Whole-Genome Sequencing of Pseudomonas koreensis Isolated from Diseased Tor tambroides.

Unlike environmental P. koreensis isolated from soil, which has been studied extensively for its role in promoting plant growth, pathogenic P. koreensis isolated from fish has been rarely reported. Therefore, we investigated and isolated the possible pathogen that is responsible for the diseased state of Tor tambroides. Herein, we reported the morphological and biochemical characteristics, as well as whole-genome sequences of a newly identified P. koreensis strain. We assembled a high-quality draft genome of P. koreensis CM-01 with a contig N50 value of 233,601 bp and 99.5% BUSCO completeness. The genome assembly of P. koreensis CM-01 is consists of 6,171,880 bp with a G+C content of 60.5%. Annotation of the genome identified 5538 protein-coding genes, 3 rRNA genes, 54 tRNAs, and no plasmids were found. Besides these, 39 interspersed repeat and 141 tandem repeat sequences, 6 prophages, 51 genomic islands, 94 insertion sequences, 4 clustered regularly interspaced short palindromic repeats, 5 antibiotic-resistant genes, and 150 virulence genes were also predicted in the P. koreensis CM-01 genome. Culture-based approach showed that CM-01 strain exhibited resistance against ampicillin, aztreonam, clindamycin, and cefoxitin with a calculated multiple antibiotic resistance (MAR) index value of 0.4. In addition, the assembled CM-01 genome was successfully annotated against the Cluster of Orthologous Groups of proteins database, Gene Ontology database, and Kyoto Encyclopedia of Genes and Genome pathway database. A comparative analysis of CM-01 with three representative strains of P. koreensis revealed that 92% of orthologous clusters were conserved among these four genomes, and only the CM-01 strain possesses unique elements related to pathogenicity and virulence. This study provides fundamental phenotypic and genomic information for the newly identified P. koreensis strain.

A new set of microsatellite markers for Phoxinus lumaireul senso lato, Phoxinus marsilii and Phoxinus krkae for population and molecular taxonomic studies.

Minnows of the genus Phoxinus are common and an often highly abundant fish species in Palearctic freshwater habitats. Phoxinus species have a complex evolutionary history, phylogenetic relationships are not well understood and there are a number of unresolved taxonomic problems. There are currently 23 different mitochondrial genetic lineages identified in the genus Phoxinus, 13 of which are recognized as valid species. The taxonomic status of these lineages requires resolution, including the degree to which they can interbreed. Suitable nuclear molecular markers for studies of population divergence and interbreeding between morphotypes and mitochondrial lineages are lacking for Phoxinus species. Therefore, the authors developed a set of microsatellite markers using genomic information from Phoxinus lumaireul and tested their suitability for this and two related species, Phoxinus krkae and Phoxinus marsilii. Out of 16 microsatellite candidate loci isolated, 12 were found to be in Hardy-Weinberg equilibrium when tested on two P. lumaireul senso lato populations. Seven loci amplified across the three species, enabling the study of intraspecific genetic diversity and population structure within P. marsilii and P. krkae. The markers were able to clearly resolve differences among the three tested species, including the recently described P. krkae, and are therefore suitable for the detection of introgression and hybridization among populations consisting of mixtures of two or more of P. lumaireul s. l., P. marsilii and P. krkae.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing.

Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)-based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease-causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83%-100%). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low- and medium-income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.

Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia: Unionoidea) macroevolution.

Using a new fossil-calibrated mitogenome-based approach, we identified macroevolutionary shifts in mitochondrial gene order among the freshwater mussels (Unionoidea). We show that the early Mesozoic divergence of the two Unionoidea clades, Margaritiferidae and Unionidae, was accompanied by a synchronous split in the gene arrangement in the female mitogenome (i.e., gene orders MF1 and UF1). Our results suggest that this macroevolutionary jump was completed within a relatively short time interval (95% HPD 201-226 Ma) that coincided with the Triassic-Jurassic mass extinction. Both gene orders have persisted within these clades for ~200 Ma. The monophyly of the so-called "problematic" Gonideinae taxa was supported by all the inferred phylogenies in this study using, for the first time, the M- and F-type mitogenomes either singly or combined. Within Gonideinae, two additional splits in the gene order (UF1 to UF2, UF2 to UF3) occurred in the Mesozoic and have persisted for ~150 and ~100 Ma, respectively. Finally, the mitogenomic results suggest ancient connections between freshwater basins of East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a continuous paleo-river system or along the Tethys coastal line, which are well supported by at least three independent but almost synchronous divergence events.

