The nearest known population of common dolphins (Delphinus sp.) to the Colombian Caribbean occurs in a fairly restricted range in eastern Venezuela. These dolphins have not been previously reported in the Colombian Caribbean, likely because of a lack of study of the local cetacean fauna. We collected cetacean observations in waters of the Guajira Department, northern Colombia (~11°N, 73°W) during two separate efforts: (a) a seismic vessel survey (December 2009—March 2010), and (b) three coastal surveys from small boats (May—July 2012, May 2013, and May 2014). Here we document ten sightings of common dolphins collected during these surveys, which extend the known range of the species by ~1000 km into the southwestern Caribbean. We also collected nine skin biopsies in 2013 and 2014. In order to determine the taxonomic identity of the specimens, we conducted genetic barcoding and phylogenetic analyses using two mitochondrial markers, the Control Region (mtDNA) and Cytochrome b (Cytb). Results indicate that these specimens are genetically closer to the short-beaked common dolphin (Delphinus delphis) even though morphologically they resemble a long-beaked form (Delphinus sp.). However, the specific taxonomic status of common dolphins in the Caribbean and in the Western Atlantic remains unresolved. It is also unclear whether the distribution of the species between northern Colombia and eastern Venezuela is continuous or disjoined, or whether they can be considered part of the same stock.

Endosymbiosis is widespread among cnidarians and is of high ecological relevance. The tropical sea anemone Aiptasia sp. is a laboratory model system for endosymbiosis between reef-building corals and photosynthetic dinoflagellate algae of the genus Symbiodinium. Here we identify the key environmental cues to induce reproducible spawning in Aiptasia under controlled laboratory conditions. We find that simulating a lunar cycle with blue-wavelength light is necessary to promote abundant gamete production and synchronous release in well-fed animals. Sexual reproduction rates are genetically determined and differ among clonal lines under similar conditions. We also find the inverse difference in rates of asexual reproduction. This study provides the requisite basis for further development of the Aiptasia model system, allowing analysis of basic cellular and molecular mechanisms in the laboratory as well as investigations of broad questions of ecological and evolutionary relevance.

We present the first complete Sweet potato virus G (SPVG) genome from sweet potato in East Timor and compare it with seven complete SPVG genomes from South Korea (three), Taiwan (two), Argentina (one), and the United States (one). It most resembles the genomes from the United States and South Korea.

Coronaviruses (CoVs) are found in a wide variety of wild and domestic animals, and constitute a risk for zoonotic and emerging infectious disease. In poultry, the genetic diversity, evolution, distribution and taxonomy of some coronaviruses have been well described, but little is known about the features of CoVs in wild birds. In this study we screened 764 samples from 22 avian species of the orders Anseriformes and Charadriiformes in Sweden collected in 2006/2007 for CoV, with an overall CoV prevalence of 18.7%, which is higher than many other wild bird surveys. The highest prevalence was found in the diving ducks—mainly Greater Scaup (Aythya marila; 51.5%)—and the dabbling duck Mallard (Anas platyrhynchos; 19.2%). Sequences from two of the Greater Scaup CoV fell into an infrequently detected lineage, shared only with a Tufted Duck (Aythya fuligula) CoV. Coronavirus sequences from Mallards in this study were highly similar to CoV sequences from the sample species and location in 2011, suggesting long-term maintenance in this population. A single Black-headed Gull represented the only positive sample from the order Charadriiformes. Globally, Anas species represent the largest fraction of avian CoV sequences, and there seems to be no host species, geographical or temporal structure. To better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance of wild birds and subsequent sequencing of detected CoV is imperative.

