The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.
Animals ; Base Composition ; DNA ; DNA Barcoding, Taxonomic ; Phylogeny ; Smegmamorpha/genetics*

Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction (mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA, five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei (H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155, 222, 292, 352, 458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.
Animals ; DNA Primers ; DNA, Mitochondrial ; Multiplex Polymerase Chain Reaction ; Smegmamorpha

Hippocampus is a precious animal medicine in Chinese herbal medicines. Numerous seahorse species possessing similar morphology were used as commercial hippocampus in herbal markets. Clarifing the zoological of commercial hippocampus in herbal markets is a crucial issue, which contributed to establish authentication and quality control standard. This study investigated 1 156 dried seahorse samples collected from eight main herbal markets using CO Ⅰ fragment DNA sequencing coupling with morphological identification. The results showed that 23 seahorse species were present in the China TCM market. Among them, five species were officially listed in China Pharmacopoeia, seven species namely winged seahorse (Hippocampus alatus), giraffe seahorse (H. camelopardalis), knysna seahorse (H. capensis), beibuwan seahorse (H. casscsio), half-spiny seahorse (H. semispinosus), Europe seahorse (H. hippocampus), zebra seahorse (H. zebra) were found in herbal markets for the first time. The present DNA sequences analysis coupling with morphological identification method could also use to survey the species origin of other Chinese herbal medicines in herbal markets.
Animals ; China ; Europe ; Sequence Analysis, DNA ; Smegmamorpha ; Surveys and Questionnaires

To know the infection status of zoonotic trematode metacercariae in brackish water fish, we surveyed mullets collected from 18 coastal areas in the Republic of Korea. The metacercariae of Pygidiopsis summa were detected in 236 (68.2%) out of 346 mullets examined. They were found in mullets from 15 areas except for those from Boseong-gun (Jeollanam-do), Pohang-si, and Uljin-gun (Gyeongsangbuk-do). Especially in mullets from Taean-gun (Chungcheongnam-do) and Geoje-si (Gyeongsangnam-do), their prevalences were 100% and 95.5%, and the average metacercarial density was more than 1,000 per fish. They were also detected in mullets from 3 coastal lakes, Gyeongpoho, Songjiho, and Hwajinpoho, in Gangwon-do, and their average densities were 419, 147, and 672 per infected fish, respectively. The metacercariae of 5 other heterophyid species, including Heterophyes nocens, Heterophyopsis continua, Metagonimus sp., Stictodora fuscata, and Stictodora lari, were found in the mullets examined. The metacercariae of H. nocens were detected in 66.7, 100, 28.6, 81.6, 3.9, 61.5, and 27.3% of mullets from Muan-gun, Shinan-gun, Haenam-gun, Gangjin-gun, and Boseong-gun (Jeollanam-do), Hadong-gun, and Geoje-si (Gyeongsangnam-do), and their metacercarial intensities were 64, 84, 119, 99, 1, 24, and 24 per fish infected, respectively. From the above results, it has been confirmed that P. summa metacercariae are heavily infected in mullets from coastal areas of Korea. It is suggested that residents who frequently consume raw mullet dish can be easily infected with heterophyid flukes.
Gangwon-do ; Heterophyidae ; Korea ; Lakes ; Metacercariae ; Prevalence ; Republic of Korea* ; Saline Waters ; Smegmamorpha* ; Trematoda

A survey was performed to investigate the infection status of zoonotic trematode (ZT) metacercariae in fish from a local market in Yangon City, Myanmar. A total of 264 fish (12 species) were collected through 4 times from December 2013 to June 2015. All collected fish were transferred to our laboratory on ice and examined by the artificial digestion method. More than 7 species of ZT metacercariae, i.e., Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp. were detected. Metacercariae of H. taichui were collected in 58 (42.3%) out of 137 fish (5 species), and their average density was 42.9 per fish infected. Metacercariae of H. pumilio were detected in 96 (49.0%) out of 196 fish (9 species), and their average density was 23.6 per fish infected. H. yokogawai metacercariae were found in 40 (50.0%) out of 80 fish (5 species), and Centrocestus spp. metacercariae in 91 (50.8%) out of 179 fish (8 species), and their densities were 306 and 25.8 per fish infected, respectively. Metacercariae of S. falcatus and P. cambodiensis were detected only in mullets, Chelon macrolepis. A total of 280 Procerovum sp. metacercariae were found in 6 out of 12 climbing perch, Anabas testudineus. Morphological characteristics of adult flukes recovered from experimental animals were described. It has been first confirmed that fish from Yangon, Myanmar are commonly infected with various species of ZT metacercariae.
Adult* ; Animals* ; Digestion ; Humans ; Ice ; Metacercariae* ; Methods ; Myanmar* ; Perches ; Smegmamorpha ; Trematoda

OBJECTIVETo clarify the commercial original species of hippocampus in the market.

METHODField survey and interview were applied to the investigation.

RESULTPresent study identified the main commercial hippocampus of 13 species, including Pharmacopoeia contained four kinds of hippocampus (without Hippocampus japonicus) listed in Chinese Pharmacopoeia, and the identification method for the 13 kinds of commercially hippocampus was established.

CONCLUSIONThe further research on hippocampus should be strengthened for the establishment of hippocampal quality control standard.

Animals ; Medicine, Chinese Traditional ; economics ; standards ; Quality Control ; Smegmamorpha ; anatomy & histology ; classification

OBJECTIVETo study the identification method of Syngnathus and its adulterants.

METHODRandom amplified polymorphic DNA (RAPD) was used to construct a dendrogram by UPGMA method based on Nei & Li's coefficient and a genetic affinity pattern for Syngnathus acus, Solenognathus hardwickii, Syngnathoides biaculeatus, Trachyrhamphus serratus, Halicampus koilomatodon, Microphis boaja.