Effects of supplementing freeze-dried Mitsuokella jalaludinii phytase on the growth performance and gut microbial diversity of broiler chickens.

Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate pens were used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3 weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p < .05) and T2 diets (p < .05), demonstrating the efficiency of MJ as a supplement to low aP diet. Nevertheless, MJ did not significantly affect the microbial population and diversity in broilers' caeca, which mainly consists of members from Bacteroidetes, Firmicutes, and Proteobacteria. Regardless, significant variations in the caecal bacterial composition were observed over time, probably due to succession as the broilers aged. This is the first reported study on the effect of MJ on the microbial diversity of broiler's caeca.

Variability of mitochondrial ORFans hints at possible differences in the system of doubly uniparental inheritance of mitochondria among families of freshwater mussels (Bivalvia: Unionida).

BACKGROUND: Supernumerary ORFan genes (i.e., open reading frames without obvious homology to other genes) are present in the mitochondrial genomes of gonochoric freshwater mussels (Bivalvia: Unionida) showing doubly uniparental inheritance (DUI) of mitochondria. DUI is a system in which distinct female-transmitted and male-transmitted mitotypes coexist in a single species. In families Unionidae and Margaritiferidae, the transition from dioecy to hermaphroditism and the loss of DUI appear to be linked, and this event seems to affect the integrity of the ORFan genes. These observations led to the hypothesis that the ORFans have a role in DUI and/or sex determination. Complete mitochondrial genome sequences are however scarce for most families of freshwater mussels, therefore hindering a clear localization of DUI in the various lineages and a comprehensive understanding of the influence of the ORFans on DUI and sexual systems. Therefore, we sequenced and characterized eleven new mitogenomes from poorly sampled freshwater mussel families to gather information on the evolution and variability of the ORFan genes and their protein products. RESULTS: We obtained ten complete plus one almost complete mitogenome sequence from ten representative species (gonochoric and hermaphroditic) of families Margaritiferidae, Hyriidae, Mulleriidae, and Iridinidae. ORFan genes are present only in DUI species from Margaritiferidae and Hyriidae, while non-DUI species from Hyriidae, Iridinidae, and Mulleriidae lack them completely, independently of their sexual system. Comparisons among the proteins translated from the newly characterized ORFans and already known ones provide evidence of conserved structures, as well as family-specific features. CONCLUSIONS: The ORFan proteins show a comparable organization of secondary structures among different families of freshwater mussels, which supports a conserved physiological role, but also have distinctive family-specific features. Given this latter observation and the fact that the ORFans can be either highly mutated or completely absent in species that secondarily lost DUI depending on their respective family, we hypothesize that some aspects of the connection among ORFans, sexual systems, and DUI may differ in the various lineages of unionids.

Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication.

BACKGROUND: The recently published complete mitogenome of the European lobster (Homarus gammarus) that was generated using long-range PCR exhibits unusual gene composition (missing nad2) and gene rearrangements among decapod crustaceans with strong implications in crustacean phylogenetics. Such atypical mitochondrial features will benefit greatly from validation with emerging long read sequencing technologies such as Oxford Nanopore that can more accurately identify structural variation. RESULTS: We re-sequenced the H. gammarus mitogenome on an Oxford Nanopore Minion flowcell and performed a long-read only assembly, generating a complete mitogenome assembly for H. gammarus. In contrast to previous reporting, we found an intact mitochondrial nad2 gene in the H. gammarus mitogenome and showed that its gene organization is broadly similar to that of the American lobster (H. americanus) except for the presence of a large tandemly duplicated region with evidence of pseudogenization in one of each duplicated protein-coding genes. CONCLUSIONS: Using the European lobster as an example, we demonstrate the value of Oxford Nanopore long read technology in resolving problematic mitogenome assemblies. The increasing accessibility of Oxford Nanopore technology will make it an attractive and useful tool for evolutionary biologists to verify new and existing unusual mitochondrial gene rearrangements recovered using first and second generation sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios.

