Haplorchis pumilio and H. taichui in Vietnam discriminated using ITS-2 DNA sequence data from adults and larvae

Samples of Haplorchis taichui and Haplorchis pumilio of different life-stages (cercariae, metacercariae and adults) and from different host species (snail, fish, dog, cat and human) were collected in Nghe An and Nam Dinh Provinces in Vietnam. Samples from Thailand were available for comparison. All adults and metacercariae were initially identified using morphological criteria. Polymerase chain reaction (PCR) assays were developed for discriminating between the species. The complete sequence for the nuclear ribosomal internal transcribed spacer-2 (ITS-2) was obtained from one adult and one metacercaria of H. taichui and three adults and three metacercariae of H. pumilio from Vietnam. Sequences from cercariae from three different snails clustered with those of H. pumilio. Intra-individual variation in the ITS-2 region was detected by sequencing of cloned PCR products. These are the first sequences from Vietnamese Haplorchis spp. to be reported and demonstrate that H. taichui and H. pumilio can be identified unambiguously from any life-stage, including the cercarial stage that is difficult to identify using morphology. Discrepancies in the literature are discussed and examples of apparent misidentification highlighted. The data provide a resource to assist in taxonomic studies on heterophyids, in the design of probes for diagnosis and for field surveys to identify infection in snails.     

Foodborne Trematode Metacercariae in Fish from Northern Vietnam and Their Adults Recovered from Experimental Hamsters

The prevalence of foodborne trematode (FBT) metacercariae was investigated in fish from 2 localities of northern Vietnam in 2004-2005. Freshwater fish (9 species) were collected from local markets in Hanoi City (n=76) and Nam Dinh Province (n=79), and were examined for FBT metacercariae using the artificial digestion technique. Adult flukes were obtained from hamsters experimentally infected with the metacercariae at day 8 post-infection. Three (Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium) and 6 (Haplorchis taichui, H. pumilio, C. formosanus, P. varium, Stellantchasmus falcatus, and Heterophyopsis continua) species of FBT metacercariae were detected in the 2 regions, respectively. Overall, among the positive fish species, H. pumilio metacercariae were detected in 104 (80.0%) of 130 fish examined (metacercarial density per infected fish; 64.2). C. formosanus metacercariae were found in 37 (40.2%) of 92 fish (metacercarial density; 14.7). P. varium metacercariae were detected in 19 (63.3%) of 30 fish (Anabas testudineus and Mugil cephalus) (metacercarial density; 247.7). S. falcatus metacercariae were found in all 10 M. cephalus examined (metacercarial density; 84.4). H. continua metacercariae (2 in number) were detected in 1 fish of Coilia lindmani. Morphologic characteristics of the FBT metacercariae and their experimentally obtained adults were described. The results have demonstrated that various FBT species are prevalent in northen parts of Vietnam.     

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

Hyperendemicity of Haplorchis taichui Infection among Riparian People in Saravane and Champasak Province,Lao PDR

In this study, we found that Haplorchis taichui, a heterophyid intestinal fluke, is highly prevalent, with heavy worm loads, among riparian people in Saravane and Champasak province, Lao PDR. Fecal specimens were collected from 1,460 people (717 men and 743 women) in 12 riparian (Mekong river) districts and were examined by the Kato-Katz fecal smear technique. The overall helminth egg positive rate was 78.8% and 66.4% in Saravane and Champasak province, respectively. The positive rate for small trematode eggs (STE), which included H. taichui and other heterophyids, Opisthorchis viverrini, and lecithodendriids, was 69.9% and 46.3% in Saravane and Champasak province, respectively. To obtain adult flukes, 30 STE-positive people were treated with 40 mg/kg praziquantel and then purged. Whole diarrheic stools were collected 4-5 times for each person and searched for fluke specimens using a stereomicroscope. Mixed infections with various species of trematodes (H. taichui, Haplorchis pumilio, O. viverrini, Prosthodendrium molenkampi, Centrocestus formosanus, and Echinochasmus japonicus) and a species of cestode (Taenia saginata) were found. However, the worm load was exceptionally high for H. taichui compared with other trematode species, with an average of 21,565 and 12,079 specimens per infected person in Saravane and Champasak province, respectively, followed by H. pumilio (41.9 and 22.5, respectively) and O. viverrini (9.4 and 1.5, respectively). These results show that diverse species of intestinal and liver flukes are prevalent among riparian people in Saravane and Champasak province, Lao PDR, with H. taichui being the exceptionally dominant species.     

