Botia

Abstract: The Botiinae have traditionally represented a subfamily of the Cobitidae. At present, the classification and phylogenetic relationships of the Botiinae are controversial. To address systematic and phylogenetic questions concerning this group, we sequenced the complete cytochrome b gene from 34 samples, of which 24 represented 13 species of the East Asian botiine fishes, while the other 10 were non-botiine loach species. For the 1140 bp sequences determined, 494 sites were variable ones, of which 424 were parsimony informative. With Myxocyprinus asiaticus as an outgroup, molecular phylogenetic trees were constructed using the neighbor-joining, maximum parsimony, maximum likelihood and Bayesian methods. All molecular phylogenetic trees revealed that botiine fishes form a monophyletic group and are distantly related to other loaches, suggesting that the Botiinae should be placed in their own family. Within the Botiinae, there are three genera; Botia, Parabotia, and Leptobotia, each genus forming a monophyletic group, with the genus Botia as the most ancestral split. Our molecular results are in agreement with morphological analyses of botiines, suggesting that Botia is the ancestral genus, while Leptobotia and Parabotia were resolved as more derived sister groups.

Abstract: The research was aimed to find the optimal level of tricainemethanesulfonate (MS-222) and density of clown loach (Botia macracanthus) intransportation. These triplicate factorial experiments consisted of the three levelsof MS-222 (0, 25, and 50 ppm) and three densities of fish (25, 50 and 75 fish L-1).The fingerlings of clown loach (3.0-3.5 cm) have been transported in 1 liter ofwater for 18 hours. The results showed that the levels of MS-222, density of fishesand interaction of the both factors were different significantly (P 0.05) with 50 fishes L-1. Level of MS-222 50 ppm and density 50 fishes L-1were the best for survival live in transportation of clown loach.

Abstract: Belonging to Cobitidae of Cypriniformes, the subfamily Botiinae is a group of small or middle freshwater fishes dwelling in slow to moderately swift waters, and many of them are common aquarium species. The classification of Botiinae fishes is controversial among different authors. Fang (1936) and Nalbant (1963) divided Botiinae into two genera. Botia and Leptobotia, while Chen (1980) separated in into three, Botia, Parabotia and Leptobotia. Up to now, there was no study conducted on the strucuture of mitochondrial DNA control region and the molecular phylogenetic relationship of the subfamily Botiinae. In the present study, the structure of the control region sequences was analyzed for fourteen species representing the three genera of Botiinae. Similar to other fishes, the control region of Botiinae is composed of three domains, the extended termination associated sequence (ETAS) domain, the Central conserved domain (CD) and the Conserved sequence block (CSB). The ETAS domain is very rich (73.3%) in the contents of A and T, while the CD domain is much rich (36.3%) in G and C. A termination-associated sequence TAS (TACATA) and its reverse complementary sequence (ATGTAT) were found in the ETAS domain, and the conserved sequence in the domain is ETAS 1. The CD domain is highly conserved, and three conserved sequences were identified, CSB-F, CSB-E, CSB-D. Three conserved sequences were identified in the CSB domain, CSB-1, CSB-2 and CSB-3.With Myxocyprius asiaticus as outgroup, compared with members of Nemacheilinae, Cobitinae and Homalopteridae as well, the phylogenetic relationship of Botiinae was analyzed using the neighbor-joining (NJ) method in MEGA2.1, the maximum parsimony (MP) method in PAUP*4.0b10 and the maximum likelihood (ML) method in TREE-PUZZLE 5.0. The results showed that the subfamily Botiinae is monophyletic and the confidence levels in the three analysis methods were 88% in NJ, 57% in MP and 97% in ML respectively. It also supported that Botiinae contains three genera, Botia, Parabotia and Leptobotia, since each of the genera formed a monophyly, which were supported with 100%, 65% and 78% of bootstrap values in NJ method and 100%, 75% and 70% of Quartet puzzling values in ML method. In MP analysis, the bootstrap value supporting monophyly of the genus Parabotia was less than 50%, while for Botia and Leptobotia, the values were 100% and 56%.Based on the results of molecular phylogenetic analysis, morphological comparison and geographical distribution of Botiinae fishes, it was suggested that Botia is the most primitive genus, Parabotia the second, and Leptobotia the most specialized.

Abstract: The egg yolk effect combined with 10% of methanol to preserve botia, Chromobotia macracanthus spermatozoa for 24 hours in liquid nitrogen has been studied. The concentration of egg yolk used in this study were, 0%, 5%, 7%, 9%, 11%, 13%, 15%, and 17%, respectively. As the positive control, the synthetic cryoprotectant carboxy methyl cellulose (CMC) was used. The sperms were diluted with the combination of 10% methanol, glucose extender and egg yolk (1 part of sample + 4 parts of solvent) and were equilibrated for 2 hours at 4°C before freezed for 24 hours in liquid nitrogen. After freezed, sample (sperms) were thawed for 13 sec at 40°C. Spermatozoa viability, motility, and abnormality, were then observed. Ten percent of methanol combined with 15% of egg yolk could preserve spermatozoa which maintain either sperm motility (96.43±1.49%) or sperm viability (84.25±1.26%) and reduced the abnormality up to 11.50±1.29%, respectively. Besides, the combination of 15% of egg yolk and 10% of methanol showed the highest fertility rate (50.64±4.37%).