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Objective:In order to investigate the genetic diversity of mitochondrial cytochrome b (Cytb) gene of Taenia asiatica ( T. asiatica) in Dali Bai Autonomous Prefecture (Dali Prefecture), Yunnan Province. Methods:From May 2019 to August 2021, a total of 131 samples of Taenia were collected from patients admitted to the Dali Prefecture Institute of Schistosomiasis Control, involving five locations (i.e., five groups), including Dali City (58 samples), Weishan Yi and Hui Autonomous County (Weishan County, 14 samples), Midu County (18 samples), Yangbi Yi Autonomous County (Yangbi County, 24 samples) and Eryuan County (17 samples). Primers were designed based on mitochondrial Cytb gene sequence, and part of the Cytb gene sequence was amplified by PCR, then sequenced and homology comparisons were performed. MEGA 7.0 and DNASP 5.10.01 were used to analyze the measured sequence, and data such as base composition, genetic distance, genetic diversity parameters, genetic differentiation index and gene flow were obtained. Results:The amplified fragments of Cytb gene in 131 samples of Taenia were 235 bp. After homology comparisons, they were all T. asiatica. The average contents of A, T, G and C bases were 23.8%, 42.3%, 24.0% and 9.9%, respectively. Of the 131 samples of T. asiatica, 12 haplotypes were defined. The haplotype diversity and nucleic acid diversity were 0.295 9 and 0.006 0, respectively. The ranges of genetic differentiation index and gene flow among the five groups were-0.053 00 to 0.050 40 and 4.710 31 to 162.087 66, respectively. The genetic distance between the five groups ranged from 0.003 5 to 0.009 0, of which the genetic distance between Midu County and Weishan County was the largest, and the genetic distance between Dali City and Yangbi County was the smallest. Conclusions:The mitochondrial Cytb gene of T. asiatica in Dali Prefecture has rich genetic diversity. There is frequent gene exchange among the five groups, and no significant genetic differentiation has been formed.
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BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.
Subject(s)
Animals , Polymerase Chain Reaction/methods , Agkistrodon/genetics , Cytochromes b/genetics , Mitochondria/genetics , Snakes , Species Specificity , DNA/analysis , Cloning, Molecular , Medicine, Chinese TraditionalABSTRACT
BACKGROUND Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.
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BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.
Subject(s)
Humans , Leishmaniasis, Cutaneous , Leishmania/genetics , Panama , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction , DNA, Protozoan/genetics , Cytochromes b/geneticsABSTRACT
Objective:To represent a new geographical record, Phlebotomus (Adlerius) kabulensis (P. kabulensis), which is suspected to be a potential vector of visceral leishmaniasis.Methods:For the first time, P. kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran. Identification of males was based on ecological, morphological, morphometric and molecular (mtDNA cytochrome b gene sequences) criteria. Generally, males have two ascoids on the 8Results:Morphometric measurement revealed that P. kabulensis specimens were the same as compared with seven other morphological characters in three provinces of the country but lengths of the coxite were significantly different. The PCR result of the cytochrome b (Cyt b)-mtDNA fragment shows 550-bp length, with its special nucleotide arrangement. The male and female of P. kabulensis were newly discovered members of the subgenus Adlerius from Iran. Initial DNA analysis indicated how distinct this species is.Conclusions:The results show that the P. kabulensis female will be identified by comparing with other Adlerius female groups regarding its morphometric characters and molecular sequencing.
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Objective: To represent a new geographical record, Phlebotomus (Adlerius) kabulensis (P. kabulensis), which is suspected to be a potential vector of visceral leishmaniasis. Methods: For the first time, P. kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran. Identification of males was based on ecological, morphological, morphometric and molecular (mtDNA cytochrome b gene sequences) criteria. Generally, males have two ascoids on the 8
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Five hundred nucleotides of cytochrome b gene was sequenced for Pakistani, Tihami, Syrian, Masri and Aardi goat breeds inhabiting Saudi Arabia. Based on two computational maximum-parsimony and neighbor-joining methods, the constructed tree clustered the studied breeds into Masri and Tihami in one group and Pakistani and Aardi came basal to this group. Syrian breed came basal to all of the studied breeds. This division was based on a few mitochondrial DNA sites and few sampling size and it therefore needs to be supported in a further molecular study. The goat breeds studied herein did not show any non-synonymous genetic variability when the protein-coding gene of the mitochondrial genome was used as a molecular maker. However, the only mitogenome maker which can show base differences among these breeds was the non-coding displacement loop (d-loop). In conclusion, using the mitochondrial genes, rather than the d-loop, for studying genetic diversity among goat breeds is with no meaning and it is better to use other markers for this purpose.