BackgroundHedera (ivies) is one of the few temperate genera of the primarily tropical Asian Palmate group of the Araliaceae, which extends its range out of Asia to Europe and the Mediterranean basin. Phylogenetic and phylogeographic results suggested Asia as the center of origin and the western Mediterranean region as one of the secondary centers of diversification. The bird-dispersed fleshy fruits of ivies suggest frequent dispersal over long distances (e.g. Macaronesian archipelagos), although reducing the impact of geographic barriers to gene flow in mainland species. Genetic isolation associated with geographic barriers and independent polyploidization events have been postulated as the main driving forces of diversification. In this study we aim to evaluate past and present diversification patterns in Hedera within a geographic and temporal framework to clarify the biogeographic history of the genus.ResultsPhylogenetic (biogeographic, time divergence and diversification) and phylogeographic (coalescence) analyses using four DNA regions (nrITS, trnH-psbA, trnT-trnL, rpl32) revealed a complex spatial pattern of lineage divergence. Scarce geographic limitation to gene flow and limited diversification are observed during the early-mid Miocene, followed by a diversification rate increase related to geographic divergence from the Tortonian/Messinian. Genetic and palaeobotanical evidence points the origin of the Hedera clade in Asia, followed by a gradual E-W Asian extinction and the progressive E-W Mediterranean colonization. The temporal framework for the E Asia - W Mediterranean westward colonization herein reported is congruent with the fossil record. Subsequent range expansion in Europe and back colonization to Asia is also inferred. Uneven diversification among geographic areas occurred from the Tortonian/Messinian onwards with limited diversification in the newly colonized European and Asian regions. Eastern and western Mediterranean regions acted as refugia for Miocene and post-Miocene lineages, with a similar role as consecutive centers of centrifugal dispersal (including islands) and speciation.ConclusionsThe Miocene Asian extinction and European survival of Hedera question the general pattern of Tertiary regional extinction of temperate angiosperms in Europe while they survived in Asia. The Tortonian/Messinian diversification increase of ivies in the Mediterranean challenges the idea that this aridity period was responsible for the extinction of the Mediterranean subtropical Tertiary flora. Differential responses of Hedera to geographic barriers throughout its evolutionary history, linked to spatial isolation related to historical geologic and climatic constraints may have shaped diversification of ivies in concert with recurrent polyploidy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0984-1) contains supplementary material, which is available to authorized users.

Glacial vicariance is regarded as one of the most prevalent drivers of phylogeographic structure and speciation among high-latitude organisms, but direct links between ice advances and range fragmentation have been more difficult to establish in marine than in terrestrial systems. Here we investigate the evolution of largely disjunct (and potentially reproductively isolated) phylogeographic lineages within the amphi-boreal kelp Saccharina latissima s. l. Using molecular data (COI, microsatellites) we confirm that S. latissima comprises also the NE Pacific S. cichorioides complex and is composed of divergent lineages with limited range overlap and genetic admixture. Only a few genetic hybrids were detected throughout a Canadian Arctic/NW Greenland contact zone. The degree of genetic differentiation and sympatric isolation of phylogroups suggest that S. latissima s. l. represents a complex of incipient species. Phylogroup distributions compared with paleo-environmental reconstructions of the cryosphere further suggest that diversification within S. latissima results from chronic glacial isolation in disjunct persistence areas intercalated with ephemeral interglacial poleward expansions and admixture at high-latitude (Arctic) contact zones. This study thus supports a role for glaciations not just in redistributing pre-existing marine lineages but also as a speciation pump across multi-glacial cycles for marine organisms otherwise exhibiting cosmopolite amphi-boreal distributions.

Biological diversity arises among genetically equal subpopulations in the same environment, a phenomenon called phenotypic heterogeneity. The life cycle of the enteric bacterium Photorhabdus luminescens involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic forms designated as primary (1°) and secondary (2°). In contrast to 1° cells, 2° cells are non-pigmented due to the absence of natural compounds, especially anthraquinones (AQs). We identified a novel type of transcriptional regulator, AntJ, which activates expression of the antA-I operon responsible for AQ production. AntJ heterogeneously activates the AQ production in single P. luminescens 1° cells, and blocks AQ production in 2° cells. AntJ contains a proposed ligand-binding WYL-domain, which is widespread among bacteria. AntJ is one of the rare examples of regulators that mediate heterogeneous gene expression by altering activity rather than copy number in single cells.

HighlightC2 photosynthesis in grasses is facilitated by organelle enrichment in tandem with enhanced levels of GDC in the carbon-concentrating cells consistent with changes in expression of a single GLDP gene.

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the “first line of defense” when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL−1 (median lethal dose) and 0.56 μg mL−1 (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant.

