Structure of cherry barb Puntius titteya (Deraniyagala 1929) egg membrane

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STRUCTURE OF CHERRY BARB PUNTIUS TITTEYA (DERANIYAGALA 1929) EGG MEMBRANE

Adam Taсski

Agata Korzelecka-Orkisz

Michaі Ogniewski

Joanna Szulc

Andrzej Sobociсski

Krzysztof Formicki

Department of Fish Anatomy and Embryology,

The Westpomeranian University

of Technology in Szczecin, Poland

ABSTRACT

The Membrane ооcyte, but then and яйцевые of the shell during incubation Puntius titteya sharply change. Oociti extracted directly from gonad possess well developed radial shell (zona radiata), which(who) then, being up daun in water quickly changes its structure and as a result набухания grows its thickness. In the work are brought results of the observations and analysis(test) this process.

Key words: cherry barb, egg membrane, oocyte

INTRODUCTION

Egg cells of oviparous animals are in an immediate threat of environmental stressors. In the course of the evolution the egg had developed egg membranes responsible for protecting the egg. Egg membranes had occurred in the phylogenic development quite early and now only few animals do not have them e.g. sponges, some coelenterata, however it is reasonable in case of these animals since they live in the marine environment which assures stable conditions. Egg membranes have various shapes and structure according to the type of environment given organism inhabits. They are made up by different parts of actual gametes or their parts or using parts of the reproductive system. It is rear that elements of the egg membrane are synthesized outside of the reproductive system, for example in fish some elements are synthesized in the liver. In fish egg membrane is called zona radiata or zona pellucida. Egg membrane is made in the ovary, however depending on the species other parts of the reproductive system can be engaged in the egg membrane synthesis as well -for example oocyte, follicular cell and less often cells of the ovary (Roњciszewska 1994).

Fish egg membranes consist of two major layers: external which is thinner - zona radiata and internal which is thicker called zona radiata interna (Jura, Klag, 2005). Egg membrane includes numerous channels, which facilitate an exchange of substances between the developing embryo and the external environment Henneguy (1889).

Current classification of the egg membranes is: primary including -zona radiata and secondary - including amorphic structures created in the oviducts. Hence, the correct name for these structures should be a sheath and not egg shell.

Egg shells fulfill many functions but their basic function is the embryo protection. They protect the embryo from the harmful environmental stressors such as physical (mechanical), chemical (poisons - egg shell functions here as a type of a molecular sieve that catches harmful substances) (Margaritis 1985). They also protect the embryo from the microbes. It is documented that the egg membranes in the teleost fishes have an active peroxydase, which enables the membrane to defend the embryo against bacteria. Egg membranes allow for the removal of the metabolic waste products of the developing embryo, for keeping ionic balance and for a gas exchange. They secure the egg from the water loss and can absorb more of it when necessary. Eggs of many aquatic animals including fish, are able to float freely at a particular depth, some of them are equipped with structures that allow them to attach to underwater features including the bottom (Riehl and Kokoscha 1993).

Puntius titteya is a small fish that measures up to 5 cm. It belongs to cyprinid family. Its origin is thought to be in Sri Lanka, where it inhabits calm shaded waters. Both sexes are easily distinguishable due to well expressed sexual dimorphism. The males are red with a gentle dark streak situated horizontally on a side of the fish. Females are paler than the males and have an additional lighter streak on the side. Their belly is white. These fish are omnivorous and are quite easy going in their behavior.

MOTIVATION OF THE STUDY

The works that have been already published and discussed the reproduction and embryology of fish, the gametogenesis etc. have discussed the topic of the egg membranes in a very general fashion. There has not yet been a study addressing the reproductive aspects of Puntius titteya so far. Hence our motivation to take a closer look at the membrane structure of this fishes eggs.

MATERIALS AND METHODS

We used eggs of Puntius titteya that we kept in an aquarium culture. We used fertilized and unfertilized eggs.

a. Unfertilized eggs:

· Non-activated, sampled by a pipette directly from the fish ovaries.

· Activated, sampled directly immediately post laying in the aquarium.

b. Fertilized eggs:

· Sampled after 0.5, 1 and 2 hours after fertilization.

Light microscopy - sample preparation and staining

Sampled eggs were fixed in 4% formaldehyde (HCOOH) for 5 days. Eggs were later dehydrated using a range of alcohol solutions of increasing concentration, beginning with 50% ethanol, and ending with 96% ethanol. Because of the small size of eggs rinses were rather short. Samples prepared this way were immersed in paraffine wax and sliced thinly using a rotating microtome HM 310. Cut slices were rid off paraffin wax and stained using hematoxylin and eosin and encapsulated in Canada balm. Preparations were analyzed histologically. They were studied using light microscopy (Nikon Eclipse TE 2000-S) using following magnifications: 2Ч, 10Ч, 20Ч, 40Ч.

