Bighead Catfish Research Articles

Development of a Monoclonal Antibody Specific to the IgM Heavy Chain of Bighead Catfish (Clarias macrocephalus): A Biomolecular Tool for the Detection and Quantification of IgM Molecules and IgM+ Cells in Clarias Catfish.

Catfish is a commonly-cultivated freshwater fish in Thailand and many Southeast Asian countries. The molecular data obtained for the IgM heavy chain (IgMH) of catfish have been useful for distinguishing monoclonal antibodies (mAbs). A mAb specific to Cμ1 of the IgMH of catfish (IgMHCμ1 mAb) was developed in a rabbit model using sequence information from bighead catfish (Clarias macrocephalus). The IgMHCμ1 mAb strongly recognized the IgM heavy chain of the tested catfish, namely, bighead catfish, African catfish (Clarias gariepinus) and their hybrid (C. macrocephalus × C. gariepinus), in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCμ1 mAb successfully recognized IgM+ cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCμ1 mAb was further used to quantify the percentage of IgM+ cells among PBLs through flow cytophotometry. The IgM+ cell percentages of healthy bighead catfish, African catfish and their hybrid were 38.0–39.9%, 45.6–53.2%, and 58.7–60.0%, respectively. Furthermore, the IgMHCμ1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish.

Genetic components of growth traits of the hybrid between ♂North African catfish (Clarias gariepinus Burchell, 1822) and ♀bighead catfish (C. macrocephalus Günther, 1864)

The hybrid between ♂North African catfish (Clarias gariepinus Burchell, 1822) (Cga) and ♀bighead catfish (C. macrocephalus Günther, 1864) (Cma) is economically important in Thailand and neighboring countries, with annual production of about 120,000 t. However, genetic approach for improvement of the hybrid has not previously been considered. In this study, five families each of fast- and slow-growing male Cga and female Cma were hybridized following a partial factorial mating scheme. The mating resulted in 40 full-sib families with 10 paternal half-sibs and 7 maternal half-sibs. They were tagged and communally reared in two earthen ponds for 77 days. Variance components for body weight (BW), total length (TL) and standard length (SL) of the hybrids were estimated at 207 days old using the PROC MIXED procedure of SAS 9.0 following a model that accounted for initial size (family mean at commencement of communal rearing) and sex of the fish as fixed effects, and for sire, dam, sire × dam, and residual as random effects. The results revealed that general combining ability (GCA) of male Cga (362.64 for BW, 0.25 for SL and 0.29 for TL) was higher than that of the Cma females (190.32 for BW, 0.23 for SL and 0.25 for TL). Specific combining ability of both males and females (63.19 for BW, 0.14 for SL and 0.08 for TL) was much lower than the GCA. These results imply that growth performance of the hybrid depends largely on additive genetic variation of the male parent followed by that of the female parent. As such, growth of the hybrid can be improved by selective breeding to enhance growth of the parental strains. Further study is also recommended to evaluate genetic correlation of a chosen trait between purebred parental strains and the hybrid, which may enable a more efficient approach in selection to improve hybrid performance.

Cân bằng vật chất dinh dưỡng trong hệ thống tuần hoàn nuôi cá trê vàng (Clarias macrocephalus)

The study was carried out with bighead catfish (Clarias macrocephalus) reared in a recirculating aquaculture system (RAS) that consisting of 100-L culture tank, 30-L swirl separator, 60-L sump tank and 70-L moving bed biofilm reactor. Four different sizes (10 g, 30 g, 70 g and 100 g) of fish was stocked at density of 100 fish/tank then fed to ad libitum rate by floating pellets (41% crude protein) for 15 days. In the total of feed supply, retention in fish accounted by 25.4-32.7% in dry matter (DM) and 28.6-42.7% in nitrogen (N); waste production by fecal loss made up 8.8-23.1% of DM and 14.7-40.0% of N; and non-fecal loss accounting for 12.8-15.3% of DM and 36.7-38.4% of N. The remaining was consumed by bacteria and lost by evaporation and seepage. Results also revealed that to produce 1 kg of fish, it was necessary to provide 780.6-1,033.7 g DM containing of 48.5-64.3 g N. Of which, nutrient accumulation in fish was 255.2-262.3 g DM and 20.0-21.1 g N; released in waste production of 525.4-771.5 g DM and 27.5-44.3 g N.

