An Identification Guide of Marine
 Fish Eggs from Taiwan


K. T. Shao, J. S. Yang, 
K. C. Chen, Y. S. Lee

Institude of Zoologe Academia Sinica

2001/12

  Know the Fish Eggs Holomorphology Egg Types Collection Identification Dignosis 

 Introduction
Almost all marine bony fishes are oviparous and a few of them are ovoviviparous and viviparous. The egg number of oviparous fishes have great differences from small numbers of hundreds, such as salmon has hundreds, to three hundred millions as in ocean sunfishes. Fishes spawn few eggs that are generally demersal eggs, which usually are attached or adhered under the gravel or in the crack of rocky reefs. Fishes those spawn many eggs are normally floating type and either drift in the ocean or coastal area by the waves. Most of the parent fishes that spawned demersal eggs have the nesting behaviors, so the identification of species can be done by the parental nursery behavior of the adult fishes. But most of the pelagic eggs found in the marine waters are too difficult to identify the species.
In the past, identification of fish eggs is by recording their continuous morphological changes from eggs hatch to larval and to juvenile fishes then to adult fishes. Some of the fish eggs are collected from the field of oceans, rivers and some of them are from the fish hatcheries. To identify these fish eggs, optical microscope is used most of the time. Fish egg classification reports are available since the beginning of 19th century. Syosi (1924) provided a checklist of 53 pelagic eggs along the coastal areas of Japan. Prof. Mito(1960a, 1960b, 1966)described 200+ fish egg forms and published the atlas and checklist of fish eggs collected from the Kyushu area of Japan from 1951 onwards. Also there were 349 types of fish egg forms described in Okiyama (1988). The book An Atlas of the Early Stage Fishes in Japan is the best tool for the egg forms and retrieve information nowadays. For the past years, Japanese scientists use hatching methods to explain over 20 types of marine fish egg forms (Suzuki et al, 1981, 1996; Suauki & Kobayashi, 1989; Kazunori et al, 1982; Tanaka, 1995, Shukei et al, 1990; Jettrey & Moyer, 1985). In mainland China, Institute of South China Sea, Chinese Academy of Sciences: Xiamen University and Yellow Sea Research Institute have added the species composition of fish eggs into their ocean ecological survey in South, East, and Yellow China Sea. Chao & Chen (1985) published Fish Eggs and Larvae in the Offshore Waters of China which includes 100 types of fish eggs. Chiang(1997)and Hu & Chiang(1991)have identified, 40 families, 26 genus, and 36 species at the central and northern parts of Taiwan Strait. However, the lack of information relating to this aspect still exists.
For twenty years, SEM is used to observe tiny structure found on the egg membrane surface and also in the micropyle region (Riehl, 1982). Later, many research reports are published relating to this topic (Hosokawa 1985, Mikodina 1987, Riehl & Koscha 1993, Hirai 1993, Britz et al 1995, Breining and Britz 2000). In Taiwan, Chen et al, (1999) used micropyle ultrastructure to classify four species of sparids, eggs and infer their phylogeny. Wu(2000)and Shih(2002)also applied this method to identify lizardfish and parrotfish eggs but add molecular techniques, and suggested that only DNA sequence could identify the species level. Shao et al (2002) discussed the advantages and defects of optical microscope, SEM and DNA sequences, and suggested that the identification using morphological characters can only identify the family or genus level. Therefore, simply using altas or diagnostic keys for identification may lead to mistakes, and this is because the characters of fish eggs are not specific enough to species identity. Many closely related species possess the same morphological characters, so when referring to the altas or the diagnostic key, some of them may key to the species, but the same characters may also belong to the characters of other species or even to whole family or genus. Even use additional observation of SEM for ultrastructures it cannot guarantee the identification to the species level. The only way to confirm the species is using DNA sequence alignment. 
Figure 1. shows the identification relations of these three methods. This reminds the readers, particularly to pay attention in advance for identification. That is when you check the form of fish eggs fitted in one of the species assigned in this book, you could only confirm it to the genus or family level, but not exactly belong to that particular species. On the other hand, the fish egg designated to that species in this book is certainly the morphology of that species. Furthermore, most of the identification in this book is according to the Alats of Okiyama (1998), Mito(1960a & 1960b) and Chao & Chang(1985)and so on. Some of the species are the first descriptions from our research in these years. It includes 22 species of fish eggs that are collected either from the known species from the fish hathery farm or from mouth-breeding cardinalfishes. The authors are responsible for allthe drawning and photos including the scanning electron microscope 
Shao et al (2002), for the first time used the mitochondrial DNA sequence of fish egg and sequence alignment of known adult fishes to identify the species. This method is also used to reconfirm the identification made in the past using fish egg atlas or diagnostic keys. Due to high cost of molecular biology facilities and the technical difficulties, it is not so easy to establish the complete molecular data of all adult fishes. However, the adult fish sequence is needed in advance to compare with the eggs. At the same time, if proceeding with the investigation of DNA sequences, one must classify the fish eggs first in order to collect adult fish sample of that particular group (taxa) to build up the species DNA sequences database. In brief, the characters observed by optical and SEM could possibly identify the order, family, and genus level: each level of identification has its own values. As a result, publication of this book has both values in terms of academic research and use.
Generally fish eggs size is small, so people wont pay much attention. In the past, fish eggs are considered as one of the zooplankton without further identification. Nevertheless, fish egg contains many important information of fish life history and fish ecology. So it plays an important role in environmental impact assessment, fishery stock analysis, fish propagation, seedling release, and fish farming. Besides, fish eggs themselves are important prey item in whole marine food web. Therefore, the identification of fish egg is as important as fish larvae.
Old Egyptians began eating fish eggs in the year of 2500. Initially fish eggs are added with salt to lengthen their editable time. Later on fish eggs become the desktop munificent objects. Many people describe the taste of fish eggs as butter flavor, fruit flavor, or special dividual seawater smell, and they even dissolved instantly when put into the mouth. Because of the different nature of fish eggs, soft, lubricate, fragrant, brittle and delicious, people love to eat it very much. Some fish eggs are even wrapped in oil membrane, which results in the delicious juicy nature. Because of high nutrition, fish eggs become routine necessity for human beings in the form of caviar, fish egg sushi, fish egg salad, mullet roe etc. Those large-sized fish eggs like salmon, trout, flying fish, etc, and many economically important fish eggs such as mullet, sailfish, cero, etc. All of these fish eggs are eaten by people in the unripe form before spawning.
However, when people eat fish eggs, why dont they think that kill chicken to get eggs will totally obstruct the primary stage of fishs life cycle. This leads to population and resource decline drastically. Especially using weaved straw mat to collect flying fish eggs or catching fishes during their spawning migration which will leads to severe depletion of the stock, e.g. massive catch of mullet or catch groupers or croakers in their spawning aggregation. Poisoning, electric fishing and blast fishing, also have destructive effect to egg laying or hatching fishes.
Taiwanese people love to eat fresh seafood very much, but the bad habit of eating rare species, fish eggs, and larval fishes should be changed. The restoration of larvae and fish eggs is one of the key factors to keep the fish resources sustainable. The conservation research must contain the long-term monitoring of the species composition and their spatial and temporal distribution of fish eggs in the coastal waters around Taiwan. All of the delimitations of Marine Protected Areas search for breeding and spawning grounds should establish the basic distribution data for fish eggs and larvae. We believe that the publication of this guidebook should be very useful for the identification of fish eggs in the future.


