Exploration of microbial communities in the guts and casts of Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida.

Earthworms and their casts have been widely used for organic waste degradation and plant growth promotion. The microbial communities that reside in the guts and casts of earthworms markedly influence both applications. In the present study, next-generation sequencing was applied to identify the microbial communities in the guts and casts of three epigeic earthworm species, Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida, reared under two different feeding conditions. A total of 580 genera belonging to 43 phyla were identified. By comparing bacterial diversity among samples divided into groups based on the earthworm species, sample types, and conditions, the beta diversity analysis supported the impact of the sample type and suggested that there was significant dissimilarity of the bacterial diversity between the gut and cast. Besides, bacterial Phylum compositions within the group were compared. The result showed that the top three high relative frequency phyla found in the casts were the same regardless of earthworm species, while those found in the gut depended on both the condition and earthworm species. Focusing on the cellulolytic and plant growth-promoting bacteria, certain cellulolytic bacteria, Paenibacillus, Comamonas, and Cytophaga, were found only in the cast. Citrobacter and Streptomyces aculeolatus were detected only in the guts of earthworms reared in the bedding containing vegetables and bedding alone, respectively. Besides, Actinomadura and Burkholderia were detected only in the gut of E. eugeniae and E. fetida, respectively. The results proved that the microbial composition was affected by sample type, condition, and earthworm species. In addition, the proportion of these beneficial bacteria was also influenced by these factors. Hence, the information from this study can be used as a guide for selecting earthworm species or their casts for more efficient organic waste decomposition and plant growth promotion.

Phylogenetic and recombinant analyses of complete coding sequences of DENV-1 from field-caught mosquitoes in Thailand.

Dengue diseases are a group of infectious diseases that have been widespread throughout the world for several decades. Dengue outbreaks have occurred in many parts of the world, including Southeast Asia. The outbreak and the severity of the diseases depend on many factors including dengue genotype. Data on the genetic variation of dengue virus is highly informative for dengue protection plans and vaccine development. In this study, we focused on the analyses of genetic variation and amino acid changes of the whole coding sequences in two dengue strains isolated from Aedes mosquitoes in Bangkok, Thailand, an endemic area. The strains were identified as belonging to dengue virus serotype 1 (DENV-1) genotype I, and have unique nucleotide sequences. In the recombinant analysis, these strains were identified as recombinants derived from Chinese counterparts for both the major (DENV-1 genotype I) and the minor (DENV-1 genotype IV) parental strains. The recombination event occurred within the prM and E genes. This corresponded with the result of multiple alignments where several amino acid residues between the recombination breakpoints in the strains were identical to those in DENV-1 genotype IV. Several of the amino acid substitutions also have changed the amino acid properties, which might affect viral infection and antigenicity. These results provide insight into the genetic variation of DENV in this endemic area, which might have been involved in the dengue outbreak and high numbers of dengue haemorrhagic fever and dengue shock syndrome cases in Thailand in 2015.

Portunus pelagicus mtDNA heteroplasmy inheritance and its effect on the use of mtCR and mtCOI sequence data.

Mitochondrial DNA (mtDNA) sequences, especially mitochondrial control region (mtCR) and mitochondrial cytochrome c oxidase subunit I (mtCOI), have been widely used in population and evolutionary genetic analyses of metazoan. The presence of mtDNA heteroplasmy - a mixture of mtDNA haplotypes - possibly affects these analyses. This study aimed to reveal mtDNA heteroplasmy in mtCR, mtCOI, and mtND2 (mitochondrial NADH dehydrogenase subunit 2) of Portunus pelagicus, and examine its effect on the use of mtCR and mtCOI sequences. The screening result showed that the probability of observing mtDNA heteroplasmy was approximately 8%. Across the three targeted regions, 92 heteroplasmic variants were observed from seven samples comprising three mothers and four offspring. Most inherited heteroplasmy presented transition and silence mutation. By comparing the proportion of shared variants among maternal relatives to that among non-relatives, the result suggested that most heteroplasmic variants observed in an individual are inherited. Statistical analyses carried out on the inter-generational differences suggested that random drift and purifying selection play roles in determining the offspring's heteroplasmy level. The size of the random shift varies according to the location of variants and the mothers. The phylogenetic analysis showed that the presence of mtDNA heteroplasmy in mtCR and mtCOI does not affect familial and species identification, respectively. This study firstly reported the mtDNA heteroplasmy in P. pelagicus, its inheritance pattern, and its effect on the use of mtDNA sequence data. This basic knowledge would be useful for the study based on mtDNA sequence data, especially in other invertebrates.

