Methods: A field survey was performed on June 29, 2017 in a pond used for culturing the shrimp Penaeus japonicus, located in the southern Yellow Sea, China. Jellyfish specimens were collected for morphological and genetic analysis. The morphological characters of the jellyfish specimens were compared to taxonomic literature. Additionally, phylogenetic analysis of the mitochondrial 16S fragments of these specimens were also conducted.
Results: These specimens had the following morphological characters: hemispherical umbrella without scapulets; J-shaped oral arms; a single larger terminal club on each arm; bluish colored with a slightly expanded white tip; and mouthlets present only in the lower half to one-third of each arm. These morphological features of the medusae indicated that the specimens found in the shrimp culture ponds belong to the genus Phyllorhiza Agassiz, 1862, but did not match with the description of any of the known species of the genus Phyllorhiza. Phylogenetic analyses of the mtDNA 16S regions revealed that these specimens, together with Phyllorhiza sp. from Malaysian coastal waters, belong to a sister group of Phyllorhiza punctata. Juveniles and ephyrae of Phyllorhiza sp. were observed in the aquaculture pond. The mean density of Phyllorhiza sp. medusa in the surface water within the pond was estimated to be 0.05 individuals/m2.
Discussion: Based on our observations of the gross morphology and molecular data, we state that the specimens collected in the aquaculture pond can be identified as Phyllorhiza sp. This is the first record of Phyllorhiza sp. in Chinese seas. Large scale dispersal through ballast water or the expansion of jellyfish aquarium exhibitions are possible pathways of invasion, but this needs to be confirmed in further studies.
Materials and Methods: The novel SSP of CYTBWB2-wb was designed by our group using PRIMERQUEST and NCBI software. DNA was extracted using propanol-chloroform-isoamyl alcohol method. The designed SSP was further subjected for validation protocols using DNA isolated from fresh meat and from meatball, which include specificity test, determination of efficiency, limit of detection and repeatability, and application of developed method for analysis of commercially meatball samples.
Results: The results showed that CYTBWB2-wb was specific to wild boar species against other animal species with optimized annealing temperature of 59°C. The efficiency of q-PCR obtained was 91.9% which is acceptable according to the Codex Allimentarius Commission (2010). DNA, with as low as 5 pg/μl, could be detected using q-PCR with primer of CYTBWB2-wb. The developed method was also used for DNA analysis extracted from meatball samples commercially available.
Conclusion: q-PCR using CYTBWB2-wb primers targeting on mitochondrial cytochrome-b gene (forward: CGG TTC CCT CTT AGG CAT TT; Reverse: GGA TGA ACA GGC AGA TGA AGA) can be fruitfully used for the analysis of WBM in commercial meatball samples.