ORIGINAL ARTICLE Aquaculture

Abnormal elongation of the lower jaw in juvenile Japaneseflounder: combined effects of a rotifer diet enrichedwith Nannochloropsis preserved by various methodsand parentage

Eitaro Sawayama • Syuichi Sakamoto •

Motohiro Takagi

Received: 19 November 2011 / Accepted: 9 March 2012 / Published online: 7 April 2012

� The Japanese Society of Fisheries Science 2012

Abstract To elucidate possible causes of skeletal mal-

formations in larval Japanese flounder, we reared larvae fed

rotifers enriched with three types of preserved Nanno-

chloropsis (fresh, refrigerated, and frozen). The incidence

of malformations at 50 days post hatch ranged from 14.5 to

38.5 % within the three experimental groups, and elonga-

tion of the lower jaw (LJ) was the most frequently observed

malformation, ranging from 68 to 89 % of total malfor-

mations. We also investigated larval parentages using

microsatellite markers. Parentage analysis of the fresh

Nannochloropsis group showed that one sire and a pair

generated significant numbers of LJ-elongated individuals.

In the refrigerated Nannochloropsis group, one dam and

two sires generated significant numbers of LJ-elongated

individuals. In the frozen Nannochloropsis group, no

broodstocks or pairs generated significant numbers of

LJ-elongated individuals. Our results suggest that LJ

elongation in Japanese flounder likely results from the

application of different types of preserved Nannochloropsis

during rotifer feeding stage. However, there is also some

level of genetic influence associated with this deformity.

Keywords Japanese flounder � Lower jaw elongation �Microsatellite DNA � Nannochloropsis � Parentage analysis

Introduction

Incidence of body malformations is a serious drawback of

fish aquaculture. Malformations can affect different aspects

of morphology, such as pigmentation [1, 2], skeleton [3–6],

and swimbladder [7–9]. The high incidence of some mal-

formations significantly reduces the market value of the

species sold as whole fish and implies additional screening

effort at farms. The high incidence of malformations also

increases costs for seed production companies, and acci-

dental mixing of malformed fish in sold products degrades

company reputation.

Japanese flounder Paralichthys olivaceus is an eco-

nomically important fish species that is widely distributed

around Japan. About 5 million juveniles are produced by

broodstock parents per year at many private companies in

western Japan [10]. Skeletal malformations, mostly in body

shape and jaw, have often occurred in seed production of

this species. Several studies have been conducted to iden-

tify the mechanisms of skeletal malformation in Japanese

flounder [11–13], and the effects of fat-soluble vitamins,

A and D, in live food on some skeletal malformations are

well characterized [1, 11–13]. However, some skeletal

malformations persist as serious problems even with the

improvements of nutritional values in live foods. Jaw

malformations, manifested as shortened upper jaw (UJ)

[14], lower jaw (LJ) elongation [15], shortened LJ [12], and

twisted jaws [16], are often found in seedlings of Japanese

flounder. These jaw malformations, especially LJ elonga-

tion, are prevalent, being found at a rate of 20–30 % of

seed production (Sawayama, unpublished). There is,

E. Sawayama

R&D Division, Marua Suisan Co., Ltd., 4472 Kamijima-Iwagi,

Ehime 794-2410, Japan

S. Sakamoto

Oriental Yeast Co., Ltd., 3-6-10 Itabashi-Azusawa,

Tokyo 174-8505, Japan

M. Takagi (&)

South Ehime Fisheries Research Center, Tarumi Branch,

Ehime University, 3-5-7 Matsuyama-Tarumi,

Ehime 790-8566, Japan

e-mail: takagi@agr.ehime-u.ac.jp

123

Fish Sci (2012) 78:631–640

DOI 10.1007/s12562-012-0494-4

therefore, a need to identify causative factors and develop

preventative methods.

The phytoplankton Nannochloropsis stores large

quantities of eicosapentaenoic acid (EPA), known to be

an essential fatty acid for marine fish [17], in the intra-

cellular region, and Nannochloropsis is commonly used

as a rotifer diet before feeding to fish larvae [18]. How-

ever, we have empirically observed that the incidence of

LJ elongation was affected by the quality of Nanno-

chloropsis (preservation at 4 and -20 �C for long peri-

ods) as rotifer enrichment material during seed production

of Japanese flounder, suggesting this as one of the caus-

ative factors.

