Corresponding author: Xiaoyun Zhou ( zhouxy@mail.hzau.edu.cn ) Academic editor: Paulo Andreas Buckup
© 2018 Dongmei Zhu, Kun Yang, Ning Sun, Weimin Wang, Xiaoyun Zhou.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhu D, Yang K, Sun N, Wang W, Zhou X (2018) Embryonic and larval development of the topmouth gudgeon, Pseudorasbora parva (Teleostei: Cyprinidae). Zoologia 35: 1-8. https://doi.org/10.3897/zoologia.35.e22162
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The topmouth gudgeon, Pseudorasbora parva (Temminck & Schlegel, 1842), is a small cyprinid fish that inhabits the littoral zones of freshwater habitats throughout Asia and Europe. It is regarded as an invasive species in Europe, but in its native range, in China, as food and as an environmental bio-indicator. In this study, the embryonic and larval development of P. parva was investigated for the first time. The eggs of P. parva are transparent, adhesive and elliptical. The mean size of fertilized eggs was (1.63×1.31) ± 0.04 mm. From fertilization to hatching, embryonic development could be divided into eight stages and 34 phases, and the incubation period lasted for 109.25 hours at 24 ± 1 °C. Newly hatched larvae were 4.1 ± 3 mm in length, and the yolk absorption was completed within six days after hatching. The first and second swim bladders formed at the third and ninth day, respectively. The pectoral fin formed before the hatching stage, followed by the caudal, dorsal, anal and ventral fin formation after hatching. About 20 days after hatching, the morphology of the fry was similar to the adult fish. These findings provide a basis for determining the complete ontogeny of P. parva, as well as facilitate the management and utilization of this fish.
Embryogenesis, invasive species, larval development
The topmouth gudgeon, Pseudorasbora parva (Temminck & Schlegel, 1842) is a small cyprinid fish (Gobioninae) native to freshwater habitats throughout China, Japan, Korea and the River Amur basin (
In China, P. parva grows quickly, with maximum length up to 11 cm; it is a batch spawner and reaches sexual maturity in the first year of its life (
Sexually mature adult P. parva (Fig.
During the incubation, water was replaced twice daily (8:00 am and 8:00 pm). Water temperature, dissolved oxygen and pH of water were measured by a multiparameter water quality analyzer (YSI Pro Plus, USA) three times a day (8:00 am, 2:00 pm and 8:00 pm), and maintained at 24 ± 1 °C, 7.85–8.03 mg/L and 7.53–7.82, respectively. Egg development was observed at 15 min intervals under a stereoscopic microscope (Olympus SZX-7, 0.8, Japan) equipped with a digital camera. Unfertilized eggs and dead embryos were removed regularly. Embryonic development time was defined as the time when 50% of fertilized eggs were hatched.
After hatching, the larvae were transferred to plastic basins (50 L) filled with aerated water and microscopically analyzed every day until the morphology of the fry was similar to the adult fish (20 days after hatching).
We measured 30 individuals at each developmental stage (embryos, larvae and fry). Statistical analyses were performed using SPSS software (Version 16.0). Data were expressed as mean ± SD.
All experimental protocols were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, the methods were carried out in accordance with the approved guidelines.
From fertilization to hatching, sequence of the most important events of the embryonic development of P. parva is shown in Table
Time and phases of embryonic development of Pseudorasbora parva at 24 ± 1 °C.
