Paragonimiasis, caused by Paragonimus species belonging to the family Paragonimidae of the suborder Xiphidiata (Platyhelminthes: Trematoda), often occurs in poor, upland, ethnic minorities, in Vietnam and the world. Asian Paragonimus species are distributed from Japan, South Korea, along with North and Southeast China, North-West and Central Vietnam, the Philippines, Thailand, Bangladesh, India, and Sri Lanka. There are various genetic variants, strains, and genotypes forming different complexes and evolutionary lineages. The 18S, 28S rDNA sequences and the intergenic transcribed spacer regions (ITS-1, ITS-2) of nuclear ribosomal transcription units are commonly used as molecular markers in genetic studies and phylogenetic analyses. We obtained a portion of 28S rDNA (domains D1–D3) of Paragonimus spp. including P. heterotremus (from Vietnam), P. ohirai (Japan), P. iloktsuenensis (Japan), and P. westermani (India and Vietnam) and conducted phylogenetic analysis for molecular evolutionary studies. The results showed that the family Paragonimidae formed the biggest cluster in a phylogenetic tree, which comprises of 46 sequences of 11 species belonging to 11 subgroups, among which the P. westermani complex of strains originating from China, Korea, Japan, India, Philippines, Malaysia, and Vietnam is present. P. westermani complex is arranged in a position of "sister" (sister group) with the subgroup P. siamensis. The P. heterotremus and P. ohirai complexes, and the P. miyazakii, P. harinasutai, P. mexicanus, P. kellicotti, and P. macrorchis complexes are clustered in a common population. P. westermani of Vietnam is in close proximity to the East Asian strains, as of which has been previously reported. P. ohirai and P. iloktsuenensis are considered “sibling” species, sharing the same clade. Phylogenetic analysis using the 28S rDNA sequences directly presented species position and their molecular evolutionary relationships in the families Paragonimidae, Troglotrematidae, Nanophyetidae, and Collyriclidae. Evolutionary analysis has also clarified a number of complex delineation problems and made a clear nomenclature for Paragonimus sp. of Vietnam, in particular, which has scientific grounds merited to recognize as that it is really the P. westermani species.
Echinostomiasis is a neglected disease caused by the intestinal flukes (family Echinostomatidae, suborder Echinostomata) and is common in communities in Asian countries, such as India, Indonesia, the Philippines, China, Malaysia, Singapore, Korea, Japan, Thailand, Laos, Cambodia and Vietnam. The genetic markers from the nuclear ribosomal transcription units are commonly used in genetic studies and phylogenetic analyses. A portion of the 28S rDNA sequence (domains D1–D3, of 1062–1067 bp for the final use) was obtained from the zoonotic Echinostoma malayanum (strain E/Amala-EMI3-TH), E. revolutum (strain Erevo-MSD15-TH), E. miyagawai (Emiya-RED11-TH), and Hypoderaeum conoideum (Hcono-RED42-TH) species; and used to perform an alignment for genetic distance estimation and phylogenetic analysis. The alignment was performed using 62 strains of 42 species from 19 genera of the family Echinostomatidae, including Echinoparyphium, Echinostoma, Artyfechinostomum, Patagifer, Neoacanthoparyphium, Hypoderaeum, Echinoparyphium, Drepanocephalus, Euparyphium, Chaunocephalus, Neopetasiger, Ribeiroia, Cathaemasia, Rhopalias, Isthmiophora, Petasiger, Moliniella, Pegosomum, and Schistosoma (family Schistosomatidae). The genetic distance estimation among 16 strains/10 species has shown a low intra-specific divergence level between strains within the same species, such as E. miyagawai (0–0.10%), E. revolutum (0.10–0.50%), and H. conoideum (0–0.10%), while between strains within the same genus it was higher (normally over 1.0%) and among strains/species between genera it was the highest (3.06–4.12%). The 28S rDNA sequence analysis and phylogenetic relationship well supported the Echinostoma/ Artyfechinostomum malayanum intergeneric taxonomy and the topology indicated clear, well-supported positions of member species in different genera in the family Echinostomatidae of the suborder Echinostomata. More sensu lato samples of the genera, are required for sequencing, particularly those of zoonotic species in the five genera: Artyfechinostomum, Echinostoma, Hypoderaeum, Echinoparyphium, and Isthmiophora. The resultant mitochondrial and nuclear data obtained from these species will be a good source to use to clearly assess the taxonomic and generic relationships.
The family Echinostomatidae includes four genera, Echinostoma, Hypoderaeum, Echinoparyphium, and Artyfechinostomum, which are of public epidemiological and pathogenic importance. The morphological similarity of species requires differentiating methods, and the use of molecular genetic markers is mainly encountered. The 28S rRNA gene sequence in the family Echinostomatidae is an important marker for the analysis of the interspecies and phylogenetic relationships. However, information about the structure and composition of the complete 28S rRNA gene in Echinostomatidae is limited. In this study, the complete 28S rRNA sequences (3,881–3,883 bp) of four species, including Echinostoma revolutum (designated Erev-MSD15-TH), E. malayanum (Emal-EMI3-TH), E. miyagawai (Emiy-RED11-TH), and Hypoderaeum conoideum (Hcon-RED42-TH) were obtained and analyzed for their genetic characteristics. All had complex secondary structures, containing numerous branches, “hairpins”, and “loops” that provide stability of the rRNA gene. The de novo structural pattern and the "skew" value in the nucleotide use of E. revolutum, E. malayanum, E. miyagawai, and H. conoideum were analyzed. The nucleotide usage of A, T, G, C and A+T and G+C of 28S rRNA in all species of the family Echinostomatidae was similar, in which A and T usage were almost equal, and G and C were slightly different in the range of 7–8%, so the skewness for AT was very low negative value (–0.029 to –0.045), and for GC was not very high (0.163 to 0.172). The genetic distance estimated among strains within a species (intra-specific level) was very low in both the Echinostomatidae (0.05%) and the Fasciolidae families (0–0.03%), and that among different species (inter-specific level) was higher. However, among species in different families (inter-familial level) the genetic distance was highest, at 3.80–4.86%, as seen between Echinostoma and Fasciola species. The 28S rRNA gene dataset obtained from four species of the intestinal flukes (family Echinostomatidae) in this study contribute to the understandings of genetic markers in the suborder Echinostomata for use in diagnostic, phylogenetic, taxonomic, epidemiological, and population genetics studies.
Spirulina is a spiral cyanobacteria with simple cell structure, making it easier to extract chlorophyll than other materials. Investigation the ultrasound assisted extraction of chlorophyll a was conducted on the following factors: type of solvent, concentration of solvent, ratio of solvent: material, time and temperature. Optimization by Design Expert the extraction of chlorophyll a from spirulina algae indicates the condition to obtain the highest amount of chlorophyll a of 15.07 mg/g was as followings: solvent ethanol concentration 99.7%, ratio solvent / material: 16/1, time 2.5 hours, temperature 52°C, ultrasonic frequency 40kHz.
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