Recently, parentage analysis based on microsatellite

DNA has been used for estimation of genetic factors

involved in several malformations [2, 19–23]. This method

involves collecting normal and malformed individuals

from the same tank and then assessing parentage using

microsatellite DNA polymorphisms. The assigned off-

spring are divided into full- and half-siblings, and inci-

dences of malformed individuals in each sibling are

compared with that of normal individuals. Hypothetically,

if particular siblings generate significant numbers of mal-

formed individuals, genetic factors are suggested as a

possible cause of the malformation [19–22]. If not, envi-

ronmental factors such as nutrition and water temperature

may be more significantly associated with the malforma-

tion [20, 23].

To understand the cause of LJ elongation, we first per-

formed rearing experiments on flounder fed rotifers enri-

ched with three types of preserved Nannochloropsis to

clarify whether preserved Nannochloropsis negatively

affect LJ elongation. Subsequent morphological observa-

tions of LJ-elongated individuals were also conducted.

Finally, we assessed parentages of the LJ-elongated indi-

viduals based on microsatellite DNA analysis to estimate

the influence of genetic factors on LJ elongation.

Materials and methods

Rearing experiment

Naturally spawned and fertilized eggs collected over 1 day

(the hatching rate was 98.8 %) from 4-year-old flounder

broodstocks (8 dams and 9 sires) cultured at Marua Suisan

Co., Ltd. (Ehime, Japan) were used for the experiments.

Eggs were divided into three groups in duplicate 500-l

polycarbonate tanks at density of 12,500 eggs/tank. Rear-

ing water was filtered and sterilized by ultraviolet (UV)

light. Water temperature was within normal parameters for

the species and ranged from 15.7 to 21.2 �C. The photo-

period was 12L:12D.

Three different storage conditions of Nannochloropsis

sp. were evaluated as follows: group A, fresh Nanno-

chloropsis cultured at Marua Suisan Co., Ltd.; group B,

refrigerated Nannochloropsis (stored at 4 �C); group C,

frozen Nannochloropsis (stored at -20 �C). The group A

Nannochloropsis originated from refrigerated Nanno-

chloropsis that was then cultured in outdoor 30-kl tanks

[24]. The refrigerated and frozen Nannochloropsis used in

groups B and C were concentrated products purchased

from a private company, and those Nannochloropsis

products were used immediately after purchase, although

the storage period at the supplier was unknown.

The rotifer Brachionus plicatilis sp. complex (S-type)

was first cultured with a 3-day batch method using con-

centrated freshwater Chlorella (V12; Chlorella Industry

Co., Ltd., Tokyo, Japan). After this culture, rotifers were

harvested and used for the secondary culture. In the sec-

ondary culture, three kinds of Nannochloropsis described

above were added to each 100-l polycarbonate tank to

reach density of 1 9 106 cells per rotifer, and harvested

rotifers were then cultured in the tanks for 8 h at density of

5 9 108 individuals/100 l. Docosahexaenoic acid (DHA)

enrichment (Bio Chromis, Chlorella Industry) was added at

concentration of 100 ml per 100 million rotifers for 12 h

with vigorous aeration. Water temperature of the secondary

culture was set at 25 �C.

Addition of rotifers to the experimental flounder tanks was

started at 4 days post hatch (dph) once or twice a day, and

feeding densities were set at 5–10 individuals/ml in accor-

dance with larval growth. In addition, the same Nanno-

chloropsis fed to rotifers were added to the experimental tank

once or twice daily to reach 5 9 105 cells/ml. From 20 dph,

Artemia nauplii enriched with the DHA enrichment material

(Bio Chromis) at concentration of 100 ml per 10 million

nauplii were added once or twice a day in all tanks. Larvae

were fed commercial aquaculture diets (Love Larvae; Hay-

ashikane Sangyo Co., Ltd., Yamaguchi, Japan) from 30 dph,

supplied manually once or twice a day.

Sample collection

All fish were collected at 50 dph, and survival was evalu-

ated by counting the individuals in each tank. For growth

analysis, a random sample of 30 individuals was taken

from each tank at 50 dph, and total length (mm) and body

weight (g) were measured. Condition factor (CF) was also

calculated according to the following formula: CF = W/

L3 9 103, where W is weight (g) and L is total length (cm).