Probiotic Effects of a Novel Strain, Acinetobacter KU011TH, on the Growth Performance, Immune Responses, and Resistance against Aeromonas hydrophila of Bighead Catfish (Clarias macrocephalus Günther, 1864).

In the present study, the novel probiotic strain Acinetobacter KU011TH with an evident lack of pathogenicity in catfish was experimented. Three practical administration routes, namely, feed additive (FD), water-soluble additive (SOL), and a combination route (FD+SOL), were applied in two sizes of catfish. After 120 days of FD+SOL administration, catfish fingerlings (15 g) exhibited a significant improvement in all tested growth performance parameters. For 15- and 30-day applications at the juvenile stage (150 g), phagocytic activity, phagocytic index, lysozyme activity, respiratory burst activity, alternative complement pathway, and bactericidal activity were significantly increased. Furthermore, probiotic-administered bighead catfish exhibited an upregulated expression of several immune-related genes in tested organs. Significant colonization by Acinetobacter KU011TH in rearing water and on skin and gills was observed among experimental groups. Histological analysis clearly indicated enhanced physical characteristics of skin mucosal immunity in the treated groups. No histopathological changes in the gills, skin, intestine or liver were observed among the fish groups. Interestingly, after challenge with Aeromonas hydrophila, the survival rates of the treated groups were significantly higher than those of the controls. In conclusion, the novel probiont Acinetobacter KU011TH provides a potent strategy for improvement in growth and disease resistance, which is an important steppingstone for sustaining catfish aquaculture.

Acinetobacter Strain KUO11TH, a Unique Organism Related to Acinetobacter pittii and Isolated from the Skin Mucus of Healthy Bighead Catfish and Its Efficacy Against Several Fish Pathogens.

The bacterial strain KU011TH was isolated from the skin mucus of healthy bighead catfish. The strain is a Gram-negative coccobacillus that is nonmotile, aerobic, catalase positive, oxidase negative, and nonhemolytic. Sequence analyses of the housekeeping genes 16S rRNA, gyrB and rpoB indicate that this strain is a new member of the Acb complex of the genus Acinetobacter and is closely related to Acinetobacter pittii and Acinetobacter lactucae. In addition, the genome relatedness-associated ANIb (<95–96%) and in silico DDH (<70%) values clearly supported the new member of the genus Acinetobacter and the Acb complex. The genome of the strain KU011TH was approximately 3.79 Mbp in size, comprising 3619 predicted genes, and the DNA G+C content was 38.56 mol%. The major cellular fatty acids were C18:1ω9c, C16:0, C16:1, C20:2, C18:2ω6c and C18:1ω9t. The whole-genome sequences and phenotypic, phylogenetic, and chemotaxonomic data clearly support the classification of the strain KU011TH as a new member in the genus Acinetobacter which is closest to A. pittii. Additionally, the new bacterial strain exhibited strong activity against a broad range of freshwater fish pathogens in vitro.

Heritability of immunity traits and disease resistance of bighead catfish, Clarias macrocephalus Günther, 1864

Disease outbreak is a major obstruction for intensive aquaculture worldwide. One of the promising solutions is genetic improvement by selective breeding, providing that a sufficient proportion of additive genetic variance (measured by heritability-h2) of disease resistance traits exists. In addition, immunity traits are of interest as potential indirect targeted traits for disease resistance. In this study, the genetic parameters of resistance to Aeromonas hydrophila were reported for the first time in the bighead catfish, Clarias macrocephalus Günther, 1864 which is an important parental species for the production of the commercially important hybrid C. macrocephalus × C. gariepinus. The analyses were performed on 736 data records obtained from 74 full-sib families (31 half-sib families) produced by factorial mating design. The results showed that the heritability of survival rate after disease (Aeromonas hydrophila) challenge (intraperitoneal injection with 0.1 ml containing 1 × 106 CFU/ml of A. hydrophila) was low to moderate (0.05 ± 0.02–0.27 ± 0.15). The immune traits (bactericidal activity-BA, lysozyme activity-LA, and alternative complement activity-ACH50) had low to moderate heritability (h2BA = 0.05 ± 0.02; h2LA = 0.16 ± 0.04; h2ACH50 = 0.31 ± 0.06) while heritability of hematocrit (Hct) was also low (h2Hct = 0.17 ± 0.04). The results suggested the possibility to improve resistance to A. hydrophila by selection, while the possibility to use immunity traits as indirect selection criteria for disease resistance is still unclear.

