FULL PAPER

Morphology of unfertilized mature and fertilized developingmarine pelagic eggs in four types of multiple spawning flounders

Xiaodong Bian • Xiumei Zhang • Tianxiang Gao •

Ruijing Wan • Siqing Chen • Yasunari Sakurai

Received: 29 December 2009 / Revised: 20 April 2010 / Accepted: 26 April 2010 / Published online: 15 June 2010

� The Ichthyological Society of Japan 2010

Abstract Starry flounder Platichthys stellatus, spotted

halibut Verasper variegates, turbot Scophthalmus maxi-

mus, and Japanese flounder Paralichthys olivaceus are four

commercially cultivated multiple spawning flounders that

spawn pelagic eggs. Through appropriate light and scan-

ning electron microscope processing, the shape and surface

structures (such as micropyle, pores, pore density, and

paten) of unfertilized mature and fertilized developing eggs

of the four species were observed and measured. First,

individual or intraspecific comparisons of the surface

structures of eggs at different developmental stages were

made. Second, interspecific differences among the four

species at the same developmental stage of unfertilized

mature eggs were statistically computed and analyzed

through one-way analysis of variance and hierarchical

cluster analysis. Eggs of the same species collected at

different stages of development tend to be different in

morphology. Smoothing of the convoluted egg envelope

surface and closure of the micropyle to serve as a final step

of the polyspermy-preventing reaction are common after

fertilization. Based on detailed morphology of micropyle of

just-mature fertilizable eggs, turbot, starry flounder, and

Japanese flounder each have a micropyle with a long canal

but no distinct micropylar vestibule, type III of Riehl and

Gotting (Arch Hydrobiol 74:393–402, 1974). In contrast,

spotted halibut has a micropyle with a distinct flat micro-

pylar vestibule and a long canal, type II. Envelope surface

microstructures, especially those in the micropyle region,

are useful characters for egg identification among the four

species. Cluster analysis using selected egg characters

indicated the highest similarity between turbot and Japa-

nese flounder and that starry flounder is obviously more

similar to turbot and Japanese flounder than to spotted

halibut.

Keywords Egg envelope � Flounders � Morphology �Multiple spawner � Ultrastructure

Introduction

Marine fish have mature eggs in which the yolk granules

are fused to one another to form a membrane-bound yolk

mass (eggs with massed yolk). Their cytoplasm and

membrane-limited round cortical alveoli are thereby con-

fined to the egg cortex as a thin layer between egg envelope

and exclusive yolk mass due to the centripetal storage of

yolk (Iwamatsu 2000; Motta et al. 2005; Otani et al. 2009).

The egg envelope is a complex and multilayered protein-

aceous shell that is a helicoidal composite of protein fibers

in a protein matrix. It consists of parallel planes or sheets of

fibrils (mono- or polymolecular) in a spiral arrangement

(Iconomidou et al. 2000). In general, its morphology dif-

fers in different fishes depending on the developmental

stage of the egg and reflects adaptations to different eco-

logical conditions (Chen et al. 1999; Fausto et al. 2004). In

striking contrast to mammalian eggs, in which the sper-

matozoon enters the egg at any site, the sperm entry point

X. Bian � X. Zhang (&) � T. Gao

The Key Laboratory of Mariculture, Ministry of Education,

Ocean University of China, Qingdao 266003, China

e-mail: gaozhang@ouc.edu.cn

R. Wan � S. Chen

Yellow Sea Fisheries Research Institute, Chinese Academy

of Fishery Sciences, Qingdao 266071, China

Y. Sakurai

Faculty of Fisheries, Hokkaido University, Hakodate,

Hokkaido 041-8611, Japan

123

Ichthyol Res (2010) 57:343–357

DOI 10.1007/s10228-010-0167-1

into teleost eggs is restricted to a canal-like micropyle in

the envelope (Coward et al. 2002; Andoh et al. 2008; Otani

et al. 2009). As micropyle plays an important role in

gamete recognition during fertilization, its morphology

may be species specific (Kobayashi and Yamamoto 1981;

Chen et al. 1999).

During the fertilization process in fish, the morpholog-

ical and biochemical changes in the extracellular matrix

following gamete fusion, from unfertilized egg envelope to

the fertilized one, are the most dynamic transformations

(Murata 2003). In addition, after the first spermatozoon

penetration, mechanisms that prevent polyspermy should

take place, because fertilization in fish is generally mono-

spermatic (Kobayashi and Yamamoto 1981). However, a

large part of these mechanisms remain unclear (Murata

2003; Ganeco et al. 2008). Some changes in the mor-

phology of the eggs are considered part of the mechanical

barriers, such as closure of the internal opening of the

micropyle (Kobayashi and Yamamoto 1981; Yamamoto

and Kobayashi 1992), formation of the fertilization cone

(Iwamatsu et al. 1991; Ganeco et al. 2008; Marques et al.

2008), activation of the cortical reaction to eliminate any

supernumerary spermatozoa (Yamamoto 1952; Iwamatsu

and Ohta 1981; Iwamatsu et al. 1991, 1993a; Andoh et al.

2008), and egg envelope hardening (Zotin 1958; Perry

1984; Oppen-Berntsen et al. 1990; Masuda et al. 1992).

These mechanical barriers, combined with the egg enve-

lope and perivitelline space, act as a permeability barrier

to establish an environment for normal embryonic devel-

opment (Yamamoto and Kobayashi 1992). These processes

of fertilization and egg activation are highly important

in fish reproductive biology. In particularly, increased

knowledge of these issues in intensively cultured species

can contribute significantly to aquaculture (Coward et al.

2002).

Comparison of the ultrastructures of egg envelope and

micropyle using electronic microscopy for species identi-

fication was attempted quite early in teleostean fishes

(Riehl and Schulte 1978). Phylogenetic relationships at the

species, genus, or even subfamily level can also be tested

using the characters of egg ultrastructure against phyloge-

nies obtained from morphological characters to determine

their congruence (Chen et al. 1999). In previous studies,

outer surface of the egg envelope and microstructure of the

micropyle were the noteworthy features for egg identifi-

cation and phylogenetic analyses in Serranidae, Sparidae,

Apogonidae, and Mugilidae (Riehl 1993; Chen et al. 1999,

2007; Li et al. 2000; Gwo 2008). However, eggs collected

in different stages of development may tend to demonstrate

different configurations in morphology (Gwo 2008).

Comparison based on the morphology developing at dif-

ferent stages with unpredictable and sometimes unidenti-

fiable changes is very problematic (Gwo 2008).

As for studies on the morphological structures of

flounder eggs, various publications aim at identifying the

development series of the embryo (Mito 1963; Zhang et al.

1965; Lei et al. 2003; Wang et al. 2008). In addition,

numerous studies are geared toward: better understanding

of ultrastructural changes in the egg envelope of the

unfertilized state and after fertilization (Hagstrom and

Lonning 1968; Lonning 1972; Perry 1984); fine structures

of the egg envelope or micropyle of fertilized eggs

obtained either by artificial insemination or collected in the

wild, for spawning ecology discussion or species identifi-

cation (Ivankov and Kurdyayeva 1973; Stehr and Hawkes

1979, 1983; Hirai 1988, 1993); morphology of sperm entry

under light microscope (Yamamoto 1952; Andoh et al.

2008); and modification of micropyles during the course of

embryonic development (Yamamoto and Kobayashi 1992).

As Hirai (1993) pointed out, the eggs of Pleuronectinae

fishes show considerable variety and lack any characteristic

structure around the micropylar canal, which may be a

common feature. According to Yamamoto and Kobayashi

(1992), in Japanese flounder, the structural modification of

the micropyle is remarkable. The canal collapses along its

whole length during the course of embryonic development

and is involved in both permanent prevention of poly-

spermy and protection of the developing embryo from

bacterial infection. However, teleost oocytes and sperma-

tozoa exhibit remarkable variety in morphology and their

own adaptations in the processes of egg activation and

fertilization (Coward et al. 2002). The distinct paucity of

focused research in this vital area of flounder reproductive

biology refers only to the morphological features of eggs.

