Genetic analysis of oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) populations based on mitochondrial cox1 and nad1 gene sequences from India and other Asian countries.

The study examined the genetic diversity and demographic history of Bactrocera dorsalis, a destructive and polyphagous insect pest of fruit crops in diverse geographic regions of India. 19 widely dispersed populations of the fly from India and other Asian countries were analysed using partial sequences of mitochondrial cytochrome oxidase I (cox1) and NADH dehydrogenase 1 (nad1) genes to investigate genetic diversity, genetic structure, and demographic history in the region. Genetic diversity indices [number of haplotypes (H), haloptype diversity (Hd), nucleotide diversity (π) and average number of nucleotide difference (k)] of populations revealed that B. dorsalis maintains fairly high level of genetic diversity without isolation by distance among the geographic regions. Demographic analysis showed significant (negative) Tajimas' D and Fu's F S with non significant sum of squared deviations (SSD) values, which indicate the possibility of recent sudden expansion of species and is further supported through distinctively star-like distribution structure of haplotypes among populations. Thus, the results indicate that both ongoing and historical factors have played important role in determining the genetic structure and diversity of the species in India. Consequently, sterile insect technique (SIT) could be a possible management strategy of species in the regions.

Investigation and genetic polymorphism analysis of rodents infected with Echinococcus in Ili Prefecture, Xinjiang Uygur Autonomous Region, China.

Introduction: Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis. Domestic animals, dogs, foxes, and small mammals constitute the circular chain of AE. To evaluate the infection, distribution, and genetic polymorphism of AE in the Ili Prefecture (Nilka, Xinyuan and Zhaosu), we conducted this survey. Methods: In June and July 2018, 267 small mammals were captured using water-infusion and mousetrap methods. Combined pathogenic and molecular biological methods were used to observe the histopathology of Echinococcus carried by rodents, amplify the mitochondrial nad1 gene of the pathogen, and investigate the genotype and haplotype diversity of Echinococcus in rodents in Ili Prefecture. Results: Morphological identification revealed that these captured small mammals belonged to three species, with Microtus gregalis being the dominant species (183/267). Pathological and molecular biological results confirmed that E. multilocularis was the pathogen of echinococcosis in small mammals, with an infection rate of 15.73% (42/267). Among the three areas sampled, the highest infection rate of rodents was 25.45% (14/55) in Nilka County. However, there was no significant difference in the infection rates between regions (χ2 = 5.119, p > 0.05). Of the three captured rodent species, M. gregalis had the highest infection rate of 17.49% (32/183), but there was no significant difference in infection rates between the rodent species (χ2 = 1.364, p > 0.05). Phylogenetic analyses showed that the nad1 gene sequences obtained in this study clustered in the same clade as isolates from China. These isolates contained 21 haplotypes (Hap_1-21); Hap_2 was the most common haplotype (9/42). Furthermore, haplotype diversity (0.925 ± 0.027) and nucleotide diversity (0.01139 ± 0.00119) were higher in the Ili Prefecture than in other regions, indicating that population differentiation was high. Tajima's D and Fu's Fs tests were negative (p > 0.10), indicating that the population had expanded. The low fixation index (Fst) ranged from 0.00000 to 0.16945, indicating that the degree of genetic differentiation was different among different populations. Discussion: In summary, Ili Prefecture is a high incidence area of AE, and Microtus spp. may play an important role in the transmission of AE in this area. The results of this study provide basic data for further study of the molecular epidemiology, genetic differences, and control of E. multilocularis in the Ili Prefecture, Xinjiang.

A Novel Designed Sandwich ELISA for the Detection of Echinococcus granulosus Antigen in Camels for Diagnosis of Cystic Echinococcosis.