Evidence of dramatic sterility in F1 male hybrid catfish [male Clarias gariepinus (Burchell, 1822) × female C. macrocephalus (Günther, 1864)] resulting from the failure of homologous chromosome pairing in meiosis I

Sterile hybrid animals exhibit spermatogenic disruptions with a decreased number and/or malformation of mature sperm. F1 hybrid catfish (2n = 55) is an important cultured fish derived from male North African catfish [Clarias gariepinus (Burchell, 1822); 2n = 56] and female bighead catfish [C. macrocephalus (Günther, 1864); 2n = 54]; they are sterile with gametogenic failure in males. Despite the generality of this phenomenon, the spermatogenic phenotype has not been well described and a comprehensive understanding of the genetic basis of the disruption remains elusive. Our observations in the F1 male hybrid showed abnormal morphology of testes with small size, while meiotic configuration indicated that meiosis succeeded in the early pachytene stage but failed to progress beyond the diplotene-diakinesis stage in primary spermatocytes. This suggests the presence of a number of degenerated spermatocytes. Histological examination recorded no postmeiotic cells and spermatozoa in F1 male hybrid testes. A high frequency of apoptotic testicular cells was also present in the F1 male hybrid, as shown by histochemical results for activated Caspase-3 and TUNEL assays. Low expression levels of Caspase-3, p53 and MCL1 (isoform 2), which activate pro-apoptotic signals in the development process, were also observed in both parental species, but significantly high expression levels were observed in the F1 male hybrid. By contrast, the level of BCL2 expression was very low in the F1 male hybrid, supporting the state of apoptosis as BCL2 inhibits the actions of pro-apoptotic proteins. These results collectively suggest that sterility in male hybrids is caused by spermatogenic disruptions in the pachytene stage, resulting from the failure of homologous chromosome pairing due to chromosomal incompatibility between parental genomes, and subsequent elimination by apoptosis leading to spermatogenic breakdown in the F1 male hybrid.

Dietary protein requirement of bighead catfish (Clarias macrocephalus Gunther, 1864) fingerling

Dietary protein requirement of bighead catfish (Clarias macrocephalus Gunther, 1864) fingerling

Regional variation in genetic diversity between wild and cultured populations of bighead catfish (Clarias macrocephalus) in the Mekong Delta

Bighead catfish (Clarias macrocephalus) is an important freshwater species for aquaculture and fisheries in Southeast Asia. The species has been domesticated for more than 30 generations in hatcheries, but wild population numbers have declined to “threatened” levels. In this study, genetic diversity was quantified in areas of intensive catfish culture and natural areas of the Mekong Delta, Viet Nam to inform hatchery genetic improvement programs and conservation actions. Fish were collected from three hatcheries and four wild populations in conservation parks (wild-conservation) and seven wild populations in rice fields of aquaculture-intensive areas (wild-farming). Results based on six microsatellite loci showed all populations had relatively high levels of genetic diversity for allelic richness (range 8.10– 10.18), and observed heterozygosity (range 0.708– 0.819). However, genetic diversity was lower in hatchery fish and wild farming populations as evidenced by significantly lower (P ≤ 0.03) allelic richness (9.16 ± 0.27 and 9.27 ± 0.66, respectively) than wild conserved populations (10.01 ± 0.19). Pairwise individuals’ genetic relatedness within three cultured populations (0.013–0.028) and five wild farming populations (0.016–0.039) indicated low potential for inbreeding in the immediate future. Genetic differentiation among populations (FST = 0.017 ± 0.002) indicated a modest level of gene flow, likely due to the expansive river network, straying of hatchery fish, and large-scale transport of wild fish to hatcheries.