Whether these observations can apply to other flounder

species remains unclear. Furthermore, no study has ever

attempted to investigate the phylogenetic relationship

directly based on the morphological characters and mea-

surements of eggs in flounder.

Turbot Scophthalmus maximus and Japanese flounder

Paralichthys olivaceus are the most important marine

species cultivated in Northern China. Cultivation of starry

flounder Platichthys stellatus and spotted halibut Verasper

variegates is also currently expanding. These four species

have all been identified as multiple spawners. They spawn

pelagic eggs, from which batches of oocytes mature and

are spawned over a protracted mating season (Spies et al.

1988; McEvoy and McEvoy 1992; Kajimura et al. 2001;

Sawaguchi et al. 2006). Starry flounder and spotted halibut

are within the subfamily Pleuronectinae in the family

Pleuronectidae, turbot is in the family Scophthalmidae, and

Japanese flounder is in the family Paralichthyidae. They all

belong to the suborder Pleuronectoidei in the monophyletic

order of Pleuronectiformes (Azevedo et al. 2008). The goal

of this present work is to perform structural and ultra-

structural analysis of the unfertilized mature and fertilized

344 X. Bian et al.

123

developing marine pelagic eggs of the four commercially

cultivated multiple spawning flounders using light

microscopy and scanning electron microscopy (SEM). We

aim to provide intact and detailed morphology of these

eggs in order to convey structural modification of egg

envelope and micropyle during the course of embryonic

development. Solid species-specific evidence and stan-

dardized empirical reference for accurate species identifi-

cation and phylogeny estimation are also presented.

Materials and methods

Sample collection. Starry flounder: Gametes were obtained

from a breeding farm in Jiaonian, Qingdao, Southeast

Shandong Province, China. One batch of eggs from one

female and milt from two or three males were hand-strip-

ped into glass Petri dishes, mixed gently, and left undis-

turbed for 1 min. Fertilized eggs were rinsed, then

transferred to incubate in filtered seawater (33% salinity,

11.5–12.5�C, DO C6 mg/l, pH 7.8–8.0). Unfertilized

mature and fertilized developing eggs (just after extrusion

out of the ovary, precell stage; 48 h post insemination,

blastopore closure stage) were sampled for light micros-

copy and SEM observation. Over 200 eggs at each stage of

development were fixed in 5% formalin-seawater for light

microscopy, while over 100 eggs were collected and

cleaned using 0.1 M phosphate buffer (PB) at pH 7.4 at

least three times. The specimens were prefixed in 2.5%

glutaraldehyde in 0.1 M PB at pH 7.4 and stored at 4�C for

SEM.

Spotted halibut: Samples were obtained at the same

breeding farm as starry flounder. Artificial insemination

and sampling followed the same method as for starry

flounder. The fertilized eggs were incubated at salinity of

33%, 8–8.5�C, DO C6 mg/l, pH 7.8–8.0. Fertilized

developing eggs were sampled and fixed 48 h post

insemination at the development stage of germ ring 1/2–3/4

down yolk.

Turbot: Eggs were collected at a breeding farm in

Rongcheng, Weihai, Northeast Shandong Province, China.

Artificial insemination and sampling methods were the

same as those for starry flounder. The fertilized turbot eggs

were incubated at salinity of 34%, 13–13.2�C, DO C6 mg/l,

pH 7.8–8.0, and fixed at advanced stages of germ ring 5/6

down yolk (72 h post insemination).

Japanese flounder: Eggs were collected at the same

breeding farm as turbot. Artificial insemination and

sampling followed the same method as for starry flounder.

The fertilized eggs were incubated at salinity of 33%,

15–15.5�C, DO C6 mg/l, pH 7.8–8.5. Fertilized developing

eggs were sampled and fixed at the embryo tail 5/8 around

the yolk stage.

Light microscopy and SEM. The samples fixed in 5%

formalin-seawater were used for light microscopy obser-

vation. A total of 100 eggs in each fixed stage of the four

species were randomly selected. Morphological analyses

(egg diameter, oil globule, perivitelline space, and char-

acters of the egg membrane at light microscope level) were

evaluated using a Nikon SMZ1500 photomicroscope

equipped with a micrometer ocular lens.

For SEM, the prefixed eggs were washed with 0.1 M PB

buffer at pH 7.4, postfixed in 1% osmium tetroxide for 2 h,

and washed in the same buffer solution. They were then

dehydrated in a graded series of ethanol at 30%, 50%, 70%,

80%, 90%, and 95% concentrations, and then two times in

baths at 100% (10 min each). Finally, they were dried with

an EMS850 critical-point dryer and then gold-coated in a

EMS500 sputter coater. The material was electron

microphotographed under SEM (JEOL-JSM-840). Six

intact eggs at each fixed stage of the four species were

selected for observation and measurement. A total of nine

surface characters of eggs under SEM were used in this

study (Table 2), five of which were measured by using

Image Pro Plus V6.0 (Media Cybernetics), including size

of the entire egg, diameter of the micropyle, diameter of

the micropyle region if it exists, and size of pores. Out-

side the range of the micropylar region, five pores in each

eggs fixed at the same development stage were randomly

selected and measured. The number of pores measured

was n = 5 9 6 = 30.

Comparative morphological studies and analysis of

species relationship. Among all collected eggs, only those

well matured or developed were chosen for further obser-

vation. Individual or intraspecific variations in morpho-

logical characters of eggs at different developmental stages

were examined and initially described by independent-

samples t test (SPSS 17.0 for Windows). For interspecific

variations, a total of 15 shape or surface characters and

measurements of unfertilized mature eggs were statistically

computed and analyzed (Tables 1, 2). Quantitative data of

egg morphometric measurements, especially the micropy-

lar ultrastructures and envelope surfaces of the four

flounder species, were compared with one another using

one-way analysis of variance (ANOVA) (Tukey’s multi-

ple-comparison test) and box plot (SPSS 17.0 for Win-

dows). Furthermore, hierarchical cluster analysis was

carried out to provide solid species-specific evidence and

standardized empirical reference for accurate species

identification and phylogeny determination in the four

species. Hierarchical cluster analysis and computation

methods followed the method of Gwo (2008) to understand

the differences in surface characters. In the first method,

observed surface characters were classified into a three-

level modified Likert scale (i.e., 1, different from; 2, sim-

ilar to; and 3, same as starry flounder eggs) using the

Morphology of multiple spawning flounder eggs 345

123

surface characters of starry flounder eggs as the basis for

comparison. In the second method, diameter of the surface

characters was measured directly from the sample. The

results of both the first and the second methods were

computed using Ward’s method (Gwo 2008) to derive

squared Euclidean distance (SPSS 17.0 for Windows).

Phylogeny (phenogram) of the four species in question was

then inferred from hierarchical cluster analysis, together

with the derived dendrogram that delineates the correla-

tions of the four clusters representing each species. All data

Table 1 Comparison of morphological characters (light microscope level) and properties of eggs in four flounders before and after fertilization

Feature/species Unfertilized mature eggs Fertilized developing eggs

Starry

flounder

Spotted

halibut

Turbot Japanese

flounder

Starry

flounder

Spotted

halibut

Turbot Japanese

flounder

Egg diameter

(n = 100; mm)

1.03 ± 0.04 1.86 ± 0.09 1.04 ± 0.03 1.02 ± 0.04 1.06 ± 0.04 2.03 ± 0.06 1.09 ± 0.05 1.06 ± 0.05

Oil globule

(n = 100; mm)

None (3) None (3) 0.20 ± 0.01

(1)

0.19 ± 0.03

(1)

None None 0.19 ± 0.02 0.20 ± 0.02

Yolk diameter

(n = 100; mm)

1.03 ± 0.04 1.86 ± 0.09 0.92 ± 0.07 0.96 ± 0.06 0.98 ± 0.04 1.91 ± 0.07 0.96 ± 0.05 0.99 ± 0.06