An increasing number of emerging tick-borne diseases has been reported in the United States since the 1970s. Using metagenomic next generation sequencing, we detected nucleic acid sequences from 2 novel viruses in the family Bunyaviridae and an emerging human rickettsial pathogen, Rickettsia philipii, in a population of the Pacific Coast tick, Dermacentor occidentalis in Mendocino County sampled annually from 2011 to 2014. A total of 250 adults of this human-biting, generalist tick were collected from contiguous chaparral and grassland habitats, and RNA from each individually extracted tick was deep sequenced to an average depth of 7.3 million reads. We detected a Francisella endosymbiont in 174 ticks (70%), and Rickettsia spp. in 19 ticks (8%); Rickettsia-infected ticks contained R. rhipicephali (16 of 250, 6.4%) or R. philipii (3 of 250,1.2%), the agent of eschar-associated febrile illness in humans. The genomes of 2 novel bunyaviruses (>99% complete) in the genera Nairovirus and Phlebovirus were also identified and found to be present in 20–91% of ticks, depending on the year of collection. The high prevalence of these bunyaviruses in sampled Dermacentor ticks suggests that they may be viral endosymbionts, although further studies are needed to determine whether they are infectious for vertebrate hosts, especially humans, and their potential role in tick ecology.

Cyclospora cayetanensis is a coccidian parasite associated with large and complex foodborne outbreaks worldwide. Linking samples from cyclosporiasis patients during foodborne outbreaks with suspected contaminated food sources, using conventional epidemiological methods, has been a persistent challenge. To address this issue, development of new methods based on potential genomically-derived markers for strain-level identification has been a priority for the food safety research community. The absence of reference genomes to identify nucleotide and structural variants with a high degree of confidence has limited the application of using sequencing data for source tracking during outbreak investigations. In this work, we determined the quality of a high resolution, curated, public mitochondrial genome assembly to be used as a reference genome by applying bioinformatic analyses. Using this reference genome, three new mitochondrial genome assemblies were built starting with metagenomic reads generated by sequencing DNA extracted from oocysts present in stool samples from cyclosporiasis patients. Nucleotide variants were identified in the new and other publicly available genomes in comparison with the mitochondrial reference genome. A consolidated workflow, presented here, to generate new mitochondrion genomes using our reference-guided de novo assembly approach could be useful in facilitating the generation of other mitochondrion sequences, and in their application for subtyping C. cayetanensis strains during foodborne outbreak investigations.

BackgroundHost-parasite relationships are expected to be strongly shaped by host specificity, a crucial factor in parasite adaptability and diversification. Because whole host communities have to be considered to assess host specificity, oceanic islands are ideal study systems given their simplified biotic assemblages. Previous studies on insular parasites suggest host range broadening during colonization. Here, we investigate the association between one parasite group (haemogregarines) and multiple sympatric hosts (of three lizard genera: Gallotia, Chalcides and Tarentola) in the Canary Islands. Given haemogregarine characteristics and insular conditions, we hypothesized low host specificity and/or occurrence of host-switching events.MethodsA total of 825 samples were collected from the three host taxa inhabiting the seven main islands of the Canarian Archipelago, including locations where the different lizards occurred in sympatry. Blood slides were screened to assess prevalence and parasitaemia, while parasite genetic diversity and phylogenetic relationships were inferred from 18S rRNA gene sequences.ResultsInfection levels and diversity of haplotypes varied geographically and across host groups. Infections were found in all species of Gallotia across the seven islands, in Tarentola from Tenerife, La Gomera and La Palma, and in Chalcides from Tenerife, La Gomera and El Hierro. Gallotia lizards presented the highest parasite prevalence, parasitaemia and diversity (seven haplotypes), while the other two host groups (Chalcides and Tarentola) harbored one haplotype each, with low prevalence and parasitaemia levels, and very restricted geographical ranges. Host-sharing of the same haemogregarine haplotype was only detected twice, but these rare instances likely represent occasional cross-infections.ConclusionsOur results suggest that: (i) Canarian haemogregarine haplotypes are highly host-specific, which might have restricted parasite host expansion; (ii) haemogregarines most probably reached the Canary Islands in three colonization events with each host genus; and (iii) the high number of parasite haplotypes infecting Gallotia hosts and their restricted geographical distribution suggest co-diversification. These findings contrast with our expectations derived from results on other insular parasites, highlighting how host specificity depends on parasite characteristics and evolutionary history.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2760-5) contains supplementary material, which is available to authorized users.