Also, a random sample of 100 individuals was taken from

each tank, and examined visually to count and categorize

malformations. Fifty each of normal individuals and LJ-

elongated individuals in groups A, B, and C were collected

and stored in 99.5 % ethanol for further morphological and

632 Fish Sci (2012) 78:631–640

123

parentage analysis. We also observed larval morphology at

4 dph (n = 30 in each tank) by stereomicroscope (SZ61;

Nikon, Tokyo, Japan).

Morphological observation of lower jaw elongation

LJ elongation (Fig. 1) was the most common malformation

in this experiment, and for a better understanding of this,

fifty each of normal individuals and LJ-elongated individ-

uals were photographed using a soft X-ray system (B-5;

Softex, Kanagawa, Japan). Juveniles were placed on an

X-ray film and exposed for approximately 15 s with soft

X-rays at 5 mA, 18 kV. From the X-radiographs, length of

UJ and LJ, and standard length of specimens were mea-

sured by stereomicroscope using NIS-Element software

(Nikon, Tokyo, Japan). Standard length was defined as the

length from the tip of snout to the end of urostyle. UJ

length was defined as the length from the tip of premaxilla

to the end of maxilla, and LJ length was defined as the

length from the tip of dentary to the end of angular. All

measurements were compared relative to standard length.

Twenty each of normal individuals and LJ-elongated

individuals collected from group A were also stained by

Alizarin Red S, as described by Kawamura and Hosoya

[25]. Data on premaxilla, maxilla, dentary, and angular

were collected by stereomicroscope and measured as

described above.

Fatty acid analysis

Rotifers were harvested with a 40-lm plankton net soon

after Nannochloropsis and DHA enrichment as described

above, then rinsed with sea water and stored at -80 �C.

Total lipids were extracted from the rotifers using the

chloroform and methanol (2:1) method [26]. Fatty acid

methyl esters were prepared and analyzed by gas–liquid

chromatography. Fatty acids were measured at Japan Food

Research Laboratories (Tokyo, Japan).

Microsatellite allele detection and parentage analysis

To better understand the causative factors of LJ elongation,

parentage analysis using microsatellite DNA was con-

ducted. Genome DNA was extracted by Wizard� genomic

DNA purification kit (Promega, WI, USA) from portions of

caudal fin of broodstocks, normal individuals, and LJ-

elongated individuals in groups A, B, and C according to

the manufacture’s protocol. The primers of four polymor-

phic microsatellite loci, Pol-1*, -3*, -4*, and -5* [27], were

amplified by polymerase chain reaction (PCR). The general

PCR protocol was: 1 ll of extracted DNA, 0.1 lM primer,

0.2 lM deoxynucleotide triphosphates (dNTPs), 0.05 ll

99 % formamide, 2.5 U Ex Taq polymerase (TAKARA

BIO, Shiga, Japan) with 109 buffers in total volumes to

5 ll. The PCR reactions were carried out on a PC816

(ASTEC, Fukuoka, Japan) using the following profile:

30 cycles of 95 �C for 30 s, 50 �C for 30 s, and 72 �C for

30 s. Forward primers were labeled with fluorescent dyes,

and reverse primers were tailed (Applied Biosystems, CA,

USA). The PCR products were separated by electropho-

resis using an ABI Prism� 310 genetic analyzer (Applied

Biosystems) for fluorescent-labeled PCR products. Alleles

were scored using GeneMapper� v4.0 software package

(Applied Biosystems).

Fig. 1 Pictures of normal individual and lower-jaw-elongated

individual, the most frequently observed malformation, taken by soft

X-rays: a normal individual; b lower-jaw-elongated individual, with

arrow indicating lower jaw elongation; c components of upper and

lower jaws; letters A–D indicate the positions of premaxilla, maxilla,

dentary, and angular, respectively. Bars represent 10 mm (a, b) or

5 mm (c)

Fish Sci (2012) 78:631–640 633

123

Potential parental pairs of each individual were explored

using the microsatellite information on the basis of the

likelihood-based parental allocation approach, which is

available in CERVUS 3.0 [28]. One microsatellite marker,

Po56* [29], was added for unassigned individuals by 4

microsatellite loci and manually allocated for the potential

parents. The PCR reaction of Po56* was the same as

described above.

