oryzias latipes belongs to the order of beloniformes and adrianichthyidae , and is known to be distributed in japan , china , and korea . in south korea , oryzias latipes are often found in freshwater areas ( e.g. , nakdong - river as well as other rivers flow into the east sea ) yet they are also discovered around brackishwater areas .
oryzias latipes are characterized with 1 ) small size , 2 ) short generation , 3 ) high resistance against water temperature , salinity , water quality , pollution , and oxygen content , and 4 ) ease of laboratory breeding hence they are widely utilized for study purposes such as genetics , physiology , and development researches ( kim & kim , 1993 ) .
there are several domestic / international studies investigated regarding taxonomy and distribution of oryzias latipes in the yunnan region of china ( chen et al . , 1989 ) , distribution areas and karyotypes of indian oryzias latipes ( uwa , 1986 ) , coefficient / measuring characteristics , and karyotypes of oryzias latipes found in southern / island areas of south korea ( kim & moon , 1987 ; magtoon & uwa , 1985 ; uwa & jeon , 1987 ) , chromosomes of oryzias latipes ( levan et al . , 1964 ) ,
two distinct population of korean oryzias latipes ( sakaizumi & jeon , 1987 ) , and list of subspecies for novel korean oryzias latipes sinensis ( kim & lee , 1992 ) ; however there is scant investigation in reference to spawning habits as well as developmental characteristics of larvae and juvenile oryzias latipes . in the present , therefore , the authors investiaged the morphology of larvae and juvenile oryzias latipes in their egg development and growth phases as a part of basic biological studies for the development of ornamental fishes , protection of oryzias latipes , and to provide preliminary data for taxonomic studies .
adult oryzias latipes and eggs laid on weeds were collected from a river in shinduck - dong , yeosu - si , jeollanam - do utilizing skimming nets ( mesh , 5.5 mm ) and dredge from may 2011 ( fig .
collected eggs were observed using a universal projector for egg sizes ( i.e. , the diameter of eggs ) while their morphological traits were investigated with a stereoscopic microscope .
hatched larvae were subsequently fed with water flea and feeding stuff for fish culture in the glass water tank ( 305030 cm ) while the temperature was maintaining 20.0~25.4c ( average 23.0c ) ; rearing water was daily changed ( morning and afternoon ; 1/2 per each time , respectively ) . in order to monitor morphological development of larvae and juvenile oryzias latipes , each part of body was observed and measured utilizing the stereoscopic microscope and universal projector from hatching to 35 days after hatching ; in each day , an average of ten fishes was anesthetized with ice and ms-222 sandoz ( tricaine methane sulfonate ) for measurements and developmental stages were determined as described elsewhere russell ( 1976 ) .
the earliest event for naturally hatched eggs was the formation of blastodisc was observed ( fig .
2 , b ) , and subsequently reached to the 4-cell stage at 1 hour 37 minutes ( fig .
the embryo reached at 8-cell stage and 32-cell stage after 2 hours 27 minutes and 3 hours 27 minutes of the blastodisc formation ( fig .
2 , g ) after 8 hours 39 minutes and 23 hours 37 minutes from the blastodisc , respectively . in turn
22 minutes of the formation of blastodisc , an elongated embryo was formed on the side of york ( fig .
2 , i ) , and then optic vesicle , kupffer 's vesicle , and myotome were formed in the embryo at 75 hours 57 minutes ( fig .
2 , j ) . the prototype of pectoral fin was form after 143 hours 37 minutes of blastodisc formation while the tail was split ; in this , the embryo started to move intermittently ( fig .
the embryo broke through the egg membrane and started to hatching from the head after 167 hours 27 minutes of formation of blastodisc ( fig .
the total length of hatched larvae was approximately 5.01 mm and swimming in a crowd near the bottom of water tank .
right after hatching , the total length of larvae was 4.95~5.10 mm ( average 5.01 mm ,
when hatched , most york was absorbed and the myotome was approximately 26~28 ( 8~9 + 18~19 ) and oil droplets were placed in the middle of the york ; dorsal fin , anal fin and the caudal fin were connected via membranes and the prototype of caudal fin was observed .
chromophores were slightly precipitated on eyes while melanophores were found on the upper part of head , back side of eyes , sides of embryo , and the air bladder with a shape of stars and dots . in this stage ,
larvae did not actively swim yet started to swim vigorously after 3~5 hours of hatching utilizing the caudal fin ( fig .
3 days after hatching , the total length of larvae was around 5.25~5.53 mm ( average 5.35 mm , n = 10 ) and active intake activities were observed ; the york was completely absorbed and the fishes were shifted to the post - larvae stage . in this stage , as the gastrointestinal tract was being developed , the anus was placed on the position of approximately 40% of the total length and a little in front of the lateral side .
while chromophores were precipitated on eyes , more melanophores were found on the head and air bladder ; these were shown along with the lateral side with shapes of stars and dots .
the caudal fin had three stems and the terminal notochord started to being bending ( fig .