Fig. 1. The subordinate relationship among three different identification methods. This figure reveals that one type of egg morphology may correspond to several different species.


The size of fish egg varied according to different species Bonitos fish eggs Oil fishs egg mass

Mullets egg mass in processing Sushi of fish eggs Cesos fish eggs

Flying fish egg is on sale at fishery market.

Every spring-summer, fisherman make weave of raw mat for collecting flying fishs eggs in the coast waters of northeastern Taiwan.

The egg mass (right side of fish body) of cardinal fish will die with the parent fish after blasting.

 

1. Get to know the fish eggs
a. Structure of fish eggs
Most fish eggs are spherical in shape and are small in size. The shape and the size of the fish eggs depend on species. Marine fish eggs are smaller than freshwater fish eggs. The range is between 0.5 mm~ 4.5mm, mostly between 1~2 mm. The size of the fish eggs also differs in each mature stage.
The structure of fish eggs from external to internal is egg membrane, perivitelline space, vitelline membrane, and ovoplasm. Egg membrane divides into two parts, jelly layer and chorion. Chorion is separated into inner and outer layer. Perivitelline space usually indicates the fertilized nature of the egg, and also absorbs water and expands to protect eggs. Egg mass includes yolk and nucleus. Yolk contains great quantities of nutrients to provide nutrition during the fish egg embryo development stage and also for the yolk-sac larvae. Yolk substance contains oil globule, yolk bubble, and yolk.(Fig.2., Fig.3.)