Infection Status of Zoonotic Trematode Metacercariae in Fishes from Vientiane Municipality and Champasak Province in Lao PDR

The infection status of fishborne zoonotic trematode (FZT) metacercariae was investigated in fishes from 2 localities of Lao PDR. Total 157 freshwater fishes (17 species) were collected in local markets of Vientiane Municipality and Champasak Province in December 2010 and July 2011, and each fish was examined by the artificial digestion method. Total 6 species of FZT metacercariae, i.e., Opisthorchis viverrini, Haplorchis taichui, Haplorchis yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium, were detected in fishes from Vientiane Municipality. The metacercariae of O. viverrini were detected in 50 (49.5%) out of 101 fishes (6 species), and their average number was 154 per fish infected. The remaining 5 species of heterophyid metacercariae were detected in 36.8%, 65.8%, 9.4%, 23.9%, and 5.1% fishes examined, and their average densities were 12, 1,038, 4, 15, and 13 per infected fish, respectively. In fishes from Champasak Province, 3 species of FZT metacercariae, i.e., O. viverrini, H. taichui, and H. yokogawai, were detected. Only 2 O. viverrini metacercariae were found in only 1 Barbonymus schwanefeldi. The metacercariae of H. taichui and H. yokogawai were detected in 60.0% and 50.0% of fishes examined, and their average densities were 47 and 28 per fish infected. By the present study, it has been confirmed that several species of FZT metacercariae are prevalent in fishes from Vientiane Municipality, with P. varium being a new member of FZT in Lao PDR. In comparison, FZT metacercariae are less prevalent in fishes from Champasak Province.     

Human Infections with Liver and Minute Intestinal Flukes in Guangxi, China: Analysis by DNA Sequencing, Ultrasonography, and Immunoaffinity Chromatography

The prevalence of liver and intestinal fluke infections was determined by surveying inhabitants of Hengxuan, Fusui, and Shanglin villages which were known to be endemic for liver flukes in Guangxi, China in May 2010. A total of 718 people were examined for helminth eggs by the Kato-Katz thick smear technique, ultrasonography, immunoaffinity chromatography, and DNA sequencing. The overall egg positive rate was found to be 59.6% (28.0-70.6%) that included mixed infections with liver and intestinal flukes. Cases showing higher than 20,000 eggs per gram of feces (EPG) were detected between 1.3% and 16.2%. Ultrasonographic findings exhibited overall 28.2% (72 of 255 cases) dilatation rate of the intrahepatic bile duct. Clonorchis sinensis infection was detected serologically in 88.3% (38 of 43 cases) among C. sinensis egg positive subjects by the immunoaffinity chromatography using a specific antigen for C. sinensis. For differential diagnosis of the liver and intestinal flukes, more precise PCR and nucleotide sequencing for copro-DNA were performed for 46 egg positive cases. Mixed infections with C. sinensis and Metagonimus yokogawai were detected in 8 of 46 egg positive cases, whereas 29 specimens were positive for Haplorchis taichui. Ultrasonographic findings and immunoaffinity chromatography results showed usefulness, even in a limited way, in figuring out of the liver fluke endemicity.     

A large scale survey of Populus nigra presence and genetic introgression from non-native poplars in Switzerland based on molecular identification

Populus nigra is considered a rare and threatened tree species in Switzerland because of dramatic habitat loss owing to river regulations during the last two centuries and because of potential gene introgression from non-indigenous P. deltoides through planted P. x canadensis hybrids. The significance of introgression as an endangerment to P. nigra, however, is controversial. The aims of the present study were (1) to assess how abundant P. nigra trees are in Switzerland and (2) to assess potential gene introgression. We present data from a molecular survey of 1372 putative P. nigra trees from Switzerland, using both chloroplast DNA and nuclear DNA markers. The results show that P. nigra is more abundant in Switzerland than hitherto thought. Furthermore, we detected a low frequency of gene introgression.     

Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation,molecular characterization,and potential use for strain identification

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.     

Trouble with lichen: the re-evaluation and re-interpretation of thallus form and fruit body types in the molecular era

Following discussions of the definition of the terms ‘lichen’ and ‘thallus’, the role of lichenization in the evolution of asco- and basidiomycetes, and divergence and convergence in fruit body types, the morphogenetic interpretation of types of thallus form in lichens is reviewed. Attention is drawn to the various morphogenetic hypotheses proposed to explain the lichen thallus, but it is concluded that it is best interpreted as a novel phenotype with no exact homologue. Similar ascomatal and thallus types are found in lichen-forming fungi of different orders and families, as now revealed by molecular phylogenetic studies. These are interpreted as examples of convergent evolution, strategies by which unrelated fungi either display captured algae to maximize photosynthetic opportunities, or to attach themselves to a substratum. Phenotypic evolution of fruit body and thallus types in the major orders and clades is summarized, and the thallus types known in each order are tabulated. An hypothesis relating the evolution of these structures to hygroscopic movements is proposed, and the critical position of lichens in developing an integrated approach to ascomycete evolution is emphasized.     

The use of RAPD markers for detecting genetic diversity,relationship and molecular identification of Chinese elite tea genetic resources [<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze] preserved in a tea germplasm repository

The genetic diversity, relationship and molecular identification of 15 well known, widely planted traditional Chinese elite tea genetic resources [Camellia sinensis (L.) O. Kuntze] preserved in the China National Germplasm Hangzhou Tea Repository in the Tea Research Institute of the Chinese Academy of Agricultural Sciences located in Zhejiang province, China, were investigated using RAPD markers. A total of 1050 bands with an average of 52.5 bands per primer, 70 bands per genetic resource were generated by the 20 selected primers from the 15 tea genetic resources. In the total of 137 amplified products, 129 were polymorphic, corresponding to 94.2% genetic diversity. The relative frequency of polymorphic products was from 0.24 to 0.83, with an average of 0.47. In general, this average frequency was relatively high. The genetic distances among the genetic resources were from 0.16 to 0.62, with an average of 0.37. The 15 tea genetic resources were grouped into three groups by UPGMA cluster analysis based on RAPD data. By using the presence of 20 unique RAPD markers and the absence of 11 unique markers, all the 15 investigated tea genetic resources could be easily identified. RAPD markers provided a practical method not only to evaluate the genetic diversity and relationship, but also to identify tea genetic resources.     

New insights to the molecular phylogenetics and generic assessment in the Rhacophoridae (Amphibia: Anura) based on five nuclear and three mitochondrial genes, with comments on the evolution of reproduction

The phylogenetic relationships among 12 genera of treefrogs (Family, Rhacophoridae), were investigated based on a large sequence data set, including five nuclear (brain-derived neurotrophic factor, proopiomelanocortin, recombination activating gene 1, tyrosinase, rhodopsin) and three mitochondrial (partial 12S and 16S ribosomal RNA and the complete valine t-RNA) genes. Phylogenetic analysis of the nuclear gene sequences resolved three major clades. The first group included Philautus, Pseudophilautus, Kurixalus, Gracixalus, and Theloderma moloch; Pseudophilautus and Kurixalus were sister taxa. The second group consisted of Nyctixalus and Theloderma. The third group contained Feihyla, Polypedates, Rhacophorus, and Chiromantis vittatus; Polypedates and Feihyla were sister taxa. Analyses of the nuclear and mitochondrial genes supported the following results: (1) Genus Liuixalus formed the sister group of all other rhacophorines. (2) Philautus, Theloderma, and Chiromantis were not resolved as monophyletic genera. Four groups, including Philautus ocellatus and P. hainanus, P. longchuanensis and P. gryllus, P. banaensis, and P. quyeti nested well within the genera Liuixalus, Pseudophilautus, Kurixalus, and Gracixalus, respectively. (3) Theloderma moloch and Chiromantis vittatus did not cluster with other species of Theloderma and Chiromantis, respectively. Foam nesting evolved only once, as did laying eggs in a jelly-like matrix containing some bubbles. Terrestrial direct development evolved twice in the Rhacophoridae.     