Character of egg

membrane

Heavy

striations (3)

Heavy

striations (2)

Slight

striations (1)

Slight

striations (2)

Slight

striations

Slight

striations

Smooth Smooth

Perivitelline space None (3) None (3) Indistinct (1) Indistinct (1) Narrow Narrow Moderate Moderate

Property of egg Pelagic (3) Pelagic (3) Pelagic (3) Pelagic (3) Pelagic Pelagic Pelagic Pelagic

Values are means ± SD for the indicated number of samples

Number in parentheses of the unfertilized mature eggs indicates transferred measurement in modified three-level Likert scale (i.e., 1, different

from; 2, similar to; and 3, same as starry flounder eggs) computed for hierarchical cluster analysis

Table 2 Comparison of microstructural characters of the eggs in four flounders before and after fertilization

Feature/species Unfertilized mature eggs Fertilized developing eggs

Starry

flounder

Spotted

halibut

Turbot Japanese

flounder

Starry

flounder

Spotted

halibut

Turbot Japanese

flounder

Diameter of micropyle

opening (lm)

5.08 ± 0.35 5.97 ± 0.37 4.18 ± 0.39 6.18 ± 0.47 6.68 ± 0.79 8.84 ± 0.42 6.83 ± 0.50 8.15 ± 1.28

Type of micropyle III (3) II (1) III (3) III (3) III II III III

Number of micropylar

ribs

8–9 (8.5) 13–15 (14) 11–13 (12) 7–8 (7.5) 6 Unclear Unclear 7

Convoluted direction

of ribs (from bottom

to opening)

Clockwise

(3)

Clockwise

(3)

Clockwise

(3)

Clockwise

(3)

Clockwise Clockwise Clockwise Clockwise

Diameter of pore

canals (lm)

0.82 ± 0.11 0.53 ± 0.11 0.34 ± 0.06 0.36 ± 0.07 0.40 ± 0.08 0.60 ± 0.10 0.40 ± 0.06 0.48 ± 0.06

Presence of distinct

micropyle region

(diameter in lm)

No (3) Yes (1) No (3) No (3) No Yes No No

5.91 ± 0.57 8.87 ± 0.39 5.49 ± 0.40 7.59 ± 0.67 Vanished 11.57 ± 0.99 Vanished Vanished

Arrangement of pore

canals

Hexagonal

(3)

Hexagonal

(3)

Hexagonal

(3)

Hexagonal

(3)

Hexagonal Hexagonal Hexagonal Hexagonal

Pores distribution

density (pores/

100 lm2)

23.62 ± 1.89 20.01 ± 0.53 29.95 ± 1.29 35.98 ± 4.07 30.05 ± 5.75 15.48 ± 1.78 26.62 ± 0.41 33.99 ± 8.95

Diameter of the fixed

egg (mm)

0.86 ± 0.04 1.56 ± 0.07 0.92 ± 0.06 0.87 ± 0.35 0.9 ± 0.04 1.70 ± 0.11 1.07 ± 0.01 0.95 ± 0.06

Values are means ± SD of each species in the respective unfertilized mature and fertilized developing stages (n = 6, except for the diameter of

pore canals n = 30)

Number in parentheses of the unfertilized mature eggs indicates transferred measurement in modified three-level Likert scale (i.e., 1, different

from; 2, similar to; and 3, same as starry flounder eggs) computed for hierarchical cluster analysis

346 X. Bian et al.

123

are presented as mean ± standard deviation (SD) in the

text.

Results

Morphological characters of the eggs under light

microscopy. Observed by light microscopy, the unfertil-

ized mature eggs of each species were colorless,

transparent, buoyant, and nonadhesive with a large homo-

geneous central yolk mass and a negligible perivitelline

space (Fig. 1a–d). The egg envelope was smooth in

appearance, but closer inspection revealed striations or

reticulations. Starry flounder (Fig. 1a) and spotted hali-

but (Fig. 1b) both produce eggs without oil globules, but

turbot (Fig. 1c) and Japanese flounder (Fig. 1d) eggs both

contain a conspicuous, moderately large oil globule in the

yolk.

In the fertilized developing eggs, the egg envelope ele-

vates and separates from the underlying cytoplasm and a

visible fluid-filled cavity designating the perivitelline space

is formed between them. The perivitelline space is very

Fig. 1 Unfertilized mature and

fertilized developing eggs of

four species of multiple

spawning flounders fixed in 5%

formalin-seawater (light

microscopy). a Starry flounder,

unfertilized mature egg.

b Spotted halibut, unfertilized

mature egg. c Turbot,

unfertilized mature egg.

d Japanese flounder, unfertilized

mature egg. e Starry flounder,

blastopore closure stage egg.

f Spotted halibut, germ ring

1/2–3/4 down yolk stage egg.

g Turbot, germ ring 5/6 down

yolk stage egg. h Japanese

flounder, embryo tail 5/8 around

yolk stage egg. Each scale bar300 lm. PVS perivitelline space

Morphology of multiple spawning flounder eggs 347

123

narrow, and the egg envelope still reveals slight striations

in starry flounder fixed at the blastopore closure stage

(Fig. 1e) and in spotted halibut fixed at the germ ring

1/2–3/4 down yolk stage (Fig. 1f). Moderate perivitelline

space, and smooth and almost transparent egg envelope

with no specialized membrane structures are observed in

turbot fixed at the germ ring 5/6 down yolk stage (Fig. 1g)

and in Japanese flounder fixed at the embryo tail 5/8 around

the yolk stage (Fig. 1h). The formation of perivitelline

space also causes the diameter of the spherical eggs to

expand from 1.03 ± 0.04 to 1.06 ± 0.04 mm in starry

flounder (t = 4.848, P \ 0.01; n = 100), 1.86 ± 0.09 to

2.03 ± 0.06 mm in spotted halibut (t = 14.761, P \ 0.01;

n = 100), 1.04 ± 0.03 to 1.09 ± 0.05 mm in turbot

(t = 7.106, P \ 0.01; n = 100), and 1.02 ± 0.04 to 1.06 ±

0.05 mm in Japanese flounder (t = -6.798, P \ 0.01;

n = 100). Measurements of each character are given as

average values in Table 1.

Ultrastructural characters on the surface of eggs and

micropyles among the four species. Starry flounder:

Under SEM inspection, the outer envelope surface of

unfertilized mature starry flounder eggs is characterized

by a criss-cross pattern of depressions. These depressions

radiate in all directions across the envelope surface,

creating a heavy wrinkled appearance (Fig. 2a, b). A

number of flush pores, 0.82 ± 0.11 lm in diameter

(n = 30), are characterized by a hexagonal pattern of

distribution scattered over the outer envelope surface

(i.e., each pore canal is surrounded by six others of the

same size) (Fig. 2b). Pore density is 23.62 ± 1.89 pores/

100 lm2 (n = 6) (Table 2). In fertilized developing eggs,

the wrinkles on the outer surface of the envelope are

indistinct and the pores are thickened and elevated

(compare Fig. 2c with b). The shape of the outer surface

of the envelope has a slightly depressed lip as it cir-

cumvents the openings of pore canals (Fig. 2c). The pores

are still uniform in diameter, 0.40 ± 0.08 lm (n = 30),

and statistically significant decreases in pore diameter

occur after fertilization (t = 16.49, P \ 0.01; n = 30).

The distribution pattern of the pores remains unchanged

at a density of 30.05 ± 5.75 pores/100 lm2 (t = 0.38,

P [ 0.05; n = 6) (Table 2).