Use of microsatellite markers in differentiating catfish hybrids (Clarias macrocephalus x C. larias gariepinus) from their parental species

This study aimed to develop microsatellite markers that can be used to differentiate catfish hybrids (bighead catfish Clarias macrocephalus x African catfish C. larias gariepinus) from their parental species. Microsatellite primers developed from previous studies were screened and optimized. Six microsatellite markers were selected to amplify samples of bighead catfish (n = 37), African catfish (n = 29), and the hybrids (n = 30). Results show that 4 of 6 loci differ in allele numbers and sizes between the bighead and African catfish. In the other two loci, these two species share one allele that overlaps at a portion of the size range with different allele frequencies. Hybrids’ genotypes are intermediate between those of the parental species. The genetic divergence between bighead and African catfish is high with Fst = 0.59 and Rst = 0.95. Therefore, these 6 microsatellites were applied to successfully distinguish hybrids from their parental species with high accuracy.

Molecular characterization and expression analysis of Cyclin B and Cell division cycle 2 in gonads of diploid and triploid bighead catfish, Clarias macrocephalus Günther, 1864

This study investigated the differential expression of genes associated with reproduction in sterile triploid and normal diploid bighead catfish (Clarias macrocephalus Günther, 1864). The triploid fish were produced using cold shock and were reared in the same conditions as the diploid counterpart. The histomicrographs showed completely retarded triploid gonads across the samples aged 2–12 mth, whereas the gonads of the diploids were in developing stages during 2–4 mth, reached the early maturing stage at 6 mth, matured at 8 mth and showed signs of atresia at 10–12 mth. In parallel, the full-length cDNAs of cyclin B1 (CmCcnb1; 1539 bp in length with an open reading frame (ORF) of 1194 bp corresponding to 397 amino acids) and cell division cycle 2 (CmCdc2; 1355 bp, an ORF of 909 bp, 302 amino acids) of bighead catfish (C. macrocephalus Günther, 1864) were isolated. Phylogenetic analysis revealed that the newly characterized CmCcnb1 should be regarded as a member of cyclin B1 rather than cyclin B2. The expression level of CmCcnb1 mRNA was limited in different stages of the ovaries and testes of triploids. In diploid ovaries, its expression was significantly higher than that in triploid ovaries in fish aged 2 mth (513.43 ± 82.22 fold) and in fish aged 8 mth (2430.87 ± 900.06 fold). The CmCcnb1 level in the testes of diploids was significantly greater than that in triploids in fish aged 2 mth (928.85 ± 208.72 fold). Similarly, expression of CmCdc2 mRNA was also reduced in triploids. Its expression was significantly lower than that in diploid females aged 2 mth (7.66 ± 3.42 fold), 4 mth (59.42 ± 10.50 fold) and 8 mth (42.74 ± 8.36 fold). In males, significantly greater expression of CmCdc2 was observed at age 6 mth (58.61 ± 34.64 fold) and 8 mth (72.70 ± 4.36 fold) diploids compared to triploids. The results illustrated that CmCcnb1 and CmCdc2 are functionally involved in oogenesis and spermatogenesis and reduced expression levels of these transcripts affected the reproductive development of triploid C. macrocephalus.

Lack of introgressive hybridization by North African catfish (Clarias gariepinus) in native Vietnamese bighead catfish (Clarias macrocephalus) populations as revealed by novel nuclear and mitochondrial markers

The widespread farming of catfish (genus Clarias) hybrids between bighead catfish (C. macrocephalus, Cm) and the introduced North African Catfish (C. gariepinus, Cg) in the Mekong Delta, where flooding is common, has raised concerns of introgression of Cg into the native catfish due to backcrossing by escaped hybrids with wild Cm. This study employed novel PCR-RFLP analyses of a mitochondrial (Cytochrome c oxidase subunit I, COI) and two nuclear (Rhodopsin and Tropomyosin) markers and six microsatellite loci to differentiate hybrid individuals from their parental species, and evaluate whether introgression has occurred in wild and cultured Cm populations in the region. Results of marker screening showed that sizes and frequencies of microsatellite alleles differed between the two parental species. Four loci exhibited fixed species-specific differences in allele frequency (monomorphic in Cg and highly polymorphic in Cm). Levels of sequence variation varied at the three genes examined, ranging from 3 to 8% (sequence length 652bp for COI, 802bp for Rhodopsin and 925bp for Tropomyosin). Based on variable sites identified that were not shared between the two species, 3 restriction enzymes (SpeI, XcmI and PflMI) were selected to digest PCR products at species-specific sites within the COI, Rhodopsin and Tropomyosin genes, respectively. Results of PCR-RFLP analyses confirmed that Cm is the maternal lineage of cultured hybrids, while nuclear genes (Rhodopsin and Tropomyosin) and microsatellite loci revealed that hybrids possessed admixed multi-locus genotypes relative to the two parental species. Analyses of six microsatellite loci and PCR-RFLP of two nuclear genes in 473 individuals collected from 11 wild and three cultured populations revealed one F1 hybrid in the wild but no evidence of widespread introgression into native bighead catfish. These results differ from findings in Thailand where intensive catfish culture and deliberate species hybridization is practiced.

