with the eggs from the adults by setting up a protective net. Hatching occurs just after 24-36 hours from the fertilization. From one hatching one can receive up to 250 larvae (Mariani 2005). As mentioned before to culture these fish from the time of their hatching till their maturity is not difficult (Jakubowski and Ring 1983).

The literature discussing the embryogenesis including early developmental stages, which are key to understand the reproductive biology and ecology of this fish species, motivated us to undertake this study. The duration of embryogenesis and the morphogenesis during that time was the main goal of this study. We hoped to be able to study the embryology of the cherry barb in the wider context of its biology. We wanted to underline the differences and show morpho-mechanical and physiological trends that occur as well as chemical and physical parameters of the environment in which these fish reproduce.

MATERIALS AND METHODS

The study was conducted from December of 2007 till April of 2008 in the labs of the Embryology and Anatomy of Fish at the Agricultural University of Szczecin. We used eggs of fish that have been cultured in our lab.

One week prior to planned experiments the fish were separated according to their sex. Barbs were fed with dry flake fish food (Tropical, Ichtiovit) twice a day. After the transfer of the fish to the spawning tanks they were fed with frozen food. They were fed with the larvae of chironomid insects, in order to speed the ripening of the fish. We equipped the bottoms of the spawning tanks with two bunches of Java moss (Vesicularia dubyana) with few small rocks on top of the moss. The moss was used as a substrate for laid eggs. The bottom of the tank was surrounded by the black plastic foil to reduce the stress. Tanks prepared that way were filled with water half way, which had the pH of 7.4, water hardness (carbonate hardness aka non-permanent) of TwW = 11; permanent water hardness TwN =3; general hardness

TwO = TwW + TwN = 14. Water temperature was 26°C.

The observation of the reproduction was made by using the setup that had consisted of digital camera (SONY) connected to the microscope joined with video camera, monitor and VCR (JVS). Images recorded on the VHS tape were analyzed using Multiscan v.13.01. Size of eggs, egg cells and larvae were measured. The parameters estimated based on the previous measurements included volume (V), surface area (S), volume to surface area factor (S/V) of eggs and egg cells.

Other measurements included:

§ The number of contractions during a minute: in the heart buds, fully formed heart as well as in heart of freshly hatched larvae.

§ Number of somatic motions of the embryos during one minute

§ Total length of larvae and the yolk sac used later for the estimation of the yolk sac volume (V = РЧl ЧhІ)

RESULTS

Characterization of the fish eggs

Eggs size of eggs from one female is not variable. The diameter of eggs ranges from 1.19 to 1.24 mm (1.23±0.04mm; ±SD) which results in a volume of 0.88-0.98 mm3 (0.97 ± 0.09). The egg cells within the eggs measure 0.86-0.92 mm (0.91 ± 0.05 mm), which translates into the volume of 0.34 do 0.40 mm3 (0.40 ± 0.06). Egg cells take up ~40% of the egg volume and the rest of the volume (60%) is constituted by perivitelline space, which creates proper respiratory conditions and allows for easy movement of the embryo. S/V factor for these eggs is 4.91 and for the egg cells 6.5 (Table 1).

Table 1. Measurements of eggs and egg cells of cherry barb (Puntius titteya)

(±SD)

Course of the embryogenesis

Embryonic development of cherry barb lasts 19 hours in the water of 26°C, which translates into thermal units of 495.3 H°.

Just after 15 minutes after the fertilization one can observe presence of the fertilization cone on the animal pole (Fig. 1), where the blastodisc originates, which undergoes multiple divisions. First divisional groove along the blastodisc appears after 45 minutes (19.5 H°) since the fertilization. There are 2 blastomers connected with yolk above it. After following 15 minutes (26.3 H°) one can observe the next groove and along with it four blastomers. At 34.7H° (80 minutes) there is the next division and hence eight-celled embryo of cherry barb. After 90 minutes (39 H°) thick-celled morula stage is present and 10 minutes later small-celled morula appears. In the following development morula becomes into blastula and it starts overgrowing - the epiboly. At 65 H° the eggs are at the half epiboly. The gastrulation ends with the closure of the blastopore at 87.1 H°.

Fig. 1. Fertilized egg and appearing fertilization cone (fc) on the animal pole, yolk sphere (ys), periviltelline space (ps), egg shell (es)

After 6 hours since the fertilization the shape of an embryo can be seen and the head and tail parts are distinguishable. In the abdominal part segmentation can be observed, first 6 myomers are created just after 169 H° (6.5 h).

After 8 hours (208.4 H°) the embryo of cherry barb has already an outline of the eye cavities and there are already ~12 myomers. During further development there is narrowness of the yolk sac, which divides it into two passages: proximal - in front and caudal - in the back.