, the post larvae had the total length of 5.45~5.60 mm ( average 5.52 mm , n = 30 ) with eight stems formed on the caudal fin . on the front lateral side
the post larvae represented the total length of 6.61~6.87 mm ( average 6.73 mm , n = 30 ) while melanophores were getting more clear and obvious on the head as well as air bladder .
the prototypes of pectoral fin and anal fin were shown , and rod - shaped bones on tail were developed ( fig .
the total length of post - larvae was 8.94~10.30 mm ( average 9.51 mm , n = 30 ) and 3 stems on the dorsal fin were formed .
the prototype was appeared on the ventral fin and melanophores were getting clearer along with the back side of fishes ( fig .
after 22 days of hatching , the post larvae had the 10.81~12.80 mm of the total length ( average 11.58 mm , n = 30 ) and dorsal fin as well as anal fin were being formed ; there were 6~7 stems of dorsal fin , 15~18 stems of anal fin , and 17~20 stems of caudal fin were formed ( fig .
the total length of post larvae was 11.50~13.50 mm ( average 12.47 mm , n = 30 ) and the ventral fin was being formed yet the primordial finfold was remained ( fig .
after 35 days of hatching , the total length of the post larvae was 13.95~15.30 mm ( average 14.64 mm , n = 30 ) .
all fins and numbers of fins were fixed , indicating that the juvenile stage was reached .
the earliest event for naturally hatched eggs was the formation of blastodisc was observed ( fig .
2 , b ) , and subsequently reached to the 4-cell stage at 1 hour 37 minutes ( fig .
the embryo reached at 8-cell stage and 32-cell stage after 2 hours 27 minutes and 3 hours 27 minutes of the blastodisc formation ( fig .
2 , g ) after 8 hours 39 minutes and 23 hours 37 minutes from the blastodisc , respectively . in turn
22 minutes of the formation of blastodisc , an elongated embryo was formed on the side of york ( fig .
2 , i ) , and then optic vesicle , kupffer 's vesicle , and myotome were formed in the embryo at 75 hours 57 minutes ( fig .
2 , j ) . the prototype of pectoral fin was form after 143 hours 37 minutes of blastodisc formation while the tail was split ; in this , the embryo started to move intermittently ( fig .
the embryo broke through the egg membrane and started to hatching from the head after 167 hours 27 minutes of formation of blastodisc ( fig .
the total length of hatched larvae was approximately 5.01 mm and swimming in a crowd near the bottom of water tank .
right after hatching , the total length of larvae was 4.95~5.10 mm ( average 5.01 mm , n = 10 ) while both anus and mouth were opened . when hatched , most york was absorbed and the myotome was approximately 26~28 ( 8~9 + 18~19 ) and oil droplets were placed in the middle of the york ; dorsal fin , anal fin and the caudal fin were connected via membranes and the prototype of caudal fin was observed .
chromophores were slightly precipitated on eyes while melanophores were found on the upper part of head , back side of eyes , sides of embryo , and the air bladder with a shape of stars and dots . in this stage ,
larvae did not actively swim yet started to swim vigorously after 3~5 hours of hatching utilizing the caudal fin ( fig .
3 days after hatching , the total length of larvae was around 5.25~5.53 mm ( average 5.35 mm , n = 10 ) and active intake activities were observed ; the york was completely absorbed and the fishes were shifted to the post - larvae stage . in this stage , as the gastrointestinal tract was being developed , the anus was placed on the position of approximately 40% of the total length and a little in front of the lateral side .
while chromophores were precipitated on eyes , more melanophores were found on the head and air bladder ; these were shown along with the lateral side with shapes of stars and dots .
the caudal fin had three stems and the terminal notochord started to being bending ( fig .
the post larvae had the total length of 5.45~5.60 mm ( average 5.52 mm , n = 30 ) with eight stems formed on the caudal fin .
the post larvae represented the total length of 6.61~6.87 mm ( average 6.73 mm , n = 30 ) while melanophores were getting more clear and obvious on the head as well as air bladder .
the prototypes of pectoral fin and anal fin were shown , and rod - shaped bones on tail were developed ( fig .
the total length of post - larvae was 8.94~10.30 mm ( average 9.51 mm , n = 30 ) and 3 stems on the dorsal fin were formed .
the prototype was appeared on the ventral fin and melanophores were getting clearer along with the back side of fishes ( fig .
after 22 days of hatching , the post larvae had the 10.81~12.80 mm of the total length ( average 11.58 mm , n = 30 ) and dorsal fin as well as anal fin were being formed ; there were 6~7 stems of dorsal fin , 15~18 stems of anal fin , and 17~20 stems of caudal fin were formed ( fig .
the total length of post larvae was 11.50~13.50 mm ( average 12.47 mm , n = 30 ) and the ventral fin was being formed yet the primordial finfold was remained ( fig .
the total length of the post larvae was 13.95~15.30 mm ( average 14.64 mm , n = 30 ) .
all fins and numbers of fins were fixed , indicating that the juvenile stage was reached .
when it comes to fish , morphological changes , observed in the development of larvae and juvenile fishes , have been widely utilized as important traits to identify classification such as family and genus ( okiyama , 1979 ) .