RESULTFour primers, LJ04, LJ09, LJ16 and LJ19, from 18 random primers were used in the dendrogram which can differentiate Syngnathus in genus level and showed a great consistence with the appearance identification. The genetic affinity pattern based on primers LJ09 and LJ19 could be used to identify Syngnathus from its adulterants.

CONCLUSIONRAPD is suitable to identify Syngnathus and its adulterants.

Animals ; China ; DNA Primers ; genetics ; Phylogeny ; Polymorphism, Genetic ; Random Amplified Polymorphic DNA Technique ; Smegmamorpha ; classification ; genetics

OBJECTIVETo establish a RP-HPLC method for simultaneous determination of thymine, hypoxanthine and uracil contents in medical pipefish.

METHODSamples were extracted with distilled water by ultrasonic wave and separated on Waters C18 column eluted with a mobile phase of 0.05 mol x L(-1) KFI2PO4-acetonitrile (97:3). The flow rate was 0.6 mL x min(-1). The determination wavelength was 260 nm and the column temperature was set at 40 degrees C.

RESULTThe method had good linearity in the range of 0.033-0.660 (r = 0.9996), 0.620-12.400 (r = 0.9999), 0.048-0.960 microg (r = 0.9995), with average recoveries of 98.67% (RSD 1.6%), 99.03% (RSD 0.74%), 98.65% (RSD 1.8%), for thymine, hypoxanthine and uracil respectively.

CONCLUSIONThe simultaneous determination method of thymine, hypoxanthine and uracil in medical pipefish is established by RP-HPLC for the first time. The contents of the three constituents in different kinds of medical pipefish are significantly different. The method is simple, rapid and sensitive, and can be used for control the quality of medical pipefish.

Animals ; Chromatography, High Pressure Liquid ; methods ; Hypoxanthine ; analysis ; Medicine, Chinese Traditional ; Smegmamorpha ; Thymine ; analysis ; Uracil ; analysis

The infection status of digenetic trematode metacercariae (DTM) was investigated in fishes from coastal lakes in Gangwon-do, the Republic of Korea (Korea). All fishes collected in 5 lakes were examined with the artificial digestion method. More than 10 species, i.e., Metagonimus spp., Pygidiopsis summa, Centrocestus armatus, Metorchis orientalis, M. taiwanensis, Clinostomum complanatum, Echinostoma spp., Stictodora spp., Diplostomum sp. and Diplostomid No. 1. by Morita (1960), of DTM were detected in fishes from 5 coastal lakes in Gangwon-do. Metagonimus spp. metacercariae were found in 52 (41.3%) out of 126 sea rundace, Tribolodon hakonensis, from 5 lakes, and their density was 14.6 per fish infected. P. summa metacercariae were detected in 48 (84.2%) out of 57 mullets from 5 lakes, and their density was 316 per fish infected. C. armatus metacercariae were detected in 7 (14.6%) T. hakonensis and 3 (15.0%) Tridentiger brevispinis from Hyang-ho, and 5 (19.2%) Acanthogobius flavimanus from Gyeongpo-ho. Stictodora spp. metacercariae were found in 4 fish species, i.e., Tridentiger obscurus, Tridentiger trigonocephalus, Chelon haematocheilus, and Acanthogobius lactipes, from Gyeongpo-ho. Total 15 C. complanatum metacercariae were detected in 2 (9.1%) crucian carp, Carassius auratus, from Songji-ho. M. taiwanensis metacercariae were found in T. hakonensis from Hyang-ho and Gyeongpo-ho and in Pseudorasbora parva from Gyeongpo-ho. Total 11 M. orientalis metacercariae were detected in 3 (6.3%) T. hakonensis from Hyang-ho. From the above results, it was confirmed that various species of DTM are infected in fishes from coastal lakes in Gangwon-do, Korea.
Carps ; Digestion ; Echinostoma ; Fishes ; Gangwon-do ; Goldfish ; Heterophyidae ; Korea ; Lakes ; Metacercariae ; Methods ; Republic of Korea ; Smegmamorpha

Stellantchasmus falcatus (Digenea: Heterophyidae) is first reported from Cambodia through recovery of the metacercariae from mullet fish and adult flukes from an experimentally infected hamster. We purchased 7 mullets, Chelon macrolepis, in a local market of Phnom Penh, Cambodia, and each of them was examined by the artificial digestion method on May 2010. The metacercariae of S. falcatus were detected in all mullets (100%) examined, and their average density was 177 per fish. They were elliptical, 220×168 μm in average size. They were orally infected to an hamster to obtain adult flukes. Adults recovered at day 10 post infection were observed with a light microscope and a scanning electron microscope (SEM). They were small, 450×237 μm in average size, had a small oral sucker (41×50 μm), subglobular pharynx (29×21 μm), slender esophagus (57 μm), long and thick-walled expulsor (119×32 μm), spherical ovary (58×69 μm), and 2 ovoid testes (right: 117×74 μm; left: 114×63 μm). Eggs were small, yellow, and 23×12 μm in average size. In SEM observations, tegumental spines were densely distributed on the whole tegument, and single small type I sensory papillae were distributed around the lip of oral sucker. The small ventral sucker was dextrally located and had 8 type I sensory papillae on the left margin. It has been first confirmed in the present study that the mullet, C. macrolepis, is playing the role of a second intermediate host of S. falcatus in Cambodia.
Adult* ; Animals ; Cambodia* ; Cricetinae* ; Digestion ; Eggs ; Esophagus ; Female ; Humans ; Lip ; Metacercariae* ; Methods ; Ovary ; Ovum ; Pharynx ; Smegmamorpha* ; Spine ; Testis ; Trematoda*