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Background & objectives: Pipistrellus ceylonicus bat species is widely distributed in South Asia, with additional populations recorded in China and Southeast Asia. Bats are the natural reservoir hosts for a number of emerging zoonotic diseases. Attempts to isolate bat-borne viruses in various terrestrial mammalian cell lines have sometimes been unsuccessful. The bat cell lines are useful in isolation and propagation of many of the viruses harboured by bats. New stable bat cell lines are needed to help such investigations and to assist in the study of bat immunology and virus-host interactions. In this study we made an attempt to develop a cell line from P. ceylonicus bats. Methods: An effort was made to establish cell line from embryo of P. ceylonicus species of bat after seeding to Dulbecco’s modified eagle medium (DMEM) supplemented with 10 per cent foetal bovine serum; a primary cell line was established and designated as NIV-BtEPC. Mitochondrial DNA profile analysis was done using cyt-b and ND-1 gene sequences from the cell line. Phylogenetic tree was constructed using neighbour-joining algorithm for cyt-b and ND-1 genes with 1000-bootstrap replicates. Results: NIV-BtEPC cell line was susceptible to Chandipura (CHPV) and novel adenovirus (BtAdv-RLM) isolated from Rousettus leschenaulti from India but did not support multiplication of a number of Bunyaviruses, Alphaviruses and Flavivirus. This might be useful for isolation of a range of viruses and investigation of unknown aetiological agents. Interpretation & conclusions: In this study, a new bat cell line was developed from P. ceylonicus. This cell line was successfully tested for the susceptibility to Chandipura and BtAdv-RLM virus isolated from bats. The approach developed and optimised in this study may be applicable to the other species of bats and this established cell line can be used to facilitate virus isolation and basic research into virus-host interaction.
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Tigers are one of the most endangered species over the world and protected internationally or locally. However, they are still traded illegally for the Traditional East Asia Medicine or souvenirs / mementos as well as fake products. In the present study, to identify the species of the specimen known as the tiger's genitalia that was sold for mementos in China, two approaches of genetic tools and morphological comparisons were applied. On the basis of the entire sequences of mitochondrial cytochrome b gene (1,140 bp), the result of nucleotide comparisons showed that the specimen examined here is matched with banteng, Bos javanicus (Identities = 1,138/1,140; 99%). In addition, the examination of external morphological characters revealed that the genitalia of the specimen is much more similar to the one of cows, not to tigers. The outcomes highlighted that there are still some fake products of tigers traded in market and molecular genetic tools could help identifying species of wildlife products.
Subject(s)
Base Sequence , China , Cytochromes b , DNA , Endangered Species , Genitalia , Medicine, East Asian Traditional , Molecular Biology , Sequence Analysis, DNA , TigersABSTRACT
Pimelodus albicans Valenciennes, 1840 (common name "moncholo" or "bagre blanco") is an endemic species of the family Pimelodidae in the río de la Plata basin. Phylogenetic approach based on cytochrome b sequences was performed to test the existence of a unique evolutionary lineage in P. albicans and to discriminate populations units or subpopulations related to a migration behavior of this taxon in the río de la Plata basin. This study included 34 samples of P. albicans of different collecting sites in the río de la Plata estuary and in the río Arrecifes belonging to the río Paraná basin. Among 614 base pairs in the cytochrome b sequence data set, 203 were variable and 120 were phylogenetically informative sites in P. albicans. A total of twenty haplotypes, nucleotide diversity (p) = 0.032 and haplotype diversity = 0.941 were found. Tajima's test showed significant value D= -1.88 (p<0.05) rejecting the neutral mutation hypothesis for the P. albicans data set. All phylogenetic approaches showed that P. albicans included four monophyletic assemblages that were supported by high bootstrap and Bayesian posterior probability values. Minimum spanning network corroborated these groups for P. albicans haplotypes. High genetic structure was found in P. albicans by means of AMOVA analysis showing that the río Arrecifes samples constitute an isolated lineage. Moreover, the high value of genetic divergence (10 percent) between the río de la Plata and the río Arrecifes populations could suggest that P. albicans may be conformed by a sibling species complex. On the other hand, a degree of genetic structuring was detected among different sites of the río de la Plata. A partial isolation of the 760 site may suggest that P. albicans could migrates to different tributaries for reproduction, generating different schools of haplotypes which could mix in the río de la Plata estuary. The high nucleotide diversity found in the 765 site and the existence of...