Serial number | Development stage | Time |
1 | Fertilized egg | 0:00 |
2 | Blastodisc formation | 0:34 |
3 | Cleavage stage | 0:54 to 3:30 |
4 | Blastula stage | 3:44 to 6:49 |
5 | Gastrula stage | 8:44 to 11:44 |
6 | Neurulation stage | 11:59 to 12:51 |
7 | Organ differentiation stage | 18:11 to 95:07 |
8 | Hatching stage | 109:15 |
Embryonic development of Pseudorasbora parva: (2) Fertilized egg; (3) The fully-swollen egg (A, blastoderm); (4) Blastodisc formation; (5) The first cleavage furrow (B, cleavage furrow); (6) 2-cell phase; (7) The second cleavage furrow (B, cleavage furrow); (8) 4-cell phase; (9) 8-cell phase; (10) 16-cell phase; (11) 32-cell phase; (12) 64-cell phase; (13) Cellulouse phase; (14) Morula phase; (15) Early blastula phase; (16) Mid-blastula phase; (17) Late blastula phase (C, cells epiboly); (18) Early gastrula phase (D, germ ring); (19) Mid-gastrula phase (D, germ ring); (20) Late gastrula phase (D, germ ring); (21) Neural embryo formation (E, blastopore); (22) Blastopore closed phase; (23) Somites appearance (F, somite); (24) Optic vesicle appearance (G, optic vesicle); (25) Optic capsule appearance; (26) Notochord appearance (H, notochord); (27) Tail bud appearance (I, tail bud); (28) Otic vesicle appearance; (29) Crystalline lenses formation; (30) Muscle function phase; (31) Heart bud appearance (J, heart bud); (32) Heartbeat phase; (33) Otolith appearance (K, otolith); (34) Eye pigment appearance; (35) Pectoral fin bud appearance (L, pectoral fin bud); (36) Body pigment appearance (M, body pigment); (37) Hatching prophase; (38) Hatching phase (N, yolk sac); (39) Newly hatched larva. Scale bars: 2–37 = 0.2 mm, 38–39 = 0.5 mm.
The mature eggs of P. parva were (1.63×1.31) ± 0.04 mm in size, transparent, adhesive and elliptical (Fig.
About 34 min after the fertilization, the egg protoplasm moved and aggregated towards the animal pole, forming the primary primordium (Fig.
The cleavage in P. parva is meroblastic, as there is plenty of yolk in the egg. About 54 min after fertilization, the first cleavage furrow occurred in the topmost blastoderm (Fig.
Along with the successive cleavages, there was a progressive increase in the number of cells with a reduction in size. About 3 h and 44 min after the fertilization, the animal pole formed a cap-like structure atop the yolk, and the morula entered the blastula stage. In the early blastula stage, the vegetal pole started to shift slightly (Fig.
As a result of epiboly and involution of blastoderm, the germ ring was completed around the margins of blastoderm about 8 h and 44 min after the fertilization, marking the beginning of the gastrula stage (Fig.
At 11 h and 59 min post-fertilization, the neural groove began to form in the dorsal midline of the embryo, and the blastoderm entered the neurula stage. During this stage, blastoderm covered about 4/5 of the yolk sac. Simultaneously the blastopore and the yolk plug appeared (Fig.
Two somites in the mid-part of the embryo could be observed about 18 h and 11 min after the fertilization (Fig.
At 95 h and 7 min post-fertilization, before the hatching stage, the tip of the tail passed over the brain of the embryo and the tail was flattened (Fig.
The pyriform yolk sac of the newly hatched larva was about 1.8 ± 0.12 mm in length, and the head of the larvae was bent toward the yolk sac. One day after the hatching, the larvae were 5.8 ± 0.09 mm in length. At this stage, the mouth was not formed but the larva had a streamlined body. Therefore, the larvae were still completely reliant on endogenous nutrients in this period (Fig.
Two days after hatching, the larvae were about 6.0 ± 0.11 mm in total length, the yolk sac contracted, and the first swim bladder began to aerate. The intestinal tube appeared and larvae’s swimming ability improved (Fig.
Three days after hatching, the total length of the larvae was 7.2 ± 0.12 mm and the first swim bladder aerated completely (Fig.
Four days after hatching, the total length of the larvae was 7.4 ± 0.09 mm, the yolk sac almost disappeared and the liver formed. The pectoral fin fold became easily observable at this stage (Fig.
Five days after hatching, the total length was about 8.3 ± 0.10 mm. The rudiment of the dorsal fin formed, and some osseous fin rays in the under-lobe of the caudal fin appeared (Fig.