Fig. 2 Structures of starry

flounder egg surface observed

by SEM. a Entire unfertilized

mature egg. b Wrinkled

unfertilized egg envelope

surface with flush pores

(arrowheads) distributed in

hexagonal pattern. c Fertilized

egg envelope surface with pore

canals (arrowheads) distributed

in hexagonal pattern.

d Micropyle region of the

unfertilized mature egg.

e Unfertilized egg micropyle

shaped with helicoid ribs

(arrow) to micropylar bottom,

unequal sized pores or cavities

(arrowheads) scattered in the

peripheral micropylar region.

f Micropyle of the fertilized

developing egg. MC micropylar

canal, MR micropyle region,

MV micropyle vestibule

348 X. Bian et al.

123

In unfertilized mature starry flounder eggs, there is no

clearly distinguishable micropylar region (Fig. 2d). The

envelope surface at the animal pole region appears

smoothened, and the micropyle detected in the center

appears cylindrical, possessing a small shallow micropyle

vestibule, 5.91 ± 0.57 lm in diameter (n = 6) (Fig. 2e).

The outer opening of the micropylar canal is 5.08 ±

0.35 lm in diameter (n = 6), with eight to nine counter-

clockwise arrangements of helicoid ribs (from opening to

bottom) traversing the thickness of the envelope (Fig. 2e,

Table 2). Pores and shallow cavities of various sizes are

scattered in the peripheral region of the micropyle vestibule

(Fig. 2e). A remarkable modification in the micropyle

structure is recognized in fertilized eggs fixed at the blas-

topore closure stage. The micropyle appears to have been

stretched and, similar to a closed funnel, increased in

roughness (Fig. 2f). The micropylar canal, with its inner

lumen completely blocked, consists of six counterclock-

wise arrangements of spiral-shaped ridges (from outer to

inner) tapering toward its terminal. The outer opening of

the micropylar canal is 6.68 ± 0.79 lm in diameter

(n = 6), statistically significantly larger than that before

fertilization (t = -4.58, P \ 0.01; n = 6) (Table 2). The

micropylar vestibule vanishes with the peripheral region,

lacking further structures (Fig. 2f). In addition, the enve-

lope decreases in thickness, particularly in the micropylar

region (compare Fig. 2f with e).

Spotted halibut: The envelope surface of unfertilized

mature spotted halibut eggs are also wrinkled (Fig. 3a, b).

Unlike the unfertilized eggs of starry flounder, there are no

apparent pores visible at low magnification (Fig. 3b, c).

At higher magnification, a number of uniform flush pores

converge in the peripheral micropyle region (Fig. 3d). The

pores, 0.53 ± 0.11 lm in diameter, are characterized by a

hexagonal pattern of distribution at a density of 20.01 ±

0.53 pores/100 lm2 (n = 6) (Fig. 3d, Table 2). The outer

surface of the envelope appears to have been stretched

during fertilization, and faint wrinkles are still visible. The

whole surface appears rough at the germ ring 1/2–3/4 down

yolk stage, which is likely due to deposition of some

materials (Fig. 3e). Although the distribution pattern of the

pores remains unchanged, they appear hexagonal (Fig. 3e);

Fig. 3 Structures of spotted

halibut egg surface observed by

SEM. a Entire unfertilized

mature egg. b Wrinkled

unfertilized egg envelope

surface. c Micropyle region of

the unfertilized mature egg.

d Micropyle shaped with a

significant shallow micropylar

vestibule with uniform flush

pores (arrowheads) distributed

in peripheral micropyle region;

micropylar canal shaped with

helicoid rib (arrow) to

micropylar bottom. e Fertilized

egg envelope surface with pore

canals (arrowheads) distributed

in hexagonal pattern.

f Fertilized egg micropyle with

the inner portion of the

micropylar canal blocked by a

massive granular substance with

the appearance of a bacterial

colony (arrow). MC micropylar

canal, MR micropyle region,

MV micropyle vestibule

Morphology of multiple spawning flounder eggs 349

123

however, the distribution density of the pores decreases

significantly to 15.48 ± 1.78 pores/100 lm2 (t = 4.22,

P \ 0.05; n = 6) (Table 2). Meanwhile, the pore size

becomes significantly larger than that of unfertilized eggs,

with diameter of 0.60 ± 0.10 lm (t = 2.13, P \ 0.05;

n = 30) (Table 2).

In unfertilized mature spotted halibut eggs, the outer

surface of the envelope at the animal pole region presents a

significant shallow pit (i.e., the micropylar vestibule)

(Fig. 3c). It extends over an 8.87 ± 0.39 lm area adjacent

to the outer opening of the micropylar canal (n = 6), which

is unique to that region of the egg. The micropyle appears

as a flattened crater with the micropylar canal detected in

the center of the vestibule (Fig. 3d). The micropylar canal

shape has 13–15 counterclockwise arrangements of annular

ribs piercing the thickness of the envelope, with an outer

opening 5.97 ± 0.37 lm in diameter (n = 6) (Fig. 3d,

Table 2). A number of flush pores, the same size as the rest

of the egg surface, are distributed in the peripheral region

of the micropylar vestibule (Fig. 3d). In fertilized devel-

oping eggs, the micropyle increases in roughness with most

of the viewable part showing some granular or bacterial

appearance (Fig. 3f). The whole micropyle seems to be

stretched heavily, with a significantly larger deformed

micropylar vestibule, 11.57 ± 0.99 lm in diameter (t = 6.27,

P \0.01; n = 6) (Fig. 3f, Table 2). The inner parts of

the micropylar canal narrowed, but its outer opening is

8.84 ± 0.42 lm in diameter (n = 6), a size significantly larger

than that of unfertilized ones (t = 13.6, P \0.01; n = 6)

(Table 2).

Turbot fish: In unfertilized turbot eggs, the envelope

surface conveys a slight undulating status (Fig. 4a, b). The

pores, many of which are scattered uniformly over the

surface, are characterized by a hexagonal pattern of dis-

tribution, with a density of 29.95 ± 1.29 pores/100 lm2

(n = 6) (Table 2). Pore openings are 0.34 ± 0.06 lm in

diameter (n = 30) (Table 2). In fertilized eggs, the enve-

lope appears to have been stretched tangentially, with no

undulating status of the outer surface detectable at the germ

ring 5/6 down yolk stage; however, it appears to be rougher

(Fig. 4c). Pores, of uniform size, are significantly larger

than those of the unfertilized eggs (0.40 ± 0.06 lm in

Fig. 4 Structures of turbot egg

surface observed by SEM.

a Entire unfertilized mature

egg. b Undulating status of

unfertilized egg envelope

surface. c Fertilized egg

envelope surface with pore

canals (arrowheads) distributed

in hexagonal pattern.

d Micropyle region of the

unfertilized mature egg.

e Unfertilized egg micropyle

shaped with helicoid rib (arrow)

to micropylar bottom, unequal

sized pores or cavities

(arrowheads) scattered in the

peripheral micropylar region.

f Micropyle of the fertilized

developing egg. MC micropylar

canal, MR micropyle region,

MV micropyle vestibule

350 X. Bian et al.

123

diameter; t = -3.99, P \ 0.01; n = 30). The distribution

pattern of pores remains unchanged at a density of

26.62 ± 0.41 pores/100 lm2 (t = 3.92, P \ 0.05; n = 6)

(Table 2).

Similar to the starry flounder, there is no clearly dis-

tinguishable micropylar region found in the egg of this

species (Fig. 4d). In unfertilized turbot eggs, the outer

surface of the envelope is slightly elevated at the animal

pole region and contains the micropyle (Fig. 4d, e). The

micropyle appears as a cylindrical hole (4.18 ± 0.39 lm

diameter, n = 6), with a small shallow vestibule (5.49 ±

0.40 lm diameter, n = 6) (Table 2). The micropylar canal

is detected in the center of the vestibule, which is rein-

forced by 11–13 indistinct counterclockwise annular ribs

perforating the thickness of the envelope (Fig. 4e). Surface

pores or cavities found in the peripheral region of the

micropyle vary in diameter from those scattered on the rest

of the envelope surface (Fig. 4d, e). In fertilized develop-

ing eggs, the whole micropyle region appears to have been

stretched heavily and shows increased roughness (Fig. 4f).