Origin of prehistoric cattle excavated from four archaeological sites in central and northeastern Thailand.

Cattle have been domesticated in Southeast Asia, including Thailand, for thousands of years, but the history of cattle domestication in the region remains unclear. To date, only genetic studies of modern Thai cattle DNA have been reported. To gain some insight into cattle domestication in the country, a total of 56 cattle remains excavated from four archaeological sites (dated to between 3550 and 1700 years before present (YBP)) in northeastern and central Thailand were analysed in this study. Of 56, the 157-bp D-loop fragment was successfully generated from 26 samples, all of which belonged to Bos taurus in haplogroup T/T3. One haplotype contained 19 members from all four archaeological sites and clustered with the ancient B. taurus from Iran, Turkey and China. Other haplotypes have not shared haplotype with B. taurus from other countries but they showed close relationship to those from China. This represents the first genetic evidence that B.taurus was domesticated in Thailand between 3550 and 1700 YBP. In addition, the close relationship among ancient Thai, Iranian and Chinese taurines suggests that cattle from the Near East were introduced into North China, and were subsequently brought into Thailand thousands of years ago.

Enhancement of the efficiency of Cd phytoextraction using bacterial endophytes isolated from Chromolaena odorata, a Cd hyperaccumulator.

Phytoextraction is a technique using a hyperaccumulator to remove heavy metals from soil. The efficiency of heavy metal uptake can be enhanced by the inoculation of endophytes. In this study, we isolated and identified 23 endophytes from Chromolaena odorata, a cadmium (Cd) hyperaccumulator that consisted of 19 bacteria, 2 actinomycetes and 2 fungi. All bacteria and fungi could produce at least 1 plant growth promoting factors. However, only 4 bacterial isolates; Paenibacillus sp. SB12, Bacillus sp. SB31, Bacillus sp. LB51, and Alcaligenes sp. RB54 showed the highest minimum inhibitory concentration (MIC) value (2.9 mM), followed by Exiguobacterium sp.RB51 (1.7 mM). Then, these 5 high-MIC bacteria and 1 low-MIC bacterium, Bacillus sp. LB15 were inoculated onto sunflower grown in soil supplemented with 250 mg/kg of Cd. After 60 days, all inoculated plants accumulated significantly higher Cd concentration than the non-inoculated counterparts, and those inoculated with strain LB51 showed the highest Cd accumulation and growth. Interestingly, strain LB15 with low MIC also enhanced Cd accumulation in plants. The results suggest that these bacteria, particularly strain LB51, could be applied to improve Cd accumulation in plants, and that bacteria with low MIC also have the potential to enhance the efficiency of phytoextraction.

Phylogenetic analyses of DENV-3 isolated from field-caught mosquitoes in Thailand.