De Novo Transcriptome Characterization and Growth-Related Gene Expression Profiling of Diploid and Triploid Bighead Catfish (Clarias macrocephalus Günther, 1864).

To enhance understanding of triploid gene expression, the transcriptome information from bighead catfish (Clarias macrocephalus Günther, 1864) was studied using the paired-end Illumina HiSeq™ 2000 sequencing platform. In total, 68,227,832 raw reads were generated from liver tissues and 53,149 unigenes were assembled, with an average length of 765bp and N50 length of 1283bp. Of these unigenes, 33,428 (62.89%) could be annotated according to their homology with matches in the NCBI non-redundant (Nr), NCBI nucleotide (Nt), Swiss-Prot, Clusters of Orthologous Groups (COG), gene ontology (GO), or Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Relative expression of liver genes between diploid and triploid bighead catfish revealed more than 90% of the annotated unigenes similarly expressed, regardless of ploidy, whereas 362 upregulated and 83 downregulated with at least a twofold change in triploid relative to diploid. Quantitative real-time PCR of 15 differentially expressed growth-related genes showed consistency between the expression profiles of those genes with the results from RNA-seq analysis. Our results showed that genes in C. macrocephalus liver responded independently to triploidy with the majority showing similar expression levels between diploid and triploid (a dosage compensation phenomenon). The underlying mechanism of the varying gene expression patterns was discussed. Notably, 5 of the top 20 upregulated genes associated with stress response and thus may reflect stress caused by triploidy. The present study adds a substantial contribution to the sequence data available for C. macrocephalus and hence provides valuable resources for further studies. Furthermore, it gives information that may enhance understanding of triploid physiology.

Effects of strains on growth performances of triploid bighead catfish, Clarias macrocephalus Günther, 1864

Despite its importance, limited empirical information is available on the effects of parental strains on the performance of triploid aquatic animals, especially of tropical species. The present study was conducted to evaluate the impacts of strains on the growth performance of the triploid bighead catfish (Clarias macrocephalus Günther, 1864) which was produced using cold shock (7 °C, 25 min shock duration), applied to newly fertilized eggs obtained from all possible crosses between two catfish strains. The fingerlings of each group were reared until they were age 240 d. At age 60 d, the triploid fish had a lower specific growth rate (SGR), absolute growth rate (AGR), body length and body weight than the diploid counterparts. However, at harvest (age 240 d) body length and body weight were not different between the two groups while SGR of the cold shock group was higher than the diploid during age 121–240 d. The results revealed that parental strains did not show significant direct effects on the growth performance of triploids, but the interaction between paternal strain × shock was significant for the following traits: body length at age 90 d, body weight at age 90 d and 120 d, AGR for age 61–90 d, and SGR for age 61–90 d and 91–120 d. The interaction between paternal × maternal strain × shock was significant for body length, body weight at age 180 d and AGR for age 121–180 d. These results suggested that the growth performance of triploid C. macrocephalus cannot be enhanced by improving the parental strains; rather, improvement may be accomplished by selecting the parental strains based on triploid performance.

Isolation of microsatellites in the bighead catfish, Clarias macrocephalus and cross-amplification in selected Clarias species

The present study documents the isolation of eight polymorphic microsatellite markers from the bighead catfish, Clarias macrocephalus and cross-amplification in two other catfish species. The number of alleles per locus in C. macrocephalus ranged from 2 to 21. The most polymorphic locus was NCm-G12 with 21 alleles while the least polymorphic locus was NCm-H2 with only two alleles. Locus NCm-F8 significantly deviated from Hardy-Weinberg equilibrium (P value <0.05) after Bonferroni correction. Linkage disequilibrium was non-significant in all loci comparisons. The observed and expected heterozygosities varied from 0.033 to 0.967 and from 0.033 to 0.942, respectively. Mean polymorphic information content for the eight loci was 0.765. Cross-amplification was successfully performed with two other catfish species, C. batrachus and C. meladerma for all eight loci. Locus NCm-D8 was monomorphic in both species while NCm-F8 was monomorphic only in C. batrachus. These newly developed markers would be useful for better management and conservation of the economically important C. macrocephalus species.