One More Decade of Agrobacterium Taxonomy.

This chapter presents a historical overview of the development and changes in scientific approaches to classifying members of the Agrobacterium genus. We also describe the changes in the inference of evolutionary relationships among Agrobacterium biovars and Agrobacterium strains from using the 16S rRNA marker to recA genes and to the use of multilocus sequence analysis (MLSA). Further, the impacts of the genomic era enabling low cost and rapid whole genome sequencing on Agrobacterium phylogeny are reviewed with a focus on the use of new and sophisticated bioinformatics approaches to refine phylogenetic inferences. An updated genome-based phylogeny of ninety-seven Agrobacterium tumefaciens complex isolates representing ten known genomic species is presented, providing additional support to the monophyly of the Agrobacterium clade. Additional taxon sampling within Agrobacterium genomovar G3 indicates potential exceptions to interpretation of the concept of bacterial genomics species as ecological species because the genomovar G3 genomic cluster, which initially includes clinical strains, now also includes plant-associated and cave isolates.

Climate-driven mitochondrial selection: A test in Australian songbirds.

Diversifying selection between populations that inhabit different environments can promote lineage divergence within species and ultimately drive speciation. The mitochondrial genome (mitogenome) encodes essential proteins of the oxidative phosphorylation (OXPHOS) system and can be a strong target for climate-driven selection (i.e., associated with inhabiting different climates). We investigated whether Pleistocene climate changes drove mitochondrial selection and evolution within Australian birds. First, using phylogeographic analyses of the mitochondrial ND2 gene for 17 songbird species, we identified mitochondrial clades (mitolineages). Second, using distance-based redundancy analyses, we tested whether climate predicts variation in intraspecific genetic divergence beyond that explained by geographic distances and geographic position. Third, we analysed 41 complete mitogenome sequences representing each mitolineage of 17 species using codon models in a phylogenetic framework and a biochemical approach to identify signals of selection on OXPHOS protein-coding genes and test for parallel selection in mitolineages of different species existing in similar climates. Of 17 species examined, 13 had multiple mitolineages (range: 2-6). Climate was a significant predictor of mitochondrial variation in eight species. At least two amino acid replacements in OXPHOS complex I could have evolved under positive selection in specific mitolineages of two species. Protein homology modelling showed one of these to be in the loop region of the ND6 protein channel and the other in the functionally critical helix HL region of ND5. These findings call for direct tests of the functional and evolutionary significance of mitochondrial protein candidates for climate-associated selection.

More evolution underground: Accelerated mitochondrial substitution rate in Australian burrowing freshwater crayfishes (Decapoda: Parastacidae).

To further understand the evolutionary history and mitogenomic features of Australia's highly distinctive freshwater crayfish fauna, we utilized a recently described rapid mitogenome sequencing pipeline to generate 24 new crayfish mitogenomes including a diversity of burrowing crayfish species and the first for Astacopsis gouldi, the world's largest freshwater invertebrate. Whole mitogenome-based phylogeny estimates using both Bayesian and Maximum Likelihood methods substantially strengthen existing hypotheses for systematic relationships among Australian freshwater crayfish with evidence of pervasive diversifying selection and accelerated mitochondrial substitution rate among the members of the clade representing strongly burrowing crayfish that may reflect selection pressures for increased energy requirement for adaptation to terrestrial environment and a burrowing lifestyle. Further, gene rearrangements are prevalent in the burrowing crayfish mitogenomes involving both tRNA and protein coding genes. In addition, duplicated control regions were observed in two closely related Engaeus species, together with evidence for concerted evolution. This study significantly adds to the understanding of Australian freshwater crayfish evolutionary relationships and suggests a link between mitogenome evolution and adaptation to terrestrial environments and a burrowing lifestyle in freshwater crayfish.

ORDER within the chaos: Insights into phylogenetic relationships within the Anomura (Crustacea: Decapoda) from mitochondrial sequences and gene order rearrangements.