Model of fish egg structure


b. Formation of fish eggs
Usually the formation of fish eggs accompanies with many ultrastructure changes. Oocyte surface has numerous microvilli. Egg membrane is formed on microvilli surface, especially on the pore or pit in mirovilli. Each pore contains one microvilli, and follicle cells breaks out between pore and pore assemblages to help oocyte development. When ovulating, pore will be blocked by secrete substances of follicle cells and become pore plug or knobs on egg membrane surface. In general, jelly layer and outer layer of egg membrane surface are composed of polyssacrodise. Inner layer and intestinal membranes are formed by protein.
c. Structure and function of egg membrane
Structure and thickness of egg membrane can respond to fish eggs adaptation to the ecological environment. Generally egg membrane of marine demersal eggs are thicker than pelagic eggs. Egg membrane can prevent fish eggs to change in shape. Egg membrane secretion can aggregate all fish eggs in order to protect eggs not to be affected by bacteria and virus. The function of pores or pits is for fish eggs nutrition and metabolite exclusion. Also, it can provide respiration. The liquids surrounding perivitelline space can provide the protection for the embryo
d. Micropyle
Generally, animal pole of the fish eggs surface has one opening called micropyle. It is developed from both oogonial central micropylar cell and granulose cells. Micropyle only allows a few sperms to enter inside to complete fertilization.
The diameter of micropyle just fit the size of its conspecific sperm head. They are speciesspecific. Thus, micropyle plays an important role either for isolating mechanism in sympatric speciation or to screen the sperm to avoid hybridization Most bony fishes have only one micropyle, but there are exceptions. For example, loach has three micropyles and sea wolfish has five. Micropyle usually has three different types: cylindrical canal such as for cod; conical canal such as for mullet; funnel like such as for sea bream. Most pelagic fish eggs are funnel type.

Fig.3. The draft morphology drawing of a fish egg and an early hatched larvae

 

2. Holomorphology of fish (Fig. 4.)
Fish eggs start cleavage after fertilization is completed. Usually the nutrition is supplied by vegetal pole. In the animal pole changes also occurs. Generally marine fishs end-yolk has cleavage regulation. Equal cleavage type in general is from oil globule stage, 2-cells stage, 4-cells stage, morula stage etc. till the formation of embryo. The anterior portion of the embryo becomes head and the posterior becomes tail. The middle portion of embryo starts somite formation. After that, the heart begins to beat. Melanophores appear gradually, they follow by lens of optic vesicle and auditory vesicle. At this stage embryo start moving and twising. Some species of eggs will secrete a little hormone for larvae to hatch out. There are various shape of hatching openings, such as short bar 一 shape, round shape, or square shape.
The hatching time is different for different types of eggs, and the influences of salinity and water temperate are quite prominent. Generally, pelagic eggs need shorter time than demersal eggs. At the early stage of hatching, yolk supplies nutrition. It is also called yolk-sac larval stage. When yolk is absorbed gradually, the larvae develop their swimming ability. Some species have special structure or filamentous extension to increase floating ability. Usually, fishes will experience two different kinds of habitat in their whole life, i.e. in the coastal waters and in the open sea. It is called bipartite life history to depict the larval fishes either drifting close to shore like deep-sea or pelagic species or offshore like coral reef fish during their hatching and drifting stage. After post flexion, they will settle down or enter to open sea or coastal waters where their parent or adult stage live. Then it will gradually grow to become juveniles. Many post flexion larval will change their morphology drastically, i.e., through metamorphosis to get into juvenile stage. They will grow up to become young and adult. Because of weak swimming ability, juvenile fishes like to live near coastal area until they mature, and then swim to deeper place for reproduction to complete their life cycle.

Fig.4. The holomorphology of fish from its egg to adult.

3. Different fish egg types in different habitats
Fish eggs can be classified into two types:demersal eggs such as damselfishes, and pelagic eggs, such as butterflyfishes. Demersal eggs are not easy to collect, so most fish egg studies are in pelagic eggs. According to different depths, pelagic eggs can be further divided into two types, epipelagic egg and bathypelagic egg. Base on their status, we can divide them into agglutinated pelagic eggs and isolated pelagic eggs. Most researches are working on isolated pelagic eggs. It is also possible to divide eggs based on their adhesion capability, such as flying fish has adhesive silk which can attach fish eggs to the floating materials on the surface water, and gobies can firm their eggs in cave using adhesive silk.
The types of fish eggs included in this book are mostly surface and isolated pelagic eggs except mouth breeding eggs of several species of cardinal fishes. 