Pimelodus albicans Valenciennes, 1840 (popularmente conhecida como moncholo ou bagre branco) é uma espécie endêmica da família Pimelodidae na bacia do rio da Prata. Estudos filogeográficos baseados nas seqüências do citocromo b mitocondrial foram realizados para testar a existência de uma única linhagem evolutiva in P. albicans e para discriminar unidades populacionais relacionadas ao comportamento migratório desse táxon na bacia do rio da Prata. Um total de 34 amostras de P. albicans provenientes de diferentes lugares de coleta no estuário do rio da Prata e rio Arrecifes na bacia do rio Paraná foram analisados. Entre as 614 pares de bases do citocromo b no conjunto de dados, 203 deles variaram e 120 foram sítios filogeneticamente informativos para P. albicans. No presente estudo foi encontrado um total de vinte haplótipos, diversidade de nucleotídeos (p) = 0,032 e diversidade de haplótipos = 0,941. O teste de Tajima mostrou valores significativos D= -1,88 (p<0,05) rejeitando a hipótese de mutação neutra para os dados de P. albicans. Todas as análises filogenéticas mostraram que o clado P. albicans apresenta quatro grupos monofiléticos com um forte suporte estatístico e uma elevada probabilidade posterior Bayesiana. A rede de haplótipos para P. albicans mostrou uma forte estrutura desses quatro grupos. Uma grande estruturação genética foi observada em P. albicans nas análises de AMOVA mostrando que as amostras do rio Arrecifes constituem uma linhagem isolada. A alta divergência (10 por cento) encontrada entre as populações do rio da Prata e rio Arrecifes sugere que P. albicans pode constituir um complexo de espécies crípticas. Foi verificada também a ocorrência de estrutura genética na bacia do rio da Prata. A localidade 760 apareceu parcialmente isolada sugerindo que P. albicans migra para procriar em os afluentes do rio da Prata gerando diferentes cardumes de haplótipos que poderiam se misturar no estuário do rio da Prata. A alta diversidade...
Subject(s)
Animals , Biodiversity , Species Specificity , Genetic Variation , Catfishes/classificationABSTRACT
Cutaneous leishmaniasis (CL) is an emerging disease in Sri Lanka, more than 400 cases having been reported since 2001. However, the morphology and taxonomic status of the Sri Lankan strain of <I>Leishmania</I> is not known yet. Therefore, it is important to study the morphology and to analyze the phylogenetic position to predict the risk and expansion of the disease and thereby to develop an effective control programme. Morphology of the amastigote of the Sri Lankan isolate was checked by light microscopy and electron microscopic observation. Presence of amastigotes within macrophages was confirmed in skin biopsy samples. The promastigote had the characteristic appearance of a kinetoplastid cell in cultures. The kinetoplast minicircle DNA has been used for diagnosis of <I>Leishmania</I> for a long time and also for phylogenetic studies on trypanosomatid flagellates. The kinetoplast minicircle was amplified using PCR and subsequently sequenced from samples obtained from Sri Lankan patients with cutaneous lesions. Mitochondrial cytochrome b gene has been recently shown to be useful for identification and phylogenetic analysis of the genus <I>Leishmania</I>. The nucleotide sequence of the cytochrome b gene of Sri Lankan <I>Leishmania</I> was determined using the semi-nested PCR and 620 bp of this gene obtained. Phylogenetic analysis using these sequences unambiguously indicated that Sri Lankan isolate of <I>Leishmania</I> belongs to <I>L. donovani</I> complex. However, the Sri Lankan isolate forms a distinct lineage within the complex and probably represents a new branch.