Echinococcus spp. are important cosmopolitan zoonotic parasitic tapeworms that cause a disease called hydatidosis or cystic echinococcosis (CE), which has remarkable economic losses. The objective of our study was to develop a specific IgG polyclonal antigen-based ELISA (Sandwich ELISA; capture ELISA) method for the detection of circulating Echinococcus granulosus (E. granulosus) antigens in camels infected with hydatid cysts before slaughtering and its application in serodiagnosis of CE in animals to assess the positive rate of hydatidosis in camels slaughtered in Giza governorate abattoirs in Egypt. In this study, molecular identification of Echinococcus sp. isolate was performed based on the NADH dehydrogenase subunit 1 (NAD1) gene, revealing the isolate (GenBank: OQ443068.1), which is identical to the G6 E. granulosus sensu lato genotype. The positive rate of hydatid cysts was determined in slaughtered camels' organs (n = 587). The results revealed that hydatid cysts were found in 46.5% (273/587) of the examined camels. Pulmonary echinococcosis was significantly more prevalent in the slaughtered camels (60%, 164/273) than hepatic echinococcosis (39.9%, 109/273), (p = 0.001, Chi Square = 11.081). Cyst fertility rates were higher in hepatic (90.8%, 99/109) than in pulmonary cysts (83.5%, 137/164) and the most viable protoscoleces were recorded from fertile the hepatic cysts (67.85 ± 12.78). In this study, hydatid cyst germinal layer antigen (GlAg) was isolated and used for the immunization of rabbits to raise IgG polyclonal antibodies (anti-Echinococcus GlAb IgG). These IgG polyclonal antibodies were purified by affinity chromatography using a protein A column, then labeled with horseradish peroxidase. Electrophoretic analysis of IgG polyclonal antibodies and crude GlAg was performed in 10% polyacrylamide gels. The SDS-PAGE revealed four bands at molecular weights of 77 kDa, 65 kDa, 55 kDa, and 25 kDa. The Sandwich ELISA was performed to evaluate the sensitivity and specificity and cross-reactivity of the prepared IgG polyclonal antibodies. The circulating hydatid antigen was found in 270 out of the 273 samples with hydatidosis, with a sensitivity of 98.9% (270/273), a specificity of 94.9% (296/312) and a diagnostic efficacy of 96.8%. Regarding the cross reactivity, anti-Echinococcus GlAb IgG showed a low cross-reactivity with Fasciola gigantica infected camel sera (3/8), and Myiasis (Cephalopina titillator larvae; 3/20). No cross-reactivity was recorded with uninfected camel sera (negative sera for E. granulosus), and no cross-reactivity was found with antigens of Eimeria spp., Toxoplasma gondii, Cryptosporidium sp., and Hyalomma dromedarii (ticks' infestation). Then, Sandwich ELISA was conducted again to detect E. granulosus antigen in all the collected camel sera, which resulted in a 48.7% (286/587) positive rate of CE compared to 46.5% (273/587) using a postmortem inspection (PM diagnosis) (p = 0.5, Chi Square = 0.302). In conclusion, the Sandwich ELISA technique introduced in this study appears to be a sufficiently sensitive diagnostic assay for the detection of camels' echinococcosis using anti-Echinococcus GlAb IgG. In addition, it might offer a significant medical and veterinary importance in helping the early detection of hydatidosis, as well as its early treatment.

[In silico cloning and comparative analysis of NAD1 gene in three common human parasites].

OBJECTIVE: To in silico clone the NAD1 gene of three common parasites and analyze their bioinformatics, so as to lay the foundation for further research on the NAD gene. METHODS: By using the in silico cloning method, the full length cDNA (s) of NAD 1 genes of Clonorchis sinensis, Ascaris lumbricoides and Schistosoma japonicum were got, then their physical and chemical properties, compositions of amino acids, subcellular localizations, binary and ternary structures were contrastively analyzed. RESULTS: The three kinds of NAD1 proteins were similar in the relative molecular weight, subcellular localization, and physical and chemical properties. The NAD1 proteins were highly similar in binary and ternary structures of A. lumbricoides and S. japonicum. The phylogenetic analysis showed that C. sinensis, A. lumbricoides and S. japonicum belonged to the different evolutionary branches with a certain of genetic distance. CONCLUSIONS: The three NAD1 genes got from C. sinensis, A. lumbricoides and S. japonicum by in silico cloning belong to the same gene of different species, which can be widely used in the researches of heritable variation of parasites.

[Genotyping and gene nucleotide polymorphism of epidemic strain of Echinococcus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province].