Scanning microscopy SEM

SEM analysis used eggs in the same development stages as those used in light microscopy. Samples were dehydrated on increasingly concentrated alcohol and later in acetone. Dried eggs were mounted onto the base and saturated with a thin layer of silver and copper alloy. Prepared samples were studied using scanning electron microscope (FEI Quanta 200), images of eggs were saved as a visual documentation.

RESULTS

The analysis of the gonads showed that despite the ripeness of fish and their readiness for egg laying, the oocytes were at different stages of development. Most of

Fig. 1. Oocyte sampled directly from the gonad

the oocytes however were identified as the last stage of development (Fig. 1). Egg membranes were not spherical and smooth on their surface but rather folded. At the surface of the egg one can observe a sticky layer, which is thought to appear as a result of the cortical reaction and water absorption by an egg. This layer can be observed on images included in this publication. This layer enables the egg to stick to underwater flora as well as eggs to each other.

The use of the scanning microscopy enabled us to investigate precisely some of the egg structure which otherwise could not be possible when using only light microscopy. The oocytes of Puntius titteya which have been sampled directly from the gonad were significantly more folded (Fig. 2).

The external surface of the egg membrane soon post the activation (when the cortical reaction sets on) becomes more regular and less folded, the internal side of the membrane has a more regular structure with few small irregularities and dents.

After half an hour from the activation, the thickness of the membrane was between 2.0 and 3.0 µm. There has not been a clearly defined border between external and internal layer of the egg membrane. The internal part had numerous openings of variable shape and size distributed unevenly.

On the external site a well defined, regular structure could be observed. Because of the irregular egg membrane structure and its roughness eggs attachment to the substrate, onto which eggs are laid, is easier (Fig. 3).

In an hour since eggs were laid the structure of the egg membrane was quite changed, as a result of water saturation and reached the turgor. External surface of an egg became quite smooth.

Thickness of the whole egg membrane was not quite 2 µm (Fig. 4), however the difference between the two layers was clear. The internal layer was thicker. The surface of the internal layer was slightly folded and covered by numerous pores (Fig. 5).

DISCUSSION

The results of this study show the changes which the egg of Puntius titteya undergoes. Data from the histological analysis as well as the images from SEM portrait the exceptionally dynamic structural changes in the egg membrane of this fish. Based on our observations we can conclude that such a structure can guarantee a full function during fertilization as well as during embryogenesis as a protection. It is worthwhile to pay attention to the functions of the primary membrane such as: structural and physical adaptation to the external environment conditions (e.g. seasonal thermal conditions at the place where eggs are laid).

The substrate and the conditions due to the type of the substrate onto which the eggs are laid has a significant influence on the variable egg membrane structure (Winnicki and Bartel, 1967), and it has influence on the functions of these egg membrane structures (Gray 1928, Hayes 1949, Zotin 1961, Winnicki 1968). Elements of the structure demand several solutions on the micro and more over on ultrastructure level.

The development of the attachment structures on the surface of the egg membranes has also the function of storage e.g. gelatinous tape of european perch eggs does not only function as a attaching and structural form but also functions as water storage (Korzelecka et al., 1999).

Eggs of cherry barb similarly to the eggs of other cyprinids are surrounded by a thin casing, which secures the embryo from potentially harmful environmental stressors (Esmaeili and Johal 2005). Study of the egg membrane structure of silver carp (Hypophthalmichthys molitrix) conducted by Esmeaili and Johal show that the surface of the egg membrane is folded with regularly distributed round openings, which is similar to the cherry barb. The difference between eggs of these two fish species lays in the presence of the attaching organs in eggs of the cherry barb and the eggs of the silver carp. Those structures are present in the egg membranes of the micropylar region of the eggs of the silver carp and in the cherry barb these were not observed. Similar conclusions addressing the attachment structures were made by Guimaraes and Quagio-Grassiotto (2001) who studied egg membranes of Serrasalamus spilopleura from Characidae family of fish, who observed attachment forms on eggs of these fish. Dumas et al. (2004) who studied histology of Lutjanus peru (family: Lutjanidae, order: Perciformes) concluded that the oocytes of the last development stage present in the gonads of these fish had a well developed yet thin egg membrane and well developed yolk sac, which is similar to our study of the oocytes sampled directly from the gonads of the cherry barb.

In the discussion of the structure and function of the egg membrane it is important to mention the secondary egg membrane which is the sheath. One of its functions is the antibacterial. This function is important and it would be difficult to imagine any possibility of the embryo development in the aquatic environment highly enriched in various sorts of pathogenic microbes.

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