Statistical analysis

Data are presented as mean ± standard deviation. Tukey’s

test was used as the post hoc test to examine differences in

growth, survival, and incidence of malformations. Stu-

dent’s t test was used to examine differences in upper and

lower jaw length and jaw bone measurements. Parentage

data relative to LJ elongation were analyzed by contin-

gency table analysis using v2 test. For LJ-elongated indi-

viduals, individual numbers in each broodstock were

compared with numbers of normal individuals.

All statistical analyses were performed using GraphPad

Prism 5.0 software (GraphPad Software, CA, USA). Dif-

ferences from equilibrium were accepted as significant

when P \ 0.05.

Results

Larval growth, survival rate, and incidence

of malformations

Growth performance at 50 dph was represented by the

following means: total length, body weight, condition

factor, survival rates, and incidence of malformation, as

summarized in Table 1. Total length, body weight, and

condition factor of fish in groups A, B, and C were not

significantly different (P [ 0.05). Survival rates were also

not significantly different in groups A, B, and C

(P [ 0.05). Incidences of malformations in group B and C

were significantly higher than in group A (P \ 0.05).

The malformations categorized in this experiment are

summarized in Table 2. LJ elongation was commonly

observed and dominant in all experimental groups. Most of

the other malformations observed occurred below 10 %,

except for missing opercula which occurred at 17.1 % in

group C. No obvious malformed individuals were observed

at 4 dph.

Measurements of jaw and bone lengths

Lengths of UJ and LJ (as %SL) in the fresh Nannochlor-

opsis group are shown in Fig. 2. Mean ratios of UJ were

18.08 % (±0.97) in normal individuals and 18.19 %

(±1.11) in LJ-elongated individuals (P [ 0.05). Mean

ratios of LJ were 17.09 % (±0.95) in normal individuals

and 18.95 % (±0.97) in LJ-elongated individuals

(P \ 0.05).

We also measured premaxilla and maxilla (Fig. 3),

dentary, and angular (Fig. 4), which are components of UJ

and LJ, to determine if the LJ was elongated. Mean ratios

of the premaxilla on the ocular and blind sides were

10.24 % (±1.04) and 10.47 % (±0.52) in normal individ-

uals and 10.29 % (±0.54) and 10.16 % (±0.50) in LJ-

elongated individuals (P [ 0.05). Mean ratios of the

maxilla on both sides were 11.99 % (±0.55) and 11.89 %

(±0.53) in normal individuals and 11.71 % (±0.57) and

11.77 % (±0.49) in LJ-elongated individuals (P [ 0.05).

Mean ratios of the dentary were 11.17 % (±0.60) and

Table 1 Growth performance of experimental groups at 50 dph

Experimental group TL (mm) BW (g) CF Survival (%) Malformation (%)

A (fresh) 22.57 ± 0.77 0.11 ± 0.01 9.70 ± 0.22 7.66 ± 1.88 14.5 ± 0.50b

B (refrigerated) 23.66 ± 0.63 0.13 ± 0.01 9.54 ± 0.38 7.07 ± 0.32 34.5 ± 1.50a

C (frozen) 20.58 ± 0.70 0.10 ± 0.01 11.14 ± 0.28 2.50 ± 0.94 38.5 ± 0.50a

TL, BW, and CF were measured using 30 individuals in each experimental tank. Survival was evaluated by counting the numbers of all

individuals in each experimental tank. Malformation was calculated using 100 individuals in each experimental tank. All values represent

mean ± standard deviation (SD) of two tanks in each experimental group. Subscripts in malformation indicate Tukey’s test comparing groups

(P \ 0.05, a [ b)

Table 2 Malformations: type and incidence (%) among experi-

mental groups at 50 dph

Malformation Experimental group

A (fresh) B (refrigerated) C (frozen)

Lower jaw elongation 86.7 ± 1.4 82.4 ± 0.7 65.8 ± 2.8

Shortened lower jaw 6.7 ± 0.0 2.7 ± 1.4 2.6 ± 0.0

Twisted jaws 0.0 8.1 ± 1.4 9.2 ± 0.7

Body curvature 6.7 ± 1.4 1.4 ± 0.7 0.0

Shortened body 0.0 0.0 5.3 ± 1.4

Missing opercula 0.0 5.4 ± 0.0 17.1 ± 0.7

Malformations were categorized using 100 individuals in each

experimental tank. All values represent mean ± SD of two tanks in

each experimental group

634 Fish Sci (2012) 78:631–640

123

11.79 % (±0.60) in normal individuals and 12.03 %

(±0.36) and 12.61 % (±0.40) in LJ-elongated individuals,

and significant differences were observed (P \ 0.05).