The micropyle appears similar to a closed funnel with the

micropylar vestibule vanishing (Fig. 4f). Most parts in the

outer opening of the micropyle are occupied by material

secreted from the perivitelline space. The inner end of the

micropylar canal narrows or closes its lumen (Fig. 4f). The

outer opening of the micropylar canal is 6.83 ± 0.50 lm in

diameter, significantly larger than that of unfertilized eggs

(t = -13.64, P \ 0.01; n = 6) (Table 2). The pores and

cavities in the peripheral region of the micropyle all

decrease in depth (Fig. 4f).

Japanese flounder eggs: The outer envelope surface of

Japanese flounder eggs, fixed shortly after stripping from

mature individuals, has numerous pores and wrinkles

(Fig. 5a, b). The smooth pores are also characterized by a

hexagonal distribution pattern with a density of 35.98 ±

4.07 pores/100 lm2 (n = 6) (Table 2). Pore openings are

0.36 ± 0.07 lm in diameter (n = 30) (Table 2). The

envelope surface is no longer wrinkled but looks rougher at

the tail of the embryo 5/8 around yolk stage (Fig. 5c).

Pores, with uniform size, are significantly larger than those

of unfertilized eggs (0.48 ± 0.06 lm diameter; t = -7.12,

P \ 0.05; n = 30) (Table 2). Pore distribution density is

Fig. 5 Structures of Japanese

flounder egg surface observed

by SEM. a Entire unfertilized

mature egg. b Undulating status

of the unfertilized egg envelope

surface. c Fertilized egg

envelope surface with pore

canals (arrowheads) distributed

in hexagonal pattern.

d Micropyle region of the

unfertilized mature egg.

e Unfertilized egg micropyle

shaped with helicoid rib (arrow)

to micropylar bottom, unequal

sized pores or cavities

(arrowheads) scattered in the

peripheral micropylar region.

f Fertilized egg micropyle with

a small elevated ridge (arrow) is

surrounded by stretched pore

canals (arrowheads). MCmicropylar canal, MR micropyle

region, MV micropyle vestibule

Morphology of multiple spawning flounder eggs 351

123

33.99 ± 8.95 pores/100 lm2 (t = 0.35, P [ 0.05; n = 6),

with the distribution patterns unchanged (Table 2).

There is no clearly distinguishable micropylar region in

the egg of this species, the same as in starry flounder and

turbot (Fig. 5d). The envelope surface at the animal pole

region shows slight elevation, and the micropyle appears as

a cylindrical hole detected in the center of the elevation

(Fig. 5d, e). The micropylar canal consists of seven to eight

counterclockwise annular ribs penetrating the thickness of

the envelope (ribs 6.18 ± 0.47 lm in diameter at the outer

opening, n = 6) (Fig. 5e, Table 2). A shallow indistinct

vestibule (7.59 ± 0.67 lm diameter, n = 6) surrounds the

outer opening of the micropylar canal (Fig. 5e, Table 2).

Surface pores or cavities of different sizes are arranged in

concentric circles around the micropyle (Fig. 5e). After

fertilization, the micropyle increases in roughness and

appears as a closed funnel, with an outer opening

8.15 ± 1.28 lm in diameter (n = 6), significantly larger

than that of unfertilized eggs (t = -3.56, P \ 0.01; n = 6)

(Fig. 5f, Table 2). The micropylar canal collapses along its

whole length with the inner half closed (Fig. 5f). The

micropylar vestibule is unclear; a small elevated ridge is

present on the edge of the outer micropyle with pores

and cavities in the peripheral region that are heavily

stretched (Fig. 5f). Thinning of the envelope is also distinct

in the micropylar region of this species (compare Fig. 5f

with e).

Comparative morphological studies of unfertilized

mature eggs and their use in phylogenetic inference.

Based on the morphological characters of the unfertilized

mature eggs of the four species using one-way ANOVA

and a box plot, egg size places the four species into two

groups (Fig. 6a). Spotted halibut eggs are the largest, with

mean diameter of 1.56 ± 0.07 mm (n = 6), followed by

turbot, starry flounder, and Japanese flounder, with their

egg sizes showing no significant difference (Tukey’s

multiple-comparison test, P [ 0.05) (Fig. 6a). Although

there is no significant difference in diameter of the mic-

ropylar canal between spotted halibut and Japanese floun-

der (Tukey’s multiple-comparison test, P [ 0.05), canal

size differs significantly between them and the other two

species (Tukey’s multiple-comparison test, P \ 0.05)

(Fig. 6b). The diameter of the micropylar vestibule dif-

ferentiates the three groups (Fig. 6c), a pattern similar to

the diameter of the micropylar canal. The mean diameter of

the outer opening of the pore canal is greatest in starry

flounder, followed by spotted halibut. The third group,

turbot and Japanese flounder, has the smallest size of pore

canals (Fig. 6d). There is also significant difference in pore

canal densities. The mean density of pore canals in Japa-

nese flounder is the largest, significantly larger than in

turbot (Tukey’s multiple-comparison test, P \ 0.05)

(Fig. 6e), with the spotted halibut and starry flounder

showing the lowest distribution. This is contrary to the

distribution of pore diameter.

Fifteen surface characters and measurements of unfer-

tilized mature eggs were used in the hierarchical cluster

analysis (Tables 1, 2). After computing using Ward’s

method (Gwo 2008), the final value of squared Euclidean

distance from proximal matrix derived for hierarchical

cluster analysis was obtained for each species pair

(Table 3). As shown in the dendrogram derived from

hierarchical cluster analysis, the phylogenetic relationship

of the four species in question was deduced: turbot showed

higher similarity to Japanese flounder than to either starry

flounder or spotted halibut in the egg characters (Fig. 7).

Although starry flounder and spotted halibut are in the

same Pleuronectidae family, starry flounder was obviously

more similar to turbot and Japanese flounder, which belong

to the two different families of Scophthalmidae and Par-

alichthyidae, respectively. Turbot and Japanese flounder

Fig. 6 Box plot of unfertilized mature flounder egg morphometrics.

a Egg diameters fixed for SEM (n = 6); b outer opening diameters of

the micropylar canal (n = 6), c micropylar vestibule diameters

(n = 6); d pores diameters on the envelope (n = 30); e pore densities

on the envelope (n = 6). Bars inside the boxes are medians. Barsoutside the box show range of data. Box associated with the same

lower-case letter above bars in the same characters tested are not

significantly different by one-way ANOVA (Tukey’s multiple-

comparison test) (P [ 0.05)

352 X. Bian et al.

123

showed high similarity to each other, even though they

belong to different families.

Discussion

Different configurations of flounder eggs before and

after fertilization. Light microscopic observations:

Through light microscopic observations, the unfertilized

mature eggs of the four flatfishes were pelagic, round, and

nonadhesive, with a large homogeneous central yolk mass

and negligible perivitelline space. The characters of eggs

showing greatest differences are the egg size and the

presence or absence of an oil globule. Spotted halibut eggs

are the largest in size, followed by turbot, starry flounder,

and Japanese flounder with their egg sizes showing no

significant difference (Tukey’s multiple-comparison test,

P [ 0.05) (Table 1). Starry flounder and spotted halibut

eggs both lack an oil globule, while turbot and Japanese

flounder both contain a moderately large oil globule in

the yolk.

The most striking phenomenon observable after fertil-

ization under light microscope is the detachment and sep-

aration of the egg envelope from the egg proper and the

formation of the visible perivitelline space. The rupture of

cortical alveoli triggers egg envelope elevation and con-

sequent enlargement of perivitelline space (Laale 1980),

leading to a significant increase in the egg diameter of

each species studied. This perivitelline space and the

perivitelline space liquid confined by the hardened and

semipermeable envelope ensure optimal conditions for

development of the embryo and protect it against adverse

environmental factors during the prehatching critical for-

mative stage (Laale 1980; Yamamoto and Kobayashi

1992).