4. The collection of fish eggs
The field works are often affected by weather and sea condition especially while collecting in the sea. Our laboratory collected quite a lot of eggs from both northern and southern parts of Taiwan from 1995 to 2001 as well as some eggs from fish hatchery farm. To collect and sort out fish eggs in field and at laboratory is a hard job since researchers must sort out the eggs from massive zooplankton sample and to do hatching observation immediately after the eggs are collected. Because the fixative method is different for different identification purposes including hatching, light microscope, SEM or DNA analysis. So the eggs will be treated differently as shown in Fig.6.

(1) From fish farm: 
To match up the different reproductive stages of cultivated fish species, the eggs were collected in unfertilized and fertilized stages, and also during different hatching stages from the farm. 10% formalin was used separately for fixation, and then brought back to the laboratory for the morphology observation and identification. The fish eggs used for scanning electron microscopy were fixed using 5% glutaraldehyde and 4% paraformaldehyde.

((2) From wild:
Fishing boats were rented to collect fish eggs in the north-eastern parts (Yenliao) and southern parts of Taiwan (Nanwan and Wanlitung) (Fig.5.). The net was 1.27m diameter, 4m length with 1/3 1.0 mm mesh size in the front, 2/3 0.5mm in the rear part. The net was towed at the surface water about 10 minutes each time . The collected eggs were fixed in 10% formalin or in special fixative for SEM examination. Additional parts of collected eggs were kept in fresh and placed in an aerated container then brought back to the lab for hatching observation. Mouth- breeding eggs of some cardinal fish species were collected by SCUBA diving.

A: Light microscope (LM): Fish eggs collected from fish farms and from the sea were fixed at 10% formalin first and then brought back to laboratory. Under dissection microscope, fish eggs were sorted out into different types according to their morphological characters, and then identify the species. Additionally, some fresh eggs were brought back to the laboratory with aeration for following hatching observation. Each character change were observed, photographed, and recorded. Different and each development stages.
B. Scanning electron microscope: Eggs collected from farm and ocean were cleaned up first to remove the impurity parts on the egg surface. Second, 5% glutaraldehyde and 4% paraformaldehyde were added into the SEM fixed sample and brought back to laboratory and then passed through 1% OsO4 . After passing a series of alcohol dehydration, they were dried by critical point dryer. Finally, plated with gold by gilding machine then placed under SEM for observation and photograph.

C: Molecular sequencing: In order to preserve the collected fresh fish eggs for molecular work, the eggs were preserved in 70﹪alcohol. The preserved eggs were used for DNA extraction. Using the polymerase chain reaction (PCR) the specific sequences of control region was amplified (D-loop). Through auto sequencing reaction (Big Dye terminator kit (ABI)), the DNA sequence was read by auto-sequencer(Applied Biosystems model 377)in order to identify the species.

Hatching the eggs in the laboratory

 

5. Identification methods of fish eggs 
In the past, species identification of fish eggs were mostly use light microscope. The following morphological characters are mainly used: (1) egg shape; (2) egg size; (3) thickness and color of egg membrane or chorion, and special structure on egg membrane surface, and other ornamentation; (4) presence or absence of oil globule, its position, number, color, size etc.; (5) the size of perivitelline space; (6) color of yolk, and with bubbles and segments or not; (7) the type of hatching opening ( 1938); (8) embryo development in the late period, myotomes of fish body, the distribution or position of pigmentation etc. (Matarese & Sandknop, 1984).
However, as a result of light microscope, still fairly confusion exists in identification. Most fish eggs can only be identified to family or genus level, and is unable to confirm to the species level. Although in recent ten years, few scholars have applied the scanning electron microscope (SEM) to find more ultrastructures especially on micorpyle and egg membrane to improve species identification. However, species identification by using SEM cannot be guaranteed. Not many papers have been published either (Boehleri, 1984), so the available reference is very limited. Thus, we have tried our best to provide as more SEM photos as we can in this book.Because of the tiny size and less distinguishable characters of fish eggs, it is difficult to identify them to species level. Recently our labaratory has successfully used DNA sequence analysis to idnetify the species level. Till now, we have already completed the sequence of adult lizardfishes and parrotfishes in Taiwen for eggs comparisons (吳 2000 , 施 2002). In these two master theses, they also proved that only using molecular sequence, the eggs can be identified to species level. Using light microscope or SEM, the fish eggs collected from wild and their parent species are unknown, they only can be identified to family or genus level at the best(Shao et al 2002).Furthermore, we are accumulating the morphological character data of all fish eggs in hoping that the phylogenetic tree of higher classification categories of fishes can be constructed based on the egg morphology to compare the phylogenies obtained from adult morphogical characters.

 

6. Dignosis of fish egg to categories of family or order