Mean ratios of the angular were 11.26 % (±0.58) and

11.71 % (±0.73) in normal individuals and 12.35 %

(±0.64) and 12.67 % (±0.41) in LJ-elongated individuals,

and significant differences were observed (P \ 0.05).

Fatty acid composition of rotifers

The fatty acid composition of rotifers used in the rearing

experiment is presented in Table 3. Crude lipids ranged

from 11.7 % (group C) to 13.2 % (group A). Total n-3

highly unsaturated fatty acids of rotifers ranged from

2.70 % (group C) to 4.83 % (group A) on dry matter basis,

and the content of DHA also ranged from 1.71 %

(group C) to 3.27 % (group A) on dry matter basis. The

EPA content in rotifers used in group A was highest

(0.92 %), and that in group C was lowest (0.53 %).

10

12

14

16

18

20

22

10 15 20 2510

12

14

16

18

20

22

10 15 20 25

Standard length (SL; mm)

LJ le

ngth

(%

SL)

UJ

leng

th (

% S

L)

Fig. 2 Comparisons of relative length of upper (UJ) and lower (LJ)

jaws in normal individuals and LJ-elongated individuals in the fresh

Nannochloropsis group. Open circles indicate normal, closed circlesindicate LJ-elongated individuals

7

8

9

10

11

12

13

10 15 20 25

premaxilla (ocular)

7

8

9

10

11

12

13

10 15 20 25

premaxilla (blind)

10

11

12

13

14

10 15 20 25

maxilla (ocular)

10

11

12

13

14

10 15 20 25

maxilla (blind)

Standard length (SL; mm)

Leng

th (

% S

L)

Fig. 3 Comparisons of relative length of bones of the upper jaw in

normal individuals and LJ-elongated individuals in the fresh Nanno-chloropsis group. Open circles indicate normal, closed circlesindicate LJ-elongated individuals

10

11

12

13

14

10 15 20 25

dentary (ocular)

10

11

12

13

14

10 15 20 25

dentary (blind)

10

11

12

13

14

10 15 20 25

angular (ocular)

10

11

12

13

14

10 15 20 25

angular (blind)

Standard length (SL; mm)

Leng

th (

% S

L)

Fig. 4 Comparisons of relative length of bones of the lower jaw in

normal individuals and LJ-elongated individuals in the fresh Nanno-chloropsis group. Open circles indicate normal, closed circlesindicate LJ-elongated individuals

Fish Sci (2012) 78:631–640 635

123

Parentage analysis

The four loci used in the microsatellite analysis allowed the

assignment of 296 out of the 300 genotyped offspring

(98.7 %), and adding Po56* allowed the assignment of 1

out of the 4 unassigned offspring, allowing genotyping of

99.0 % of offspring. The representations of the offspring in

the different families are given in Table 4. In group A,

normal individuals from dams 3, 4, 6, and 7 were observed

(22.4, 20.4, 28.6, and 16.3 %, respectively). In addition,

LJ-elongated individuals from dams 1, 3, 5, and 6 were

observed (14.6, 37.5, 18.8, and 16.7 %, respectively). No

significant differences were observed between numbers of

normal individuals and LJ-elongated individuals from each

dam (P [ 0.05). Normal individuals from sires 3, 4, 6, 7,

and 8 were observed in group A (20.4, 12.2, 12.2, 26.5, and

20.4 %, respectively). Also, LJ-elongated individuals from

sires 3, 6, and 7 were observed (14.6, 16.7, and 50.0 %,

respectively), and sire 7 was significantly associated with

LJ-elongated individuals (P [ 0.05). We also analyzed

interaction of sire 7 with dams, and a coupling of sire 7

with dam 3 generated only LJ-elongated individuals (sig-

nificant at P \ 0.05).