Envelope morphology before and after fertilization: The

envelope of the fish egg undergoes structural and

mechanical changes during oocyte development and after

fertilization (Meloni et al. 2004). Just after extrusion out of

the ovary, the unfertilized egg envelope surface conveys a

criss-cross pattern of depressions in starry flounder, wrin-

kled in spotted halibut, undulating in turbot, and slightly

wrinkled in Japanese flounder. Smooth pores are radially

scattered on the envelope. These wrinkles and undulating

statuses of the egg envelope correspond to the interdigita-

tions of microvilli and cellular processes from both oocyte

and follicular cells during egg envelope development (Park

et al. 1998; Ravaglia and Maggese 2003). Through

immunohistochemical and ultrastructural studies of egg

envelope development in many fish species (Ravaglia and

Maggese 2003; Fausto et al. 2004; Meloni et al. 2004;

Francisco and Medina 2005; Ortiz-Delgado et al. 2008),

the growing envelope is shown to have numerous tiny

radial canals through which finger-like microvilli from

both oocyte and granulosa cells cross one other. However,

the microvilli are withdrawn from the egg envelope prior to

ovulation, leaving radially scattered empty pores.

The fertilization envelope in each of the studied species

is regular and appears to have been stretched tangentially,

with the wrinkled and undulating status disappearing. The

envelope thickness is decreased, especially in the micro-

pylar region, while the whole envelope surface becomes

rough due to deposited materials (Figs. 2c, 3e, 4c, 5c).

Starry flounder shows decreased pore diameter, increased

pore distribution density, and a depressed lip in peripheral

openings of pore canals. The other three species demon-

strate increased pore diameters and decreased depth and

distribution densities (Table 2). All these postfertilization

changes manifest in the morphological aspect of a fertil-

ization envelope surface. According to Kudo and Teshima

(1998), following normal fertilization or artificial activa-

tion, alveoli polysialoglycoprotein content is proteolyti-

cally cleaved and released into the perivitelline space,

which contributes to transformation of the unfertilized egg

envelope into the fertilized one. The unfertilized envelope

outermost layer is then replaced by cortical alveolus

exudates. An increased turgor pressure against the inner

surface of the envelope following the formation of the

perivitelline space and shrinkage of the inner layer of the

envelope due to the exocytosis of cortical alveoli may

be responsible for the decreasing thickness of envelope

(Iwamatsu et al. 1993a).

Table 3 Proximity matrix of squared Euclidean distance among the

four species using from Ward’s method of hierarchical cluster anal-

ysis for 15 egg characters

Species Starry

flounder

Spotted

halibut

Turbot Japanese

flounder

Starry flounder 0.000 – – –

Spotted halibut 27.006 0.000 – –

Turbot 19.948 35.045 0.000 –

Japanese flounder 17.605 33.333 11.063 0.000

Fig. 7 Dendrogram from hierarchical cluster analysis using Ward’s

method, based on squared Euclidean distances from 15 egg characters

(Tables 1, 2, 3) of starry flounder, spotted halibut, turbot, and

Japanese flounder

Morphology of multiple spawning flounder eggs 353

123

Micropyle morphology and their structure modification

after fertilization: Riehl and Gotting (1974) classified the

shapes of micropyle of most fish eggs into three types:

type I, micropyle with deep micropylar vestibule and short

canal; type II, micropyle with flat micropyle vestibule but

relatively longer micropyle canal; and type III, micropyle

with no micropyle vestibule but with micropyle canal

widened at its upper end. Judging from the SEM images,

there are no clear micropylar vestibules in all unfertilized

mature micropyles of starry flounder, turbot, and Japanese

flounder. In addition, long micropylar canals appear as

cylindrical helicoid ribs varying in species and number

(type III) (Figs. 2e, 4e, 5e). However, the micropyle of

spotted halibut, considered as type II and which has a

distinct shallow vestibule in the micropylar region and the

long micropyle canal, appears to have cylindrical helicoid

ribs (Fig. 3d). Several authors have reported that, until the

initiation of oocyte maturation, the micropylar canal is

plugged with cytoplasmic protrusion of a large, mushroom-

shaped micropylar cell (Takano and Ohta 1982; Kobayashi

and Yamamoto 1985; Iwamatsu et al. 1993b; Nakashima

and Iwamatsu 1994). Bodies of the micropylar cell and

nearby granulosa cells exert mechanical pressure on the

external surface of the growing oocyte and thus participate

in the formation of the micropylar vestibule. The cyto-

plasmic process of the micropylar cell forms a passive

barrier for the deposition of material to the egg envelope in

the animal pole. This results in the formation of the mic-

ropylar canal. The helicoid rib appearance of the canal wall

might therefore be due to the presence of layers in the

envelope (Kobayashi and Yamamoto 1981). After the

envelope is completely formed, cytoplasmic protrusion of

the micropylar cell is withdrawn from the micropylar canal

during oocyte maturation or ovulation.

Remarkable modifications of the micropyle in fertilized

eggs of each flounder species have been discovered in this

research. Fertilized micropyles in starry flounder and Jap-

anese flounder appear as closed funnels, exhibiting spiral-

shaped ridges tapering toward their terminal, with the inner

part of their micropylar canals completely closed. Spotted

halibut and turbot both appear narrow in their inner portion

and block the outer opening of the micropylar canals using

deposited materials. The outer opening of micropyle in

each species appears to have been stretched heavily; the

micropylar vestibule has disappeared in starry flounder,

turbot, and Japanese flounder and is deformed in spotted

halibut. Factors inducing structural modification of the

micropyle have been discussed in a few studies (Yamamoto

and Kobayashi 1992; Iwamatsu et al. 1993a; Kobayashi

and Yamamoto 1993). At fertilization, due to the shrinkage

(decrease in thickness) of the envelope in the inner layer

and the formation of the perivitelline space, which follows

an established increased turgor (pressure against the inner

surface of the envelope), the inner portion of the micro-

pylar canal appears to be closed or narrowed, and the outer

opening of the micropyle to be heavily stretched. At the

same time, parts of the perivitelline space liquid extrude

out of the eggs through the micropylar canal and parts

remain to form the blocked plug. Bacteria are widespread

in seawater and attach to eggs in large numbers (Morrison

et al. 1999), as shown in the micropylar region of fertilized

developing spotted halibut eggs (Fig. 3f). Thus, the closure

of the micropyle also functions to protect developing

embryos from infection by external pathogens such as

bacteria, viruses, and fungi, by participating in preserving

the sterility of the perivitelline space during embryogenesis

(Yamamoto and Kobayashi 1992).

Comparisons of unfertilized eggs. In the current study,

eggs of the four multiple spawning flounders at different

stages of development tend to demonstrate different con-

figurations in morphology of both envelope surfaces and

micropylar regions, the two most important features for egg

identification and phylogenetic analysis. All these factors

make the morphology of the eggs unpredictable after fer-

tilization. In addition, in fertilized eggs, the morphological

changes in the micropyle render this feature difficult to

use for species-specific egg identification. Based on the

same empirical basis of just-mature fertilizable eggs, the

current study provides intact and detailed morphological

characters of egg surface structure in the four flounders

through light and SEM processing for accurate species-

specific egg identification and phylogenetic relationship

prediction.

Surface structures unique to the micropyle region occur

in each species observed. The diameter of the micropyle

differs significantly in all four species, all shaped with

cylindrical micropylar canal and counterclockwise

arrangements of helicoidal ribs (from opening to bottom).

However, the number of ribs differs among species.

Although belonging to the same subfamily as starry

flounder, spotted halibut contains a distinct shallow vesti-

bule in the micropylar region, a unique structure in the four

species studied. Moreover, no micropyle-related studies on

other Pleuronectinae species refer to such a distinct shallow

vestibule in the micropylar region (Yamamoto 1952; Stehr

and Hawkes 1979; Hirai 1988, 1993; Andoh et al. 2008).