Embryonic motorics

When the tail end is well established and the yolk sac is well divided the embryo starts frequent and intensive active movements. First somatic movements can be observed just after 10 hours (262.2 H°). Later on the activity declines (Fig. 2).

Fig. 2. Somatic movements of the cherry barb embryo incubated at 26°C

At 377 H° (14.5 h) the heart starts a slow activity with 35 contractions per minute. The blood vessels start being visible. With time the heart action becomes faster and more rhythmic. Before hatching it reaches 72 contractions per minute 72 (Fig. 3). Towards the end of the embryogenesis, just prior hatching a specific type of movement is observed. It is called “the embryo tremble”. It is a series of short movements that repeat up to 5 times per minute, when fully developed embryo becomes ready to abandon the environment of the egg.

Fig. 3. Heart activity of the cherry barb embryo developing at 26°C

Hatching

Just after 19 hours (495.3 H°) from the time of fertilization larvae hatch. Frequent and intensive movements of the embryo as well as the proteolitic enzyme activity that dissolves the egg shell facilitate the stretching and ripping of the egg shell. First comes out the tail and after few more minutes the whole larva escapes from the egg. Freshly hatched larva of average total body length 2.91±0.15 mm (±SD) has yolk sac of the volume 0.89±0.3 mm

(±SD) (Fig. 4).

Fig. 4. Larva of cherry barb (Puntius titteya) just post hatching

Overall an individual of cherry barb laid 150 eggs. Embryonic mortality during the whole period of the embryogenesis was 60% at 26°C.

DISCUSSION

Results of the study concerning the cherry barb fertility are close to those already published where authors inform about 150 to 250 eggs laid by an individual (Mariani 2005). Our experiments yielded ~150 eggs. However in comparison to other ornamental fish cherry barb is not very fertile. Tiger barb (Puntius tetrazona) can lay ~300 eggs (Smaruj et al. 2005), and females of gold barb (Barbus schuberti) can lay up to 2000 eggs (Korwin-Kossakowski and Kamiсski 2001). Roe of cherry barb as well as tiger barb (Smaruj et al. 2005), gold barb (Korwin-Kossakowski and Kamiсski 2001), and other cyprinid species is sticky (Korzelecka and Winnicki 1998, Bonisіawska et al. 1999). The egg membrane is covered by a sticky envelope which allows it to attach to other eggs or to the underwater flora (Szulc et al. 2006). Development of the roe takes place within in the surrounding of the aquatic plants some distance from the bottom, which is frequently covered by sediment, which could be less optimal for the egg whose surface is sticky, grains of the sediment could easily stick to the egg surface. Above all mentioned egg membranes are transparent and quite thin, yet they are able to endure the potentially harmful bacteria. The roe of cherry barb is small and the egg diameter is 1.23 ±0.04 mm. This could seem so natural not only because of these fish specifications but also due to the fact that the embryonic development takes place in quite high temperatures, which is known to speed the metabolic rate. Despite the small egg size ~60% of its value is taken up by the perivitelline space that is filled with fluid. Such a vast space surrounding the yolk like in the most of cyprinid fishes (Korzelecka-Orkisz et al. 2006) betters the oxygen exchange conditions of this quickly growing embryo. It also assures a lesser constraint for movements. Embryo movements assure a better mixing of the perivitelline fluid, which allows for a faster diffusion of oxygen across the respiratory surface (Korzelecka-Orkisz 1999).

During our study, we concluded that most of the yolk sacs of the cherry barbs hold a spherical or similar to the sphere form. It sure stays intact during the whole period of embryogenesis, unlike in some other fish (carp bream, belica, common rudd and others) the shape of the yolk sac undergoes significant changes during latter organogenesis (Winnicki et al. 2001). In case of cherry barb during the embryogenesis the yolk sac became divided into two parts: proximal (in front) of spherical shape and caudal (in the back) of a cylindrical shape.

It has been concluded that the walls of the yolk sac in the caudal part of it undertake rhythmical contractions which outpace the occurrence of the heart bud pulsation. Because of the presence of the yolk sac divided into two parts the embryo can bend under an angle of 90°, which allows it for an easier departure of the egg (Korzelecka-Orkisz et al. 2006; Smaruj et al. 2005; Winnicki et al. 2001). Frequent and intensive contractions along with the activity of the proteolitic enzymes cause stretching and ripping of the egg membranes. Cherry barb, similarly to other barbs or carp is hatching with its tail first. Later it leaves the remnants of the egg shell quite quickly.

The easiness of bringing the cherry barb to spawning, spectacular mating behavior, short incubation time, make this fish a quite desirable and interesting in the circle of the aquarists. The results of this work will be used in improving the aquaculture of this species, which will lead to increased protection of wild populations and prevent the commercial overfishing.

puntius titteya deraniyagala

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