oryzias latipes is one of two fishes ( the other is oryzias sinensis ) that belongs to the oryzias genus ; these fishes are very similar yet no study has been done in regards to their spawning habits and early life history .
embryos of o. latipes are spherical transparent separate demersal eggs ; there are short hairs as well as a bunch of long hairs on the membrane ; these hairs stick together , working as sticking thread , thereby aiding to stick on weeds . in the present study ,
the average size of o. latipes eggs was ranging 1.29~1.40 mm ( average 1.35 mm , n = 50 ) which was slightly smaller than the observation of kim & kim ( 1993 ) while bigger than those of o. latipes sinensis ( 1.10~1.20 mm , average 1.15 mm ) .
also when it compares other freshwater fish , zacco koreanus with 3.090.07 mm diameter ( lee et al . ,
2013 ) showed the big difference with o. sinensis , the egg size of pseudogobio esocinus was 1.980.19 mm ( lee et al . , 2008 ) , the diameter of fertilized egg of luciogobius grandis was 2.060.23 mm ( yun et al .
, 2011 ) so that egg diameter of was smaller than other freshwater fish . in comparison with the saltwater fish , cinnamon clownfish ( amphiprion melanopus ) s fertilized size was 2.40.1 mm ( noh et al . , 2011 ) , so there was no difference , and scomber japonicus , otherwise , was smaller than o. latipes as the diameter 0.950.03 mm ( park et al . , 2008 ) .
on the other hand , we found that time took for hatching was approximately 167 hours 27 minutes from the formation of blastodisc ; this results is somewhat different compared to other study . in the study of kim & kim ( 1993 ) , it took approximately 9~10 days for hatching and such inconsistency might be due to the difference in water temperature .
the egg development of z. koreanus was completed around 68 hours after fertilization ( lee et al . , 2013 ) ,
the hatching time of p. esocinus was 164 hours ( lee et al . , 2008 ) , it was similar to o. latipes , and l. grandis was 120 hours 54 minutes ( yun et al . , 2011 ) , so it was earlier than o. latipes s hatching time .
cinnamon clownfish ( a. melanopus ) , saltwater aquarium fish , was 168 hours ( noh et al . , 2011 ) and it was also similar to o. latipes , in saltwater fish case , the time for hatching of s. japonicus was far faster than o. latipes , as 51 hours ( park et al . , 2008 ) .
right after the hatching , the total length of larvae was 4.95~5.10 mm ( average 5.01 mm , n=10 ) with opened anus and mouth . in the juvenile stage
was reached after the 35 days from hatching ; in this , the total length was found to be 14.64 mm .
freshwater fish , z. koreanus , the total length of larvae right after the hatching was 10.300.40 mm , in the juvenile stage was reached after the 13 days from hatching as the total length 16.980.56 mm ( lee et al . , 2013 ) , it was faster than o. latipes .
total length of larvae of p. esocinus was 4.610.83 mm , 16.220.65 mm after the 42 days from hatching was reached in the juvenile stage ( lee et al . , 2008 ) .
it showed similar to o. latipes , and l. grandis was 3.300.07 mm for larvae after hatching , 11.460.12 mm for juvenile after the 29 days ( yun et al . , 2011 ) .
saltwater aquarium fish , a. melanopus , showed larvae of average 3.50.3 mm , it was developed like adult after the 90 days as 41.00.3 mm ( noh et al . , 2011 ) .
the development was slower than o. latipes . in s. japonicus case , on the other hand ,
right after the hatching , the total length of larvae was average 2.750.04 mm , in the juvenile stage was reached after the 25 days from hatching ( average 55.956.74 mm ) ( park et al . , 2008 ) , so that there was difference from o. latipes .
 in order to monitor the developmental features of embryos , larvae , and juveniles of oryzias latipes ( temminck and schlegel ) , oryzias latipes was caught in river of shinduck - dong , yeosu - si , jeollanam - do , on may 2011 , and experiments were carried out in ichthyology laboratory at chonnam national university . 
 the blastodisc step was the first level for natural spawning . 
 the optic vesicle , kupffer 's vesicle , myotome began to appear 75 hours 57 minutes later . 
 after blastodisc development , the pectoral fins were made at 143 hours 37 minutes and the tail was separated started at the same time . 
 hatching was observed at 167 hours 27 minutes after blastodisc . 
 the total length of the hatched larvae was 4.95~5.10 mm ( mean , 5.01 mm ) , the mouth and anus were opened . 
 larvae used yolk completely after 3 days after hatching . 
 the total length larvae was 5.45~5.56 mm ( mean , 5.52 mm ) after 8 days after hatching , and appeared the stems for tail . the stems pectoral , anal fin were showed after 14 days and the stems dorsal , ventral fin were appeared after 19 days . for 35 days after hatching , the total length of larvae 13.95~15.30 mm ( mean , 14.64 mm ) , and at this time , fins and body were transferred like the adult oryzias latipes . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Developmental stages of eggs
Morphological development of larva and juvenile 
DISCUSSION