This is not in agreement with Hirai’s (1993) argument that

lack of any characteristic structure around the micropylar

canal in eggs of Pleuronectinae fishes may be a common

feature. Unfertilized egg envelope surfaces of the four

flounder species demonstrate the same pattern of wrinkled

and undulating statues. The pore canals show the same

hexagonal pattern of arrangement. This finding further

confirms Olivar’s (1987) argument that it is difficult to

believe that the pore canal pattern could be either species

or family specific. The species differences observed on the

354 X. Bian et al.

123

envelope surface that would be useful for species identifi-

cation are the differences in pore canal size and electron

density. From the compared results, starry flounder and

spotted halibut within Pleuronectinae in the family Pleu-

ronectidae both have larger pore diameters and lower pore

densities than Japanese flounder in the family of Paral-

ichthyidae and turbot in Scophthalmidae. This provides

further evidence in support of Hirai’s (1993) argument that

large pore size and lower density may be characteristic of

Pleuronectinae fishes. Although there is no significant

difference between turbot and Japanese flounder in pore

canal size, pore density in Japanese flounder was signifi-

cantly greater than in turbot. All these show that the outer

surface of the envelope could be used to separate the three

families, though it may not show remarkable differences in

microstructure among species at a genus or family level

(Chen et al. 1999).

Phylogenetic relationships among the four species.

To date, taxonomy of Pleuronectiformes has attracted great

attention because of its economic importance and extre-

mely specialized asymmetric morphology. The relation-

ships among families in suborder Pleuronectoidei and

among the genera of their families have also been debated

extensively; however, consensus has not been reached

(Azevedo et al. 2008). The present result of the phyloge-

netic analysis using a phenetic approach suggests that

turbot and Japanese flounder are the most closely related

and that there is great diversity between starry flounder and

spotted halibut, even though they belong to the same

family. This result is not completely in agreement with

conclusions obtained from other character suites, including

morphological, biochemical, and molecular data (Ahlstrom

et al. 1984; Nelson 2006; Azevedo et al. 2008). A possible

explanation for the anomalies is that a rich diversity in both

spawning ecology and egg morphology exists in Pleuro-

nectinae fishes (Minami 1984). Moreover, the characters of

eggs used in this study are determined by the biology of

reproduction of the species more than by their taxonomic

affinities (Lonning 1972). All these appear to weaken the

validity of using egg characters for phylogeny determina-

tion. According to Hensley and Ahlstrom (1984), the

characters of eggs have great value in phylogenetic studies

of Pleuronectiformes. In certain groups, however, the eggs

of Pleuronectiformes are still too poorly known. Therefore,

gaining accurate information on the morphological char-

acters of the eggs and applying them to understand phy-

logenetic status of the flounder species would properly be

useful. However, caution must be used when generalizing

surface structures of eggs to infer phylogenetic relation-

ships among species.

Acknowledgments The present study could not have been carried

out without the willing help of those listed below in collecting

specimens: Mr. Z. Song and Mrs. J. Zhao. Thanks to Dr. N. Anene for

proofreading the manuscript. The National Key Basic Research Pro-

gram from the Ministry of Science and Technology, P.R. China

(2005CB422306), the National High Technology Research and

Development Program from the Ministry of Science and Technology,

P.R. China (2006AA09Z418) and the National Department Public

Benefit Research Foundation from the Ministry of Agriculture,

P.R. China (200903005) supported this work. Any fieldwork in this

study complied with the current laws of P.R. China, in which it was

performed.

References

Ahlstrom EH, Amaoka K, Hensley D, Moser HG, Sumida BY (1984)

Pleuronectiformes development. In: Moser HG, Richards WJ,

Cohen DM, Fahay MP, Kendall AW, Richardson SL (eds)

Ontogeny and systematics of fishes. Special publication 1.

American Society of Ichthyologists and Herpetologists, Lawrence,

pp 640–670

Andoh T, Matsubara T, Harumi T, Yanagimachi R (2008) The use of

poly-L-lysine to facilitate examination of sperm entry into

pelagic, non-adhesive fish eggs. Int J Dev Biol 52:753–757

Azevedo MFC, Oliveira C, Pardo BG, Martınez P, Foresti F (2008)

Phylogenetic analysis of the order Pleuronectiformes (Teleostei)

based on sequences of 12S and 16S mitochondrial genes. Genet

Mol Biol 31:284–292

Chen KC, Shao KT, Yang JS (1999) Using micropylar ultrastructure

for species identification and phylogenetic inference among four

species of Sparidae. J Fish Biol 55: 288–300

Chen CH, Wu CC, Shao KT, Yang JS (2007) Chorion microstruc-

ture for identifying five fish eggs of Apogonidae. J Fish Biol

71:913–919

Coward K, Bromage NR, Hibbitt O, Parrington J (2002) Gamete

physiology, fertilization and egg activation in teleost fish. Rev

Fish Biol Fish 12:33–58

Fausto AM, Picchietti S, Taddei AR, Zeni C, Scapigliati G,

Mazzini M, Abelli L (2004) Formation of the egg envelope of

a teleost, Dicentrarchus labrax (L): immunochemical and

cytochemical detection of multiple components. Anat Embryol

208:43–53

Francisco JA, Medina A (2005) Ultrastructure of oogenesis in the

bluefin tuna, Thunnus thynnus. J Morphol 264:149–160

Ganeco LN, Franceschini-Vicentini IB, Nakaghi LS (2008) Structural

analysis of fertilization in the fish Brycon orbignyanus. Zygote

17:93–99

Gwo HH (2008) Morphology of the fertilizable mature egg in the

Acanthopagrus latus, A. schlegeli and Sparus sarba (Teleostei:

Perciformes: Sparidae). J Microsc 232:442–452

Hagstrom BE, Lonning S (1968) Electron microscopic studies of

unfertilized and fertilized eggs from marine teleosts. Sarsia

33:73–80

Hensley D, Ahlstrom EH (1984) Pleuronectiformes: relationships. In:

Moser HG, Richards WJ, Cohen DM, Fahay MP, Kendall AW,

Richardson SL (eds) Ontogeny and systematics of fishes. Special

publication 1. American Society of Ichthyologists and Herpe-

tologists, Lawrence, pp 670–688

Hirai A (1988) Fine structures of the micropyles of pelagic eggs of

some marine fishes. Jpn J Ichthyol 35:351–357

Hirai A (1993) Fine structure of the egg membranes in four species of

Pleuronectinae. Jpn J Ichthyol 40:227–235

Iconomidou VA, Chryssikos DG, Gionis V, Pavlidis MA, Paipetis A,

Hamodrakas SJ (2000) Secondary structure of chorion proteins

of the teleostean fish Dentex dentex by ATR FT-IR and

FT-Raman spectroscopy. J Struct Biol 132:112–122

Morphology of multiple spawning flounder eggs 355

123

Ivankov VN, Kurdyayeva VP (1973) Systematic differences and the

ecological importance of the membranes in fish eggs. J Ichthyol

13:864–873

Iwamatsu T (2000) Fertilization in fishes. In: Tarın JJ, Cano A (eds)

Fertilization in protozoa and metazoan animals. Springer-Verlag

Berlin, Heidelberg, pp 89–145

Iwamatsu T, Ohta T (1981) Scanning electron microscopic observa-

tion on sperm penetration in teleostean fish. J Exp Zool 218:

