Page 181 - 《水产学报》2025年第12期

张金叶，等 水产学报, 2025, 49(12): 129414

Laboratory of Animal Biology of Chongqing Muni- temperature of 72 °C for 2 min, followed by an
cipal Education Commission, China. extra extension at 72 °C for 5 min, followed by
Twelve specimens of C. auratus, which were holding at 12 °C. Subsequently, 3 μL PCR ampl-
captured by fishermen from the natural waters of icon was subjected to electrophoresis on a 1%
TM
Mei River, were purchased at Yuxiu Farmers' Mar- agarose gel stained with GoldView Nuclear Stain-
ket in Zhonghe Town (28.45º N, 108.98º E), Xiushan ing Dyes, and PCR products were purified with the
th
County, Chongqing, China on 24 June 2021. The DNA Agarose Gel Extraction Kit (OMEGA Bio-
body length of these fish specimens ranged 7.4-17.5 Tek, Norcross City, USA). The purified PCR
cm. Subsequently to euthanasia, all fish specimens products were sent to the TSINGKE (Tsingke Bio-
underwent immediate dissection. Microscopic exam- technology Co., Ltd., Chongqing) for bidirectional
ination was performed on tissues and organs, includ- sequencing.
ing the gills, muscle, liver, kidney, spleen, intestine,
1.3 Molecular and phylogenetic analysis
heart, gall bladder and so on. Many cysts of varying
Sequences were assembled and modified
sizes were isolated from the gills. The cysts were
[15]
manually by BioEdit 7.0.5.3 . Pairwise sequence
then punctured using a fine needle, and the released
alignment for sequence similarity was conducted
myxospores were diluted three times with distilled
with NCBI BLASTn. Multiple sequence align-
water and collected via centrifugation at 2 000 × g.
ments and divergences were carried out using
The morphological structures of myxospores were
[16]
MEGA 11 with default parameters .
observed, photographed and measured using a Leica
For phylogenetic relationship analysis, a total
DM6000B microscope (Leica Microsystems, CMS
of 51 additional myxosporean SSU rDNA sequences
GmbH, Germany) at a magnification of 1 000 ×.
from GenBank, exhibiting a minimum sequence sim-
Illustrations were created using CorelDRAW 11 and
ilarity of 90% to those of the present species, were
Photoshop CS6. Species identification and specimen
selected. These sequences consisted of 33 Myxo-
preservation of the parasite followed previously
reported methods [11-12] . bolus sequences, 17 Thelohanellus sequences and
one sequence from Neoactinomyxum. Two
1.2 DNA extraction, PCR amplification, and
sequences, one from Kudoa quadricornis Whipps et
sequencing
al., 2003 and the other from Kudoa alliaria Koval-
Genomic DNA was extracted from fresh eva et al., 1979, respectively, were used as out-
myxospores derived from cysts using the DNeasy groups. G-blocks were applied to exclude highly
Blood & Tissue Kit (QIAGEN, Düsseldorf, Ger- variable, potentially poor alignment, or fast-evolving
[17]
many) following the manufacturer’s instructions for positions . The best-fitting model, GTR + I + G
animal tissue. The primers for the SSU rDNA under the Akaike information criterion, was calcu-
sequences were as follows: 18E-5'-CTGGTTGAT lated and selected using Paup 4.0 and Modeltest 3.7,
[18]
CCTGCCAGT-3' and 18R-5'-CTACGGAAACCT- respectively . The base frequencies were 0.244 9,
TGTTACG-3' [13-14] . Polymerase chain reaction 0.208 9, 0.266 6 and 0.279 6, respectively, with cor-
TM
(PCR) was performed on a Veriti 9902 Thermal responding base substitution frequencies of 1.123 1,
Cycler (Applied Biosystems, Singapore) in a 20 μL 3.622 6, 1.223 7, 0.629 6, 5.102 4 and 1.000 0. The Γ
reaction system consisting of 8 μL ddH O, 6 μL 2 × distribution shape parameter (G) and the proportion
2
Taq Master Mix, 0.5 μL each primer, 5 μL genomic of invariable sites (I) were 0.388 2 and 0.255 4,
DNA. Initial denaturation at 95 °C for 90 s, the respectively. Bayesian (BI) and maximum likeli-
amplifications were carried out with 35 cycles at a hood (ML) trees were constructed using MrBayes
melting temperature of 95 °C for 30 s, an annealing 3.2.7 and PhyML 4.0, respectively. For the Bayesian
temperature of 58 °C for 30 s, and an extension trees, six independent Markov-chain Monte Carlo

中国水产学会主办 sponsored by China Society of Fisheries https://www.china-fishery.cn
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