In group B, normal individuals from female brood-

stocks 3, 6, and 7 were observed at 26.0, 36.0, and 18.0 %,

respectively. LJ-elongated individuals from dams 1, 3, 5, 6,

and 7 were observed at 16.0, 26.0, 18.0, 16.0, and 14.0 %,

respectively. Significant differences were observed

between numbers of normal individuals and LJ-elongated

individuals from dam 1 (P \ 0.05). Normal individuals

from sires 3 and 7 were observed in group B at 32.0 and

42.0 %, respectively. Among sires, LJ-elongated individ-

uals from sires 6, 7, and 8 were observed at 24.0, 28.0, and

18.0 %, respectively. LJ-elongated individuals were gen-

erated at a significantly higher rate from sires 5 and 6

(P \ 0.05). No significant difference was observed

between numbers of normal individuals and LJ-elongated

individuals in each parental pair (P [ 0.05).

In group C, normal individuals from dams 1, 3, 5, 6, and

7 were observed at 18.0, 20.0, 20.0, 16.0, and 14.0 %,

respectively. LJ-elongated individuals from dams 1, 5, and

7 were observed at 26.0, 32.0, and 20.0 %, respectively. No

significant difference was observed between numbers of

normal individuals and LJ-elongated individuals from each

dam (P [ 0.05). Normal individuals from sires 3, 6, and 7

were observed in group C at 20.0, 26.0, and 30.0 %,

respectively, and LJ-elongated individuals from sires 5, 7,

and 8 were observed at 20.0, 38.0, and 16.0 %, respec-

tively. No significant difference was observed between

numbers of normal individuals and LJ-elongated individ-

uals from each sire (P [ 0.05).

We also assessed incidence of LJ-elongated individuals

in half-sibling families between groups A, B, and C. The

number of LJ-elongated individuals from every dam was

not significantly different between each group. However,

sire 8 generated small numbers of LJ-elongated individuals

in group A (4.2 %), and significant numbers of LJ-elon-

gated individuals were observed in groups B (18.0 %) and

C (16.0 %) (P \ 0.05).

Discussion

Dietary effects on LJ elongation

The incidences of malformations in groups B and C were

significantly higher than in group A. This suggests that

rotifers enriched with Nannochloropsis concentrated and

preserved at 4 and -20 �C result in increased skeletal

malformations, especially LJ elongation. In this rearing

experiment, Artemia and formula diets were uniform across

all groups, and the only dietary variable was Nannochlor-

opsis. Therefore, the incidence of LJ elongation was pos-

sibly affected by the quality of Nannochloropsis or those

storage techniques. Suzuki et al. [30] reported that envi-

ronmental factors in early larval stages, especially during

cartilage formation, cause LJ abnormality in Japanese

flounder. In this rearing experiment, no obvious malformed

individuals were observed at 4 dph in all experimental

tanks, and this result may eliminate the possibility of

rearing conditions affecting LJ elongation before rotifer

feeding.

In this study, we did not prepare refrigerated and frozen

Nannochloropsis from the same lot of fresh Nannochlor-

opsis cultured at Marua Suisan Co., Ltd. because of facility

limitations. Several studies have shown that culture con-

ditions such as light intensity [31], temperature [31, 32],

pH [33], culture media [34], and growth phases [35] may

influence essential fatty acid levels in several microalgae

Table 3 Crude lipid and fatty acid contents of rotifers enriched with

three kinds of Nannochloropsis stored under different conditions (%

total lipid on dry matter basis)

Rotifers enriched with Nannochloropsis

Fresh Refrigerated Frozen

Crude lipid 13.2 12.1 11.7

n-3 HUFAa 4.83 3.97 2.70

AA 0.38 0.35 0.30

EPA 0.92 0.90 0.53

DHA 3.27 2.26 1.71

AA arachidonic acid, EPA eicosapentaenoic acid, DHA docosahexae-

noic acida Total highly unsaturated fatty acid (20:3n-3; 20:5n-3; 21:5n-3;

22:5n-3; 22:6n-3)

636 Fish Sci (2012) 78:631–640

123

species including Nannochloropsis. Also, longer periods of

storage possibly lead to deterioration in lipid quality of

Nannochloropsis by oxidation. Therefore, the differences

in culture conditions and storage periods between fresh,

refrigerated, and frozen Nannochloropsis might influence

the larval quality observed in this study.