261–277

Iwamatsu T, Onitake K, Yoshimoto Y, Hiramoto Y (1991) Time

sequence of early events in fertilization in the Medaka egg. Dev

Growth Differ 33:479–490

Iwamatsu T, Ishijima S, Nakashima S (1993a) Movement of

spermatozoa and changes in micropyles during fertilization in

medaka eggs. J Exp Zool 266:57–64

Iwamatsu T, Nakashima S, Onitake K (1993b) Spiral patterns in the

micropylar wall and filaments on the chorion in eggs of the

medaka, Oryzias latipes. J Exp Zool 267:225–232

Kajimura S, Yoshiura Y, Suzuki M, Aida K (2001) cDNA cloning of

two gonadotropin b subunits (GTH-Ib and II-b) and their

expression profiles during gametogenesis in the Japanese

flounder (Paralichthys olivaceus). Gen Comp Endocrinol

122:117–129

Kobayashi W, Yamamoto TS (1981) Fine structure of the micropylar

apparatus of the chum salmon egg, with a discussion of the

mechanism for blocking polyspermy. J Exp Zool 217:265–275

Kobayashi W, Yamamoto TS (1985) Fine structure of the micropylar

cell and its change during oocyte maturation in the chum salmon,

Oncorhynchus keta. J Morphol 184:263–276

Kobayashi W, Yamamoto TS (1993) Factors inducing closure of

the micropylar canal in the chum salmon egg. J Fish Biol

42:385–394

Kudo S, Teshima C (1998) Assembly in vitro of vitelline envelope

components induced by a cortical alveolus sialoglycoprotein of

eggs of the fish Tribolodon hakonensis. Zygote 6:193–202

Laale HW (1980) The perivitelline space and egg envelopes of bony

fishes: a review. Copeia 1980:210–226

Lei JL, Ma AJ, Liu XF, Men Q (2003) Study on the development of

embryo, larval and juvenile of turbot Scophthalmus maximusl.Oceanol Limnol Sin 34:9–18

Li YH, Wu CC, Yang JS (2000) Comparative ultrastructural studies

of the zona radiata of marine fish eggs in three genera in

Perciformes. J Fish Biol 56:615–621

Lonning S (1972) Comparative electronmicroscopic studies of

teleostean eggs with special reference to the chorion. Sarsia

49:41–48

Marques C, Nakaghi LSO, Faustino F, Ganeco LN, Senhorini JA

(2008) Observation of the embryonic development in Pseudo-platystoma coruscans (Siluriformes: Pimelodidae) under light

and scanning electron microscopy. Zygote 16:333–342

Masuda K, Murata K, Iuchi I, Yamagami K (1992) Some properties

of the hardening process in chorions isolated from unfertilized

eggs of medaka Oryzias latipes. Dev Growth Differ 34:545–552

McEvoy LA, McEvoy J (1992) Multiple spawning in several

commercial fish species and its consequences for fisheries

management, cultivation and experimentation. J Fish Biol

41(Suppl b):125–136

Meloni S, Mazzini M, Fausto A, Macchioni R, Taddei A, Buonocore

F, Fiani M, Baldacci A, Scapigliati G (2004) Egg envelope

organization in the icefish Chionodraco hamatus. Polar Biol

27:586–594

Minami T (1984) Early life history of flatfishes–III. Characteristics of

eggs. Aquabiology 6:46–49

Mito S (1963) Pelagic fish eggs from Japanese water–IX Echeneida

and Pleuronectida. Jpn J Ichthyol 11:81–102

Morrison C, Bird C, O’Neil D, Leggiadro C, Martin-Robichaud D,

Rommens M, Waiwood K (1999) Structure of the egg envelope

of the haddock, Melanogrammus aeglefinus, and effects

of microbial colonization during incubation. Can J Zool 77:

890–901

Motta CM, Tammaro S, Simoniello P, Prisco M, Richiari L,

Andreuccetti P, Filosa S (2005) Characterization of cortical

alveoli content in several species of Antarctic notothenioids.

J Fish Biol 66:442–453

Murata K (2003) Blocks to polyspermy in fish: a brief review. In:

Sakai Y, McVey JP, Jang D, McVey E, Caesar M (eds)

Aquaculture and pathobiology of crustacean and other species.

Proceedings of the Thirty-second U S Japan Symposium on

Aquaculture Panel Symposium, Davis and Santa Barbara,

California USA, pp 1–15

Nakashima S, Iwamatsu T (1994) Ultrastructural changes in micro-

pylar and granulosa cells during in vitro oocyte maturation in the

medaka, Oryzias latipes. J Exp Zool 270:547–556

Nelson JS (2006) Fishes of the world, 4th edn. John Wiley & Sons

Inc, New York

Olivar MP (1987) Chorion ultrastructure of some fish eggs from the

southeast Atlantic. S Afr J Mar Sci 5:659–671

Oppen-Berntsen DO, Helvik JV, Walther BT (1990) The major

structure proteins of cod (Gadus morhua) eggshell and protein

crosslinking during teleost egg hardening. Dev Biol 137:

258–265

Ortiz-Delgado JB, Porcelloni S, Fossi C, Sarasquete C (2008)

Histochemical characterization of oocytes of the swordfish

Xiphias gladius. Sci Mar 72:549–564

Otani S, Iwai T, Nakahata S, Sakai C, Yamashita M (2009) Artificial

fertilization by intracytoplasmic sperm injection in a teleost fish,

the Medaka (Oryzias latipes). Biol Reprod 80:175–183

Park JY, Richardson KC, Kim IS (1998) Developmental changes of

the oocyte and its enveloping layers, in Micropercops swinhonis(Pisces: Perciformes). Korean J Biol Sci 2:501–506

Perry DM (1984) Post-fertilization changes in the chorion of winter

flounder, Pseudopleuronectes americanus Walbaum, eggs

observed with scanning electron microscopy. J Fish Biol

25:83–94

Ravaglia MA, Maggese MC (2003) Ovarian follicle ultrastructure in

the teleost Synbranchus marmoratus (Bloch 1795), with special

reference to the vitelline envelope development. Tissue Cell

35:9–17

Riehl R (1993) Surface morphology and micropyle as a tool for

identifying fish eggs by scanning electron microscopy. Eur

Microsc Anal 5:29–31

Riehl R, Gotting KJ (1974) Zu Struktur und Vorkommen der

Mikropyle an Eizellen und Eiern von Knochenfischen (Teleosti).

Arch Hydrobiol 74:393–402

Riehl R, Schulte E (1978) Bestimmungsschlussel der wichtigsten

deutschen Susswasser-Teleosteer anhand ihrer Eier. Arch

Hydrobiol 83:200–212

Sawaguchi S, Ohkubo N, Aritaki M, Ohta K, Matsubara T (2006)

Process of final oocyte maturation and ovulation cycle in captive

spotted halibut, Verasper variegates. Aquac Sci 54:465–472

Spies RB, Rice DW Jr, Felton J (1988) Effects of organic

contaminants on reproduction of the starry flounder Platichthysstellatus in San Francisco Bay. II. Reproductive success of fish

captured in San Francisco Bay and spawned in the laboratory.

Mar Biol 98:191–200

Stehr CM, Hawkes JW (1979) The comparative ultrastructure of

the egg membrane and associated pore structures in the

starry flounder, Platichthys stellatus (Pallas), and pink salmon,

Oncorhynchus gorbuscha (Walbaum). Cell Tissue Res 202:

347–356

356 X. Bian et al.

123

Stehr CM, Hawkes JW (1983) The development of the hexagonally

structured egg envelope of the C-D sole (Pleuronichthyscoenosus). J Morphol 178:267–284

Takano K, Ohta H (1982) Ultrastructure of micropylar cells in the

ovarian follicles of the pond smelt, Hypomesus transpacificusnipponemis. Bull Fac Fish Hokkaido Univ 33:65–78

Wang B, Liu ZH, Sun PX, Wang ZL, Liu P, Teng ZJ (2008) The

morphological observation on the embryonic development of Starry

flounder, Platichthys stellatus. Acta Oceanol Sin 30:131–136

Yamamoto K (1952) Studies on the fertilization of the egg of the

f1ounder. II. The morphological structure of the micropyle and

its behavior in response to sperm-entry. Cytologia 16:302–306

Yamamoto TS, Kobayashi W (1992) Closure of the micropyle during

embryonic development of some pelagic fish eggs. J Fish Biol

40:225–241

Zhang XW, He GF, Sha XS (1965) A description of the important

morphological characters of the eggs and larvae of the two flat

fishes, Paralichthys olivaceus (T&S) and Zebrias zebra (Bloch).

Acta Oceanol Sin 7:158–173

Zotin A (1958) The mechanism of hardening of the salmonid

egg membrane after fertilization or spontaneous activation.

J Embryol Exp Morphol 6:546–568

Morphology of multiple spawning flounder eggs 357

123