日本仙人掌中仙人掌孢囊线虫形态和分子鉴定

【背景】近年来,种苗等植物通过邮寄包裹进入我国的数量越来越多,有害生物随之入侵的风险与日俱增。深圳口岸曾多次从入境包裹中截获孢囊线虫、根结线虫等。【方法】2015年10月12日,深圳口岸在对日本邮寄来的仙人掌进行检疫时,从其根部发现了一种孢囊线虫。对该线虫进行分离,结合形态学和28S r DNA序列扩增并测序的方法,对截获的线虫进行鉴定。【结果】分离到了一种孢囊线虫,其形态特征与仙人掌孢囊线虫基本吻合,28S r DNA序列与Gen Bank数据库中登录号为DQ328702的德国群体的同源性高达99%。因此,判定截获的线虫为仙人掌孢囊线虫。【结论与意义】入境包裹检疫需要引起口岸检疫部门的高度重视。采用形态学和分子生物学相结合的方法对植物寄生线虫进行鉴定,可以得到更加准确的结果。     

The tip of the tail: molecular identification of seahorses for sale in apothecary shops and curio stores in California

Millions of seahorses a year are traded internationally, with many eventually sold at retail shops as curios, as aquarium pets, and especially for use in traditional medicine. The entire genus is now protected by CITES, but conservation measures have been limited in part by difficulties with species identification and incomplete understanding of trade patterns. In this study, we use molecular techniques to make species- and population-level identifications of 56 seahorses sampled from both traditional medicine and curio shops in San Francisco and central California. Seahorses from medicinal sources included unexpectedly large numbers of the eastern Pacific Hippocampus ingens, a species poorly protected by current CITES export recommendations. Curio shops were dominated by H. barbouri, a spiny species that has been reportedly confused in trading records as H. histrix. Specific populations of origin could be inferred for several species through comparison with publicly available phylogeographic data. Our results underscore the strengths and weaknesses of current recommended export regulations, and we suggest that molecular forensics can help in verifying trade documentation and developing more effective conservation measures.     

Molecular phylogeny and biogeography of the bipolar Euphrasia (Orobanchaceae): recent radiations in an old genus

Euphrasia includes perennial or annual green parasitic plants, and has a disjunct bipolar distribution except for one transtropical connection across the high mountains of Oceania. The disjunction is coupled with strikingly contrasting patterns of morphological diversity between the southern and northern hemispheres, making it an exciting model to study processes of evolutionary diversification which shaped present floras. We inferred the relationships among 51 species representing 14 of the 15 sections of the genus based on nrDNA ITS and cpDNA trnL intron, trnL-trnF and atpB-rbcL intergenic spacers. Maximum parsimony and Bayesian inference support monophyly of the genus and of several intrageneric groups characterized by morphology, ploidy level, and geographic range. Molecular phylogenetic dating using Bayesian “relaxed” clock methods suggests that the earliest Euphrasia radiations occurred minimum 11–8 Mya with bipolarity being achieved 7–5 Mya. Biogeographic analyses using explicit model-based approach inferred Eurasia as an ancestral area for the genus. The most parsimonious reconstruction found by a dispersal-vicariance analysis requires 17 dispersals to account for the current biogeographic pattern and supports Eurasian origin for Euphrasia. Both long-distance dispersal and across land vicariance can be invoked to explain the diversification in the genus, which experienced rapid radiations driven by new ecological opportunities of the late Pliocene and Pleistocene but also retained a set of local endemic or relict species of an earlier origin.     

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.