Several studies have demonstrated the usefulness of

preserved Nannochloropsis in rotifer culture [36, 37].

Seychelles et al. [38] also reported that frozen Nanno-

chloropsis contains sufficient arachidonic acid (AA) and

EPA, and therefore they concluded that rotifer fed frozen

Nannochloropsis could be a good source of AA and EPA

Table 4 Individual distributions and rate of normal and LJ-elongated individuals in full- and half-sibling families

Sire Dam Normal (%) LJ elongated (%)

1 2 3 4 5 6 7

Group A (fresh)

1 1/0 2.0 0.0

2 1/1 1/0 4.1 2.1

3 0/2a 2/1 3/0 4/2 1/2 20.4 14.6

4 4/1 2/0 0/1 12.2 4.2

5 1/1 0/4 2.0 8.3

6 1/1 2/3 1/1 2/1 0/1 0/1 12.2 16.7

7 1/4 1/0 0/11* 2/1 1/4 7/3 1/1 26.5 50.0*

8 0/1 2/0 3/1 5/0 20.4 4.2

9 0.0 0.0

Normal (%)b 4.1 2.0 22.4 20.4 6.1 28.6 16.3 100

LJ elongated (%)c 14.6 0.0 37.5 4.2 18.8 16.7 8.3 100

Group B (refrigerated)

1 1/0 2.0 0.0

2 1/2 2.0 4.0

3 3/0 0/1 0/1 8/2 5/1 32.0 10.0

4 2/1 1/2 6.0 6.0

5 0/3 0/2 0.0 10.0*

6 1/4 6/6 3/1 0/1 0/1 0/2 10.0 24.0*

7 0/1 0/2 2/0 1/1 2/2 9/1 3/0 42.0 28.0

8 2/0 0/3 0/2 1/4 6.0 18.0

9 0.0 0.0

Normal (%) 2.0 6.0 26.0 8.0 4.0 36.0 18.0 100

LJ elongated (%) 16.0* 4.0 26.0 6.0 18.0 16.0 14.0 100

Group C (frozen)

1 1/1 0/1 2.0 4.0

2 0.0 0.0

3 1/0 2/0 1/2 4/1 2/2 20.0 10.0

4 1/0 1/0 4.0 0.0

5 2/3 1/7 8.0 20.0

6 5/4 1/0 2/1 1/0 2/0 0/1 1/0 26.0 12.0

7 1/6 0/1 4/3 1/0 5/6 3/0 1/3 30.0 38.0

8 1/0 0/3 3/5 8.0 16.0

9 1/0 2.0 0.0

Normal (%) 18.0 6.0 20.0 6.0 20.0 16.0 14.0 100

LJ elongated (%) 26.0 4.0 8.0 4.0 32.0 6.0 20.0 100

* Significant difference (P \ 0.05)a Numbers of normal individuals/LJ-elongated individualsb (Numbers of normal individuals in each dam and sire/total numbers of normal individuals) 9 100c (Numbers of LJ-elongated individuals in each dam and sire/total numbers of LJ-elongated individuals) 9 100

Fish Sci (2012) 78:631–640 637

123

for larviculture. However, the EPA contents in rotifers

enriched with both preserved Nannochloropsis used in this

rearing experiment were decreased, compared with rotifers

enriched with fresh Nannochloropsis. Especially, the EPA

content of the rotifers enriched with frozen Nannochlor-

opsis was almost half of the recommended level for larval

Japanese flounder [39]. Also, the DHA contents in rotifers

enriched with both preserved Nannochloropsis were

decreased, compared with rotifers enriched with fresh

Nannochloropsis. We observed that preserved Nanno-

chloropsis flocculated more than fresh ones in the sec-

ondary culture tank (data not shown). This suggests that

flocculation occurred due to damage to Nannochloropsis

during the concentration and preservation process with

leakage of cell metabolites. Rotifers may not eat floccu-

lated Nannochloropsis due to its size, resulting in low

vitality and affecting EPA and DHA enrichment.