The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups.

Expansion and systematics redefinition of the most threatened freshwater mussel family, the Margaritiferidae.

Two Unionida (freshwater mussel) families are present in the Northern Hemisphere; the Margaritiferidae, representing the most threatened of unionid families, and the Unionidae, which include several genera of unresolved taxonomic placement. The recent reassignment of the poorly studied Lamprotula rochechouartii from the Unionidae to the Margaritiferidae motivated a new search for other potential species of margaritiferids from members of Gibbosula and Lamprotula. Based on molecular and morphological analyses conducted on newly collected specimens from Vietnam, we here assign Gibbosula crassa to the Margaritiferidae. Additionally, we reanalyzed all diagnostic characteristics of the Margaritiferidae and examined museum specimens of Lamprotula and Gibbosula. As a result, two additional species are also moved to the Margaritiferidae, i.e. Gibbosula confragosa and Gibbosula polysticta. We performed a robust five marker phylogeny with all available margaritiferid species and discuss the taxonomy within the family. The present phylogeny reveals the division of Margaritiferidae into four ancient clades with distinct morphological, biogeographical and ecological characteristics that justify the division of the Margaritiferidae into two subfamilies (Gibbosulinae and Margaritiferinae) and four genera (Gibbosula, Cumberlandia, Margaritifera, and Pseudunio). The systematics of the Margaritiferidae family is re-defined as well as their distribution, potential origin and main biogeographic patterns.

Phylogeography of red muntjacs reveals three distinct mitochondrial lineages.

BACKGROUND: The members of the genus Muntiacus are of particular interest to evolutionary biologists due to their extreme chromosomal rearrangements and the ongoing discussions about the number of living species. Red muntjacs have the largest distribution of all muntjacs and were formerly considered as one species. Karyotype differences led to the provisional split between the Southern Red Muntjac (Muntiacus muntjak) and the Northern Red Muntjac (M. vaginalis), but uncertainties remain as, so far, no phylogenetic study has been conducted. Here, we analysed whole mitochondrial genomes of 59 archival and 16 contemporaneous samples to resolve uncertainties about their taxonomy and used red muntjacs as model for understanding the evolutionary history of other species in Southeast Asia. RESULTS: We found three distinct matrilineal groups of red muntjacs: Sri Lankan red muntjacs (including the Western Ghats) diverged first from other muntjacs about 1.5 Mya; later northern red muntjacs (including North India and Indochina) and southern red muntjacs (Sundaland) split around 1.12 Mya. The diversification of red muntjacs into these three main lineages was likely promoted by two Pleistocene barriers: one through the Indian subcontinent and one separating the Indochinese and Sundaic red muntjacs. Interestingly, we found a high level of gene flow within the populations of northern and southern red muntjacs, indicating gene flow between populations in Indochina and dispersal of red muntjacs over the exposed Sunda Shelf during the Last Glacial Maximum. CONCLUSIONS: Our results provide new insights into the evolution of species in South and Southeast Asia as we found clear genetic differentiation in a widespread and generalist species, corresponding to two known biogeographical barriers: The Isthmus of Kra and the central Indian dry zone. In addition, our molecular data support either the delineation of three monotypic species or three subspecies, but more importantly these data highlight the conservation importance of the Sri Lankan/South Indian red muntjac.

Signatures of polygenic adaptation associated with climate across the range of a threatened fish species with high genetic connectivity.