Blood flukes (Digenea: Aporocotylidae) of walking catfishes (Siluriformes: Clariidae): new genus and species from the Mekong River (Vietnam) with comments on related catfish aporocotylids

Nomasanguinicola canthoensis gen. et sp. n. infects the branchial vessels of bighead catfish, Clarias macrocephalus Günther (Siluriformes: Clariidae), in the Mekong River near Can Tho, southern Vietnam. Nomasanguinicola differs from all other genera of fish blood flukes (Digenea: Aporocotylidae) by the combination of lacking body spines and by having an anterior sucker with two flanking columns of large denticles, an intestine comprising several short papilla-like caeca, an inverse U-shaped uterus, and an ootype located near the separate genital pores. The new species has an ootype that is posterior to the level of the female genital pore. That feature most easily differentiates it from the only other putative aporocotylid species having an anterior sucker with two flanking columns of large denticles, Plehniella dentata Paperna, 1964 and Sanguinicola clarias Imam, Marzouk, Hassan et Itman, 1984, which have an ootype that is lateral (P. dentata) or anterior (S. clarias) to the level of the female genital pore. These two species apparently lack extant type materials, infect North African catfish, Clarias gariepinus (Burchell), and herein are considered incertae sedis, but likely comprise species of Nomasanguinicola. An updated list of hosts, sites of infection and geographic localities for the six species and three genera of blood flukes that mature in catfishes is provided. The new species is the first fish blood fluke recorded from Vietnam and only the third reported from a walking catfish (Clariidae).

Assessment of the invasive catfishPterygoplichthys pardalis(Castelnau, 1855) in Thailand: ecological impacts and biological control alternatives

A species of Neotropical suckermouth armored catfish (family Loricariidae), tentatively identified as the Amazon sailfin catfish Pterygoplichthys pardalis, is a highly invasive, non-native species in Southeast Asia. In Thailand, the spread of P. pardalis is an increasing concern because of the possibility that these non-native catfish are negatively impacting freshwater biological resources. In this study, we conducted a series of laboratory experiments to investigate the food preferences of P. pardalis as well as the interactions of this catfish with the native bighead catfish Clarias macrocephalus (Gunther, 1864). We also conducted laboratory experiments to evaluate the potential use of three different native piscivorous fishes as biological control agents. Laboratory results revealed that P. pardalis in aquaria conditions readily consumed the eggs and fry of Nile tilapia Oreochromis niloticus (Linnaeus, 1758) and that large specimens P. pardalis preferred O. niloticus eggs over Tubifex worms. Pterygopl...

Growth and morphological development of laboratory-reared larval and juvenile bighead catfish Clarias macrocephalus (Siluriformes: Clariidae)

Morphological development in laboratory-reared larval and juvenile bighead catfish Clarias macrocephalus is described. Body length (BL) of larvae and juveniles was 3.4 ± 0.3 (mean ± SD) mm just after hatching, reaching 11.3 ± 1.0 mm by day 16, and 24.2 ± 2.8 mm by day 40. Overall aggregate fin ray numbers (except for caudal fin procurrent rays) attained full complements by 15.2 mm BL. Gill buds appeared on day 0, those of barbels (four pairs) and primordial nostrils on day 1, and pectoral fins on day 3. All larvae began feeding by day 3. Conical teeth were observed on day 7. Notochord flexion began on day 2, the yolk being completely absorbed during days 7–9. Melanophores were scarce at hatching, increasing with growth to cover almost the entire body except the ventral surface of the abdominal cavity. Proportions of head length, pre-anal length, body depth, eye diameter, and maxillary barbel length became relatively constant after yolk absorption, those of snout length and upper jaw length increasing until ca. 12–13 mm BL and decreasing thereafter. Suprabranchial organ started developing in postflexion larval stage larger than ca. 11.0 mm BL (day 16), and air-breathing was suggested to be functional at that time.