OBJECTIVE: To understand the genotypes and nucleotide polymorphisms of Echinococcus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province. METHODS: The specific primers were designed according to the cox1 and nad1 genes of E. granulosus mitochondrial genome sequences accessed by GenBank. The primers were used to detect the cyst samples from 16 sheep and 2 humans infected with E. granulosus in Tianjun region of Qinghai Province by PCR, then the PCR amplification products were sequenced, the genotypes and nucleotide polymorphisms of the cox1 and nad1 genes were analyzed. RESULTS: The 18 isolated samples all belonged to E. granulosus G1 genotype. Among all the isolates, 9 haplotypes existed in the cox1 gene with 16 nucleotide mutation sites, and there were 0 to 5 nucleotide differences with the highest variation rate of 0.31%, whereas 7 haplotypes occurred with 15 nucleotide mutation sites, and there were 1 to 8 nucleotide differences with the highest variation rate of 0.89% for the nad1 gene. CONCLUSIONS: The epidemic genotype of E. granulosus is G1 in humans and sheep in Tianjun region of Qinghai Province, and the nucleotide polymorphisms of the cox1 gene were more abundant than those of the nad1 gene, and the resolution of the nucleotide polymorphisms of cox1 gene is higher than that of the nad1 gene used in E. granulosus isolates.

Genetic analysis of Bactrocera zonata (Diptera: Tephritidae) populations from India based on cox1 and nad1 gene sequences.

The peach fruit fly, Bactrocera zonata, is among the most serious and polyphagous insect pest of fruit crops in many parts of the world under genus Bactrocera. In the present study, the genetic structure, diversity and demographic history of B. zonata in India were inferred from mitochondrial cytochrome oxidase 1 (cox1) and NADH dehydrogenase 1 (nad1) sequences. The efficiency of DNA barcodes for identification of B. zonata was also tested. Genetic diversity indices [number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π) and average number of nucleotide differences (k)] of B. zonata populations across India maintain high level of genetic diversity without isolation by distance among the geographic regions. Non-significant negative correlation between pairwise Fst and geographic distance suggests a high level of gene flow among studied populations of B. zonata. The possibility of sudden expansion of B. zonata revealed through mismatch distribution analysis as well as negative Tajima's D and Fu's Fs values further supported by star-like network of haplotypes. DNA barcoding analysis suggests that B. zonata specimens can be clearly differentiated from other species with 100% accuracy of identification. Therefore, cytochrome oxidase 1 (cox1) barcode sequences generated in the present study could be a valuable source for the rapid identification and global population genetic study of B. zonata.

Loss of a Trans-Splicing nad1 Intron from Geraniaceae and Transfer of the Maturase Gene matR to the Nucleus in Pelargonium.

The mitochondrial nad1 gene of seed plants has a complex structure, including four introns in cis or trans configurations and a maturase gene (matR) hosted within the final intron. In the geranium family (Geraniaceae), however, sequencing of representative species revealed that three of the four introns, including one in a trans configuration and another that hosts matR, were lost from the nad1 gene in their common ancestor. Despite the loss of the host intron, matR has been retained as a freestanding gene in most genera of the family, indicating that this maturase has additional functions beyond the splicing of its host intron. In the common ancestor of Pelargonium, matR was transferred to the nuclear genome, where it was split into two unlinked genes that encode either its reverse transcriptase or maturase domain. Both nuclear genes are transcribed and contain predicted mitochondrial targeting signals, suggesting that they express functional proteins that are imported into mitochondria. The nuclear localization and split domain structure of matR in the Pelargonium nuclear genome offers a unique opportunity to assess the function of these two domains using transgenic approaches.

The first report of human-derived G10 genotype of Echinococcus canadensis in China and possible sources and routes of transmission.

Cystic echinococcosis (CE) is one of the most important parasitic zoonoses. 10 distinct genotypes, designated G1-G10 genotypes of Echinococcus granulosus sensu lato (s.l.), have been split into 4 species: Echinococcus granulosus sensu stricto (s.s.) (G1-G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and Echinococcus canadensis (G6-G10); Echinococcus felidis has also been suggested as a sister taxon of E. granulosus s.s. recently. Four genotypes belonging to two species (G1 and G3 genotypes of E. granulosus s.s., and G6 and G7 genotypes of E. canadensis) have been identified in humans and animals in China. In the present study, a human-derived hydatid cyst from a patient in northeastern China's Heilongjiang Province was identified as G10 genotype of E. canadensis based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Homology analysis showed the cox1 gene sequence of G10 genotype of E. canadensis had 100% homology with those from wolves in Mongolia and from a moose in Russia. The cytb and nad1 gene sequences of G10 genotype of E. canadensis had 100% homology with the complete sequence from a moose in Finland at an amino acid level. The infection source of the CE patient here might be primarily attributable to wolves. This is the first report of G10 genotype of E. canadensis in a human in China. The finding of G10 genotype of E. canadensis in China shows that this genotype possibly has a more wide geographical distribution than previously considered.