Morphological features of LJ elongation

LJ length of LJ-elongated individuals, the most observed

malformation in this experiment, was significantly longer

than that of normal individuals. Sawada et al. [14] reported

a deformity named ‘‘shortened UJ’’ in Japanese flounder

that has phenotypic similarity to LJ elongation, but UJ

length of normal individuals and LJ-elongated individuals

analyzed in this study were not significantly different. We

conclude, therefore, that the deformity is due to abnormal

elongation of the LJ. LJ length (in ratio to body length) is

typically[18 % in LJ-elongated individuals, and this value

will be a useful criterion in assessing LJ elongation in

20-mm-size juveniles.

We also measured the length of bones within the jaws,

and the dentary and angular of deformed individuals were

significantly longer than bones of normal individuals. This

result strongly supports the fact that the jaw deformity is

caused by abnormal elongation of bones composing the LJ,

and our research is the first to indicate this type of defor-

mity in Japanese flounder. However, LJ lengths of LJ-

elongated individuals partly overlapped with those of

normal individuals. We collected individuals with LJ

elongation by visual observation, and this might cause

some contamination by UJ-shortened individuals.

Parentage effects on LJ elongation

Because we used naturally spawned and fertilized eggs for

the rearing experiments, there was a possibility of con-

tamination by low-quality eggs. However, the hatching rate

of fertilized eggs used in this study was high (98.8 %), and

using high-quality fertilized eggs may eliminate the pos-

sibility of maternal effects such as egg quality affecting

deformity rate and survivorship [40].

Parentage analysis revealed that one sire and one parental

couple in group A, and one dam and two sires in group B,

were significantly associated with LJ-elongated individuals.

This suggests that some LJ-elongated individuals generated

from these broodstocks in groups A and B were possibly

affected by genetic factors. Similar results were observed in

previous studies [2, 19–22] and support our speculation that

genetic factors possibly affect LJ elongation in some LJ-

elongated individuals. In addition, the broodstocks signifi-

cantly contributing LJ-elongated individuals in groups A

and B were different. This suggests that genetic factors

associated with LJ-elongation traits interact with the nutri-

tional environment. Interactions of environment and genetic

factors are known to affect several malformations in fish [22,

41, 42]. This lends support to our conclusion that sensitivity

to the nutritional environment of LJ elongation may be

controlled by genetic background. However, in this study,

the relatively low number of larvae analyzed and the

experimental design did not allow us to estimate heritability,

thus our results only suggest the possibility of genetic

influences on LJ elongation.

The incidence of LJ-elongated individuals generated

from sire 8 in groups B and C was significantly higher than

that in group A. This also suggests that sire 8 has strong

sensitivity to the nutritional environment, including pres-

ervation of Nannochloropsis, and genetic background

probably controls individual variance for nutritional envi-

ronment, at least during rotifer feeding periods. During

normal rearing, it is difficult to find broodstocks that are

susceptible to inappropriate nutritional conditions. Thus,

most nutritional and feeding plans prioritize production

efficiency and ignore genetic sensitivity. No significant

difference was observed in the frequency of normal indi-

viduals and LJ-elongated individuals in group C. Within

this group, a number of malformations including twisted

jaws, missing opercula, and short body were observed at

higher frequency than in the other groups. Group C also

had the lowest survivorship. This possibly shows that, if

environmental factors are stronger than genetic influences,

genetic factors will be masked and/or hard to be detected.

Although LJ elongation in Japanese flounder is gener-

ally thought to result from an unsuitable nutritional envi-

ronment during larval stages, our findings support the

hypothesis of some additional level of genetic influence. To

prevent this malformation in Japanese flounder, use of

fresh and high-quality Nannochloropsis as rotifer enrich-

ment material is recommended. In addition to improving

the nutritional environment, additional effort is required in

selection and exclusion of broodstocks significantly asso-

ciated with LJ elongation.

Acknowledgments The authors greatly appreciate Y. Haga, Asso-

ciate Professor at Tokyo University of Marin Sciences and

638 Fish Sci (2012) 78:631–640

123

Technology, for useful advice on jaw measurement. We are also

grateful to Dr. T. Yamamoto and two anonymous reviewers for

providing constructive comments on the manuscript.

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