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Total sequence phylogenies have low information content. Ordinary misconceptions are that character quality can be ignored and that relying on computer algorithms is enough. Despite widespread preference for a posteriori methods of character evaluation, a priori methods are necessary to produce transformation series that are independent of tree topologies. We propose a stepwise qualitative method for analyzing protein sequences. Informative codons are selected, alternative amino acid transformation series are analyzed, and most parsimonious transformations are hypothesized. We conduct four phylogenetic analyses of philodryanine snakes. The tree based on all nucleotides produces least resolution. Trees based on the exclusion of third positions, on an asymmetric step matrix, and on our protocol, produce similar results. Our method eliminates noise by hypothesizing explicit transformation series for each informative protein-coding amino acid. This approaches qualitative methods for morphological data, in which only characters successfully interpreted in a phylogenetic context are used in cladistic analyses. The method allows utilizing character information contained in the original sequence alignment and, therefore, has higher resolution in inferring a phylogenetic tree than some traditional methods (such as distance methods).
Filogenias baseadas em seqüências totais têm baixo conteúdo informativo. Erros comuns são acreditar que a qualidade dos caracteres pode ser ignorada e que é suficiente confiar nos algoritmos computacionais. Apesar de ampla preferência por métodos a posteriori para a avaliação de caracteres, métodos a priori tornam-se necessários para produzir séries de transformação independentes das topologias das árvores. Propomos um método qualitativo passo a passo para analisar seqüências de proteínas. Codons informativos são selecionados, séries de transformação alternativas de aminoácidos são analisadas e as transformações mais parcimoniosas são hipotetizadas. Conduzimos quatro análises filogenéticas em cobras Phylodrininae. A árvore baseada em todos os nucleotídeos produz a menor resolução. Árvores baseadas na exclusão das terceiras posições, numa matriz de passos assimétrica, e em nosso protocolo de análise produzem resultados similares. Nosso método elimina ruído ao hipotetizar séries de transformação explícitas para cada aminoácido informativo para a codificação de proteínas. Essa abordagem se aproxima de métodos qualitativos para dados morfológicos, nos quais apenas caracteres interpretados com sucesso num contexto filogenético são usados em análises cladísticas. O método permite utilizar informação de caracteres contidos no alinhamento original da seqüência e, portanto, tem maior poder de resolução para inferir árvores filogenéticas que alguns métodos tradicionais (como métodos de distância).
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The feasibility of species identification using sequence analysis of the cytochrome B (Cyt B) gene in mitochondrial DNA was investigated. DNA was extracted from nine different animals that could be easily met in our surroundings and Cyt B gene was amplified. Direct sequencing results for the amplified PCR products were compared with each other. Human was also included. Nucleotide sequence of the Cyt B gene for each animals was also compared with the previously known ones registered in nucleotide databases, Genebank. The inter-species sequence variation was high as the percent similarity of each sequences ranged 64.6-83.5%. Compared to this the percent similarity of sequences obtained here were high when compared to the sequences of the same species registered in the database showing relatively low intra-species variation. This data shows that the nucleotide sequences of Cyt B gene in a certain biological materials can be identified at species level. The applicability of this method to the forensic field is also demonstrated by performing a casework; determination of the origin for the placentae which were commercial available as "invogorant". Points about the use of Cyt B gene in forensic field was also reviewed.
Subject(s)
Animals , Humans , Base Sequence , Cytochromes b , Cytochromes , DNA , DNA, Mitochondrial , Placenta , Polymerase Chain Reaction , Sequence AnalysisABSTRACT
The feasibility of species identification using sequence analysis of the cytochrome B (Cyt B) gene in mitochondrial DNA was investigated. DNA was extracted from nine different animals that could be easily met in our surroundings and Cyt B gene was amplified. Direct sequencing results for the amplified PCR products were compared with each other. Human was also included. Nucleotide sequence of the Cyt B gene for each animals was also compared with the previously known ones registered in nucleotide databases, Genebank. The inter-species sequence variation was high as the percent similarity of each sequences ranged 64.6-83.5%. Compared to this the percent similarity of sequences obtained here were high when compared to the sequences of the same species registered in the database showing relatively low intra-species variation. This data shows that the nucleotide sequences of Cyt B gene in a certain biological materials can be identified at species level. The applicability of this method to the forensic field is also demonstrated by performing a casework; determination of the origin for the placentae which were commercial available as "invogorant". Points about the use of Cyt B gene in forensic field was also reviewed.