Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

Background

Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.

Methods

In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.

Results

The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).

Conclusions

These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.     

Genetic diversity among geographically separated populations of Nepenthes mirabilis

The distribution of Nepenthes mirabilis ranges from Northeast (NE) to South (S) Thailand. Eleven individuals from NE, S and Suen Jatujak market in Bangkok, Central (C) Thailand, were collected and divided into four populations according to their geographical areas. These four populations were analyzed to determine a genetic diversity profile using thirteen inter-simple sequence repeat markers. The individuals produced 75.18% polymorphic banding profiles. The Shannon’s index was used to estimate genetic diversity. Total genetic diversity (H _T) and inter-population genetic diversity (H _S) were 0.854 and 0.678, respectively. The degree of genetic differentiation (G _ST) of the species populations is 0.206, whereas the gene flow (Nm) among all the various geographical area populations is 1.016. Both the dendrogram and the results of the Shannon’s diversity index suggest great genetic diversity. These results support the broad range of distribution sites of Nepenthes mirabilis, which would require high genetic diversity to adapt to the environmental variations that can be found between NE, C, and S Thailand. ANOVA shows that the genetic diversity in each population is not significantly different (P > 0.05). Mantel tests reveal that geographical distance is an important factor for affecting the genetic distances among populations.     

Molecular identification and barcodes for the genus Nymphaea

Nymphaea species, the most popular decorative plants, were collected for specificity of inter-simple sequence repeat (ISSR) analyses in species identification and differentiation of cultivars and natural populations. Dendrogram constructed from ISSR analyses separated out wild species, namely Nymphaea cyanea, N. nouchali, N. capensis, N. lotus and an outgroup N. mexicana, and cultivars. The dendrogram indicates that the cultivars should be differentiated from N. capensis, as they are sister individuals of N. capensis. The ISSR banding data and the dendrogram are concordantly concluded that wild N. capensis would be an effective type species for producing different cultivars. After plant identification by ISSR markers, DNA barcodes of all sample materials were done to provide species specific markers which can be used for rapid and accurate further plant identification without morphological characters. DNA barcoding sequence analysis indicates genetic distance values. All sequences were recorded in GenBank database.     

Two new species of Curcuma (Zingiberaceae) used as cobra-bite antidotes

Two new species of Curcuma, C. sattayasaii A. Chaveerach ＆ R. Sudmoon and C. zedoaroides A. Chaveerach ＆ T. Tanee with rhizomes traditionally used for many decades as cobra-bite antidotes are described and illustrated. Curcuma sattayasaii is similar to C. longa L., but differs in rhizome horizontally branching on ground; coma bracts pinkish-white or pinkish-pale green; corolla pale yellow with orange tip; labellum pale orange with an orange central band; anther crest very short, broadly ovate, wider than long. Curcuma zedoaroides is similar to C. zedoaria （Christm.） Roscoe, but differs in rhizome branching pattern; the protruding secondary rhizomes curved down; blades oblong to oblong-lanceolate; peduncle glabrous; fertile and coma bracts glabrous; corolla lobes pale yellow to white, lateral lobe ovate, dorsal lobe broadly ovate. The new taxa have been found in a village of Khon Kaen Province, Northeastern Thailand.     

Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%-20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands by liquid chromatography-tandem mass spectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18, 4.87 and 4.72, whereas 2-mercaptoethanol-treated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.     

Piper protrusum (Piperaceae), a new species from southern Thailand based on morphological and molecular evidence

Abstract Piper protrusum Chaveer. & Tanee, sp. nov. is described and illustrated. It dominantly comprises three branching types with three different types of leaf blades, bases, and apexes. The critical distinguishing character is the protruded receptacle having a bract and nine stamens. Individual plants have been discovered in areas of Southern Thailand since 2004 without reproductive parts. The investigated sites were revisited several times, and an individual with flowers was finally found in July 2009. Phylogenetic analysis of the new species and five similar species is carried out based on DNA fingerprinting. The genetic distances between the new species and five similar species range from 0.25 to 0.35, supporting new species designation. Molecular data conform to morphological data in validating that it is a new species. Additionally, its DNA barcodes have been provided for further identification in case the morphological data is unclear. The sequence data have been submitted to the GenBank database under accession numbers GU980898 (rpoB gene), GU980899 (rpoC1 gene), and GU980900 (psbA‐trnH region).     

Engagement of Penicillium marneffei conidia with multiple pattern recognition receptors on human monocytes

P. marneffei is a thermal dimorphic fungus which causes penicilliosis, an opportunistic infection in immunocompromised patients in South and Southeast Asia. Little is known about the innate immune response to P. marneffei infection. Therefore, the initial response of macrophages to P. marneffei conidia was evaluated by us. Adhesion between monocytes from healthy humans and fungal conidia was examined and found to be specifically inhibited by MAbs against PRR, such as MR, (TLR)1, TLR2, TLR4, TLR6, CD14, CD11a, CD11b, and CD18. To study the consequences of these interactions, cytokines were also examined by ELISA. Binding of P. marneffei conidia to monocytes was significantly inhibited, in a dose-dependent manner, by MAbs against MR, TLR1, TLR2, TLR4, TLR6, CD14, CD11b and CD18. When monocytes were co-cultured with the conidia, there was an increase in the amount of surface CD40 and CD86 expression, together with TNF-α and IL-1β production, compared to unstimulated controls. In assays containing anti-TLR4 or anti-CD14 antibody, reduction in the amount of TNF-α released by monocytes stimulated with P. marneffei conidia was detected. In addition, it was found that production of TNF-α and IL-1β from adherent peripheral blood monocytes was partially impaired when heat-inactivated autologous serum, in place of untreated autologous serum, was added to the assay. These results demonstrate that various PRR on human monocytes participate in the initial recognition of P. marneffei conidia, and the engagement of PRR could partly initiate proinflammatory cytokine production.     

Piperprotrusum (Piperaceae), a new species from southern Thailand based on morphological and molecular evidence

Piper protrusum Chaveer.& Tanee,sp.nov.is described and illustrated.It dominantly comprises three branching types with three different types of leaf blades,bases,and apexes.The critical distinguishing character is the protruded receptacle having a bract and nine stamens.Individual plants have been discovered in areas of Southern Thailand since 2004 without reproductive parts.The investigated sites were revisited several times,and an individual with flowers was finally found in July 2009.Phylogenetic analysi...     

泰国胡椒科胡椒属三新种

The genus Piper L. is estimated to contain over 1000 species which are distributed mainly in tropical regions of the world. Several species have great economic and cultural importance and are used as foods, medicines, stimulants, antiseptics and antioxidants. Moreover, their essential oil and leaf extracts possess activities against several bacterium strains and fungi. Leaves of P. hispidinervum C. DC. contain a high level of safrole (83%–93%) which is an important raw material for chemical…