Six days after hatching, the yolk sac disappeared completely and the larvae were 8.4 ± 0.07 mm-long (Fig.
Nine days after hatching, the fry was 9.7 ± 0.08 mm-long. The second swim bladder began to aerate and the dorsal fin separated completely from the fin fold. In addition, the caudal fin bifurcated in this period (Fig.
Eleven days after hatching, the total length of the fry reached 10.0 ± 0.08 mm. In this period, the rudiments of the anal fin appeared and the fry behaved agilely (Fig.
Thirteen days after hatching, the fry was 11.0 ± 0.10 mm-long. The rudiments of the ventral fin were formed, and the shape of the upper and lower lobes of the caudal fin was almost identical (Fig.
Twenty days after hatching, the total length of the fry reached 13.8 ± 0.07 mm. Only a few fin folds existed on the ventral and caudal peduncle. At this stage, the fry was morphologically similar to adult fish (Fig.
The larval development of Pseudorasbora parva: (40) One day after hatching (DAH); (41) Two DAH (O, the first swim bladder); (42) Three DAH; (43) Four DAH; (44) Five DAH (P, dorsal fin); (45) Six DAH (Q, intestinal tube wriggle); (46) Nine DAH (R, the second swim bladder); (47) Eleven DAH (S, anal fin); (48) Thirteen DAH (T, pectoral fin); (49) Twenty DAH (U, fin fold). Scale bars: 40–48 = 0.5 mm, 49 = 1.0 mm.
The fertilized egg of P. parva is a typical telolecithal egg, exhibiting discoidal meroblastic cleavage. The egg size, shape and properties are similar, but not identical, to other Gobioninae species, such as Saurogobio dabryi (Bleeker, 1871) (
Embryonic development of P. parva is similar to other freshwater teleosts. The organs of P. parva are almost completely developed before hatching, which is similar to H. maculatus (
The timing of hatching was quite different even among the eggs fertilized at the same time and developed under the same conditions. The interval from the first hatched larva (75.47 h) to the last (116.80 h) was about two days. This phenomenon was also observed in Esox lucius Linnaeus, 1758 (
In addition, during the embryonic development, rotation of embryos was a commonly observed phenomenon.
Newly hatched larvae were about 4.1 ± 3 mm in total length, and remained immobile at the bottom of the tank immediately after hatching. A series of significant changes in the external morphology, as well as some internal organs, could be seen after hatching, during the larval development. During the yolk-sac larva period, only the pectoral fins could be seen, and the larva’s ability of swimming and escaping from predators was very limited. The pectoral fins formed before hatching, followed by the caudal fin, dorsal fin and anal fin forming in that order after the hatching, while the ventral fins formed last. The first swimming bladder formed about one day after hatching, and then aerated gradually in subsequent days, while the second swim bladder appeared about nine days after hatching. The complete disappearance of the yolk sac six days after hatching marked the metamorphosis of larvae into fry, which began to feed on algae, protozoa and rotifers. Twenty days after hatching, all fins were formed, and the morphology of the fry was very similar to the adult fish.
In conclusion, the eggs of P. parva were transparent, adhesive and elliptical. From fertilization to hatching, the embryonic development lasted for about 109.25 h at 24 ± 1 °C. The newly hatched larvae were 4.1 ± 3 mm in total length. Yolk absorption was completed within six days post-hatching. About 20 days after hatching, the morphology of the fry was similar to the adult fish. These findings provide a basis for determining the complete ontogeny of P. parva, as well as facilitate the management and utilization of this fish.
This study was supported by the National Natural Science Foundation of China (grants 31472267 and 31602163), Wuhan Youth Science and Technology Plan (grant 2015071704011613), and Open Project of Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture (2015PY075). And thanks are given to Ivan Jakovlić from Bio-Transduction Lab, Wuhan Institute of Biotechnology for editing the manuscript.