Adaptive differences across species' ranges can have important implications for population persistence and conservation management decisions. Despite advances in genomic technologies, detecting adaptive variation in natural populations remains challenging. Key challenges in gene-environment association studies involve distinguishing the effects of drift from those of selection and identifying subtle signatures of polygenic adaptation. We used paired-end restriction site-associated DNA sequencing data (6,605 biallelic single nucleotide polymorphisms; SNPs) to examine population structure and test for signatures of adaptation across the geographic range of an iconic Australian endemic freshwater fish species, the Murray cod Maccullochella peelii. Two univariate gene-association methods identified 61 genomic regions associated with climate variation. We also tested for subtle signatures of polygenic adaptation using a multivariate method (redundancy analysis; RDA). The RDA analysis suggested that climate (temperature- and precipitation-related variables) and geography had similar magnitudes of effect in shaping the distribution of SNP genotypes across the sampled range of Murray cod. Although there was poor agreement among the candidate SNPs identified by the univariate methods, the top 5% of SNPs contributing to significant RDA axes included 67% of the SNPs identified by univariate methods. We discuss the potential implications of our findings for the management of Murray cod and other species generally, particularly in relation to informing conservation actions such as translocations to improve evolutionary resilience of natural populations. Our results highlight the value of using a combination of different approaches, including polygenic methods, when testing for signatures of adaptation in landscape genomic studies.

Characterization of an autotrophic bioreactor microbial consortium degrading thiocyanate.

Thiocyanate (SCN-) forms as a by-product of cyanidation during gold ore processing and can be degraded by a variety of microorganisms utilizing it as an energy, nitrogen, sulphur and/or carbon source. In complex consortia inhabiting bioreactor systems, a range of metabolisms are sustained by SCN- degradation; however, despite the addition or presence of labile carbon sources in most bioreactor designs to date, autotrophic bacteria have been found to dominate key metabolic functions. In this study, we cultured an autotrophic SCN--degrading consortium directly from gold mine tailings. In a batch-mode bioreactor experiment, this consortium degraded 22 mM SCN-, accumulating ammonium (NH4+) and sulphate (SO42-) as the major end products. The consortium consisted of a diverse microbial community comprised of chemolithoautotrophic members, and despite the absence of an added organic carbon substrate, a significant population of heterotrophic bacteria. The role of eukaryotes in bioreactor systems is often poorly understood; however, we found their 18S rRNA genes to be most closely related to sequences from bacterivorous Amoebozoa. Through combined chemical and phylogenetic analyses, we were able to infer roles for key microbial consortium members during SCN- biodegradation. This study provides a basis for understanding the behaviour of a SCN- degrading bioreactor under autotrophic conditions, an anticipated approach to remediating SCN- at contemporary gold mines.

Field calibration of blowfly-derived DNA against traditional methods for assessing mammal diversity in tropical forests.

Mammal diversity assessments based on DNA derived from invertebrates have been suggested as alternatives to assessments based on traditional methods; however, no study has field-tested both approaches simultaneously. In Peninsular Malaysia, we calibrated the performance of mammal DNA derived from blowflies (Diptera: Calliphoridae) against traditional methods used to detect species. We first compared five methods (cage trapping, mist netting, hair trapping, scat collection, and blowfly-derived DNA) in a forest reserve with no recent reports of megafauna. Blowfly-derived DNA and mist netting detected the joint highest number of species (n = 6). Only one species was detected by multiple methods. Compared to the other methods, blowfly-derived DNA detected both volant and non-volant species. In another forest reserve, rich in megafauna, we calibrated blowfly-derived DNA against camera traps. Blowfly-derived DNA detected more species (n = 11) than camera traps (n = 9), with only one species detected by both methods. The rarefaction curve indicated that blowfly-derived DNA would continue to detect more species with greater sampling effort. With further calibration, blowfly-derived DNA may join the list of traditional field methods. Areas for further investigation include blowfly feeding and dispersal biology, primer biases, and the assembly of a comprehensive and taxonomically-consistent DNA barcode reference library.

Impact of dengue virus (DENV) co-infection on clinical manifestations, disease severity and laboratory parameters.