The identification of Sect. Cruciata (Gentiana) species using mtDNA nad1/b-c and nad5/d-e fragments / 药学学报

italic>Gentiana section Cruciata (Gentianaceae) is a medicinally important section of herbs, including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji. Here, we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data. A total of 144 nad1/b-c and nad5/d-e sequences from 11 species within Gentianaceae were obtained, including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica (outgroup). The results showed that mtDNA nad1/b-c has species- level resolution within the section of Cruciata, i.e. the variable in the position 45 “C” could be used as a stable marker locus to distinguish G. robusta from other taxa; the variable in the position 352 and 353 “GA” could distinguish G. crassicaulis and G. tibetica from other taxa within the section. Intraspecies genotype variability was detected in nad1/b-c sequences of G. officinalis and G. siphonantha, respectively. These genotypes could be used as potential DNA barcode. In addition, intraspecies genotype variability was detected in nad5/d-e sequences of G. macrophylla, G. officinalis and G. siphonantha, respectively. Based on the stable marker locus, a species-specific PCR protocol was developed using the primer PF to identifying G. robusta in the section. This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana, and provide the essence for the species identification within Gentiana section Cruciata.

In silico cloning and comparative analysis of NAD1 gene in three common human parasites / 中国血吸虫病防治杂志

Objective To in silico clone the NAD1 gene of three common parasites and analyze their bioinformatics,so as to lay the foundation for further research on the NAD gene. Methods By using the in silico cloning method,the full length cDNA (s)of NAD 1 genes of Clonorchis sinensis,Ascaris lumbricoides and Schistosoma japonicum were got,then their physical and chemical properties,compositions of amino acids,subcellular localizations,binary and ternary structures were contrastively an-alyzed.Results The three kinds of NAD1 proteins were similar in the relative molecular weight,subcellular localization,and physical and chemical properties.The NAD1 proteins were highly similar in binary and ternary structures of A.lumbricoides and S.japonicum.The phylogenetic analysis showed that C.sinensis,A.lumbricoides and S.japonicum belonged to the different evolu-tionary branches with a certain of genetic distance. Conclusion The three NAD1 genes got from C.sinensis,A.lumbricoides and S.japonicum by in silico cloning belong to the same gene of different species,which can be widely used in the researches of heritable variation of parasites.

Genotyping and gene nucleotide polymorphism of epidemic strain of Echino-coccus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province / 中国血吸虫病防治杂志

Objective To understand the genotypes and nucleotide polymorphisms of Echinococcus granulosus metacestode from humans and sheep in Tianjun region,Qinghai Province. Methods The specific primers were designed according to the cox1 and nad1 genes of E.granulosus mitochondrial genome sequences accessed by GenBank.The primers were used to detect the cyst samples from 16 sheep and 2 humans infected with E.granulosus in Tianjun region of Qinghai Province by PCR,then the PCR amplification products were sequenced,the genotypes and nucleotide polymorphisms of the cox1 and nad1 genes were analyzed.Results The 18 isolated samples all belonged to E.granulosus G1 genotype.Among all the isolates,9 haplotypes ex-isted in the cox1 gene with 16 nucleotide mutation sites,and there were 0 to 5 nucleotide differences with the highest variation rate of 0.31%,whereas 7 haplotypes occurred with 15 nucleotide mutation sites,and there were 1 to 8 nucleotide differences with the highest variation rate of 0.89% for the nad1 gene.Conclusions The epidemic genotype of E.granulosus is G1 in hu-mans and sheep in Tianjun region of Qinghai Province,and the nucleotide polymorphisms of the cox1 gene were more abundant than those of the nad1 gene,and the resolution of the nucleotide polymorphisms of cox1 gene is higher than that of the nad1 gene used in E.granulosus isolates.