Dengue virus serotype 3 (DENV-3) can cause all forms of dengue diseases and is a predominant serotype in many countries. This serotype is classified into five genotypes: I-V. Genotypes I-III have widely spread throughout the world, whereas genotypes IV and V are rare. Despite the impact on the spread of dengue diseases, only a few studies have reported the characteristics of DENV present in mosquito vectors. Hence, this study aimed to identify DENV-3 genotypes and reveal genetic variation of this virus presented in field-caught mosquitoes collected from endemic areas in Thailand during 2011-2015. First, we examined the effectiveness of the E gene sequence on DENV-3 genotyping, with results supporting the use of this gene for genotype identification. Then, we sequenced this gene in ten DENV-3 strains isolated from mosquitoes. The results showed that eight and two samples were genotypes III and V, respectively, and that they are closely related to DENV-3 isolated from Southeast and East Asian samples. The translated E gene sequences showed 25 unique amino acid (AA) residues located at 23 positions. Eight out of 25 residues have different chemical properties compared to the conserved AAs that are distributed across the three domains functioning in virus-host interaction. Hence, our study reports the first DENV-3 genotype V in Thailand, with these viruses potentially influencing both the disease severity and epidemic potential of DENV-3.

Complete coding sequence of dengue virus serotype 4 isolated from field-caught mosquitoes in Thailand.

This report is the first to characterise the complete coding sequence of a dengue virus serotype 4 (DENV-4) genotype I that was isolated from field-caught mosquitoes from an endemic area in Thailand in June 2013. The sequence was assembled from high-throughput sequencing reads generated by Illumina HiSeq. Three out of four observed intra-sample variants caused an amino acid variation in C, NS2B, and NS5 genes. The C4279T variant located in the NS2B gene can indirectly affect the proteolytic activity of the NS3 protein. The sequence provided in this study might be useful for the epidemiological study of DENV-4.

Active Compounds Against Anopheles minimus Carboxypeptidase B for Malaria Transmission-Blocking Strategy.

Malaria transmission-blocking compounds have been studied to block the transmission of malaria parasites, especially the drug-resistant Plasmodium. Carboxypeptidase B (CPB) in the midgut of Anopheline mosquitoes has been demonstrated to be essential for the sexual development of Plasmodium in the mosquito. Thus, the CPB is a potential target for blocking compounds. The aim of this research was to screen compounds from the National Cancer Institute (NCI) diversity dataset and U.S. Food and Drug Administration (FDA)-approved drugs that could reduce the Anopheles CPB activity. The cDNA fragment of cpb gene from An. minimus (cpbAmi) was amplified and sequenced. The three-dimensional structure of CPB was predicted from the deduced amino acid sequence. The virtual screening of the compounds from NCI diversity set IV and FDA-approved drugs was performed against CPBAmi. The inhibition activity against CPBAmi of the top-scoring molecules was characterized in vitro. Three compounds-NSC-1014, NSC-332670, and aminopterin with IC50 at 0.99 mM, 1.55 mM, and 0.062 mM, respectively-were found to significantly reduce the CPBAmi activity.

Potential of Sonchus arvensis for the phytoremediation of lead-contaminated soil.

Sonchus arvensis is one of the pioneer plant species that were found in the abandoned Bo Ngam Pb mine in Thailand. S. arvensis was collected from three sites. The highest Pb shoot concentration was 9317 mg kg(-1) and the highest translocation factor (TF) and bioaccumulation factor (BF) values were 2.5 and 6.0, respectively. To investigate Pb uptake capacity of S. arvensis, a hydroponic experiment was performed for 15 d. S. arvensis exposed to 5 mg L(-1) Pb solution had the highest Pb shoot accumulation (849 mg kg(-1)). In a pot study, S. arvensis was grown in Pb mine soils amended with organic and inorganic fertilizers for 2 mo. The addition of organic fertilizer to the soil increased plant dry biomass sharply. All treatments with ethylene-diamine-tetra-acetic acid (EDTA) had Pb accumulation in shoots greater than 1000 mg kg(-1) and the highest Pb shoot accumulation was found in S. arvensis grown in soil amended with organic fertilizer and EDTA (1397 mg kg(-1)). In a field trial study, S. arvensis was grown at three sites in the mine area for 6 mo. S. arvensis could tolerate a total Pb of 100,000 mg kg(-1) in the soil and accumulated Pb in the shoots up to 3664 mg kg(-1) with high TF (2.19) and BF (2.38) values. These results suggest that S. arvensis is a good candidate for Pb phytoremediation.