BACKGROUND: The co-circulation of 4 DENV serotypes in geographically expanding area, has resulted in increasing occurrence of DENV co-infections. However, studies assessing the clinical impact of DENV co-infections have been scarce and have involved small number of patients. This study explores the impact of DENV co-infection on clinical manifestations and laboratory parameters. METHODS: This retrospective study involved consecutive hospitalized patients with non-structural protein 1 (NS1) antigen positivity during an outbreak (Jan to April 2014). Multiplex RT-PCR was performed directly on NS1 positive serum samples to detect and determine the DENV serotypes. All PCR-positive serum samples were inoculated onto C6/36 cells. Multiplex PCR was repeated on the supernatant of the first blind passage of the serum-infected cells. Random samples of supernatant from the first passage of C6/36 infected cells were subjected to whole genome sequencing. Clinical and laboratory variables were compared between patients with and without DENV co-infections. RESULTS: Of the 290 NS1 positive serum samples, 280 were PCR positive for DENV. Medical notes of 262 patients were available for analysis. All 4 DENV serotypes were identified. Of the 262 patients, forty patients (15.3 %) had DENV co-infections: DENV-1/DENV-2(85 %), DENV-1/DENV-3 (12.5 %) and DENV-2/DENV-3 (2.5 %). Another 222 patients (84.7 %) were infected with single DENV serotype (mono-infection), with DENV- 1 (76.6 %) and DENV- 2 (19.8 %) predominating. Secondary dengue infections occurred in 31.3 % patients. Whole genome sequences of random samples representing DENV-1 and DENV-2 showed heterogeneity amongst the DENVs. Multivariate analysis revealed that pleural effusion and the presence of warning signs were significantly higher in the co-infected group, both in the overall and subgroup analysis. Diarrhoea was negatively associated with co-infection. Additionally, DENV-2 co-infected patients had higher frequency of patients with severe thrombocytopenia (platelet count < 50,000/mm(3)), whereas DENV-2 mono-infections presented more commonly with myalgia. Elevated creatinine levels were more frequent amongst the co-infected patients in univariate analysis. Haemoconcentration and haemorrhagic manifestations were not higher amongst the co-infected patients. Serotypes associated with severe dengue were: DENV-1 (n = 9), DENV-2 (n = 1), DENV-3 (n = 1) in mono-infected patients and DENV-1/DENV-2 (n = 5) and DENV-1/DENV-3 (n = 1) amongst the co-infected patients. CONCLUSION: DENV co-infections are not uncommon in a hyperendemic region and co-infected patients are skewed towards more severe clinical manifestations compared to mono-infected patients.

Characterisation of 12 microsatellite loci in the Vietnamese commercial clam Lutraria rhynchaena Jonas 1844 (Heterodonta: Bivalvia: Mactridae) through next-generation sequencing.

The marine clam Lutraria rhynchaena is gaining popularity as an aquaculture species in Asia. Lutraria populations are present in the wild throughout Vietnam and several stocks have been established and translocated for breeding and aquaculture grow-out purposes. In this study, we demonstrate the feasibility of utilising Illumina next-generation sequencing technology to streamline the identification and genotyping of microsatellite loci from this clam species. Based on an initial partial genome scan, 48 microsatellite markers with similar melting temperatures were identified and characterised. The 12 most suitable polymorphic loci were then genotyped using 51 individuals from a population in Quang Ninh Province, North Vietnam. Genetic variation was low (mean number of alleles per locus = 2.6; mean expected heterozygosity = 0.41). Two loci showed significant deviation from Hardy-Weinberg equilibrium (HWE) and the presence of null alleles, but there was no evidence of linkage disequilibrium among loci. Three additional populations were screened (n = 7-36) to test the geographic utility of the 12 loci, which revealed 100 % successful genotyping in two populations from central Vietnam (Nha Trang). However, a second population from north Vietnam (Co To) could not be successfully genotyped and morphological evidence and mitochondrial variation suggests that this population represents a cryptic species of Lutraria. Comparisons of the Qang Ninh and Nha Trang populations, excluding the 2 loci out of HWE, revealed statistically significant allelic variation at 4 loci. We reported the first microsatellite loci set for the marine clam Lutraria rhynchaena and demonstrated its potential in differentiating clam populations. Additionally, a cryptic species population of Lutraria rhynchaena was identified during initial loci development, underscoring the overlooked diversity of marine clam species in Vietnam and the need to genetically characterise population representatives prior to microsatellite development. The rapid identification and validation of microsatellite loci using next-generation sequencing technology warrant its integration into future microsatellite loci development for key aquaculture species in Vietnam and more generally, aquaculture countries in the South East Asia region.

Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3-F5. Although inclusion of F0 and CT fractions (F1-F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW. © 2016 Society of Chemical Industry.