During recent years, extensive amounts of data have become available regarding influenza A virus (IAV) in wild birds in northern Europe, while information from southern Europe is more limited. Here, we present an IAV surveillance study conducted in western Portugal 2008–2009, analyzing 1653 samples from six different species of waterfowl, with the majority of samples taken from Mallards (Anas platyrhynchos). Overall 4.4% of sampled birds were infected. The sampling results revealed a significant temporal variation in the IAV prevalence, including a pronounced peak among predominantly young birds in June, indicating that IAV circulate within breeding populations in the wetlands of western Portugal. The H10N7 and H9N2 subtypes were predominant among isolated viruses. Phylogenetic analyses of the hemagglutinin and neuraminidase sequences of H10N7, H9N2 and H11N3 virus showed that sequences from Portugal were closely related to viral sequences from Central Europe as well as to IAVs isolated in the southern parts of Africa, reflecting Portugal’s position on the European-African bird migratory flyway. This study highlights the importance of Portugal as a migratory crossroad for IAV, connecting breeding stationary waterfowl with birds migrating between continents which enable transmission and spread of IAV.

Following aquarium releases, invasive lionfishes have colonized large areas of the Caribbean and western Atlantic, resulting in an immense ecological damage. The early stages of that invasion are poorly known. Indeed, a lag of time between the introduction and detection often preclude genetic characterization of that crucial phase. With elevated awareness, the recent invasion of Pterois miles was quickly detected in the Mediterranean Sea. We hereby show that the very first individuals establishing populations in the Mediterranean Sea display haplotypes that nest within the large genetic diversity of Red Sea individuals, thus indicating an invasion via the Suez Canal. We also show that only two haplotypes are detected in the Mediterranean Sea, suggesting that few individuals may have been involved in the invasion. Thus, we conclude that the Mediterranean invasion is the result of a movement of individuals from the Red Sea, rather than from other means, and that low genetic diversity does not seem to have a negative effect on the success and spread of lionfish into the Mediterranean Sea.

ABSTRACTWe report here the draft genome sequences of two Arthrobacter strains isolated from a holm oak forest affected by wildfire. Both strains were shown to act as plant growth promoters, with AFG20 being a member of the most abundant group found in this soil and AFG7.2 being the strain with the highest indole-3-acetic acid production level.

AbstractWe describe a new stylommatophoran land snail of the family Partulidae from Palau. The new species has a combination of morphological and ecological characters that do not allow its placement in any existing partulid genus, so we describe a new genus for it. The new genus is characterized by a large (18–23 mm) obese-pupoid shell; smooth protoconch; teleoconch with weak and inconsistent, progressively stronger, striae; last half of body whorl not extending beyond the penultimate whorl; widely expanded and reflexed peristome; relatively long penis, with longitudinal pilasters that fuse apically into a fleshy ridge that divides the main chamber from a small apical chamber; and vas deferens entering and penial-retractor muscle attaching at the apex of the penis. Unlike all other partulids, the new species is strictly associated with rocks in contact with the ground. Comparing the other three Palauan species – currently assigned to Partula – to our new genus and to other partulids makes it clear that they require their own genus because their morphology is quite different from that of true Partula and from that of all other genera. Hence, we resurrect the name Palaopartula Pilsbry for these snails.

Arabidopsis thaliana has three genes encoding type I 3-ketoacyl-CoA thiolases (KAT1, KAT2, and KAT5), one of which (KAT5) is alternatively transcribed to produce both peroxisomal and cytosolic proteins. To evaluate the potential importance of these four gene products, their evolutionary history in plants and their expression patterns in Arabidopsis were investigated. Land plants as a whole have gene lineages corresponding to KAT2 and KAT5, implying conservation of distinct functions for these two genes. By contrast, analysis of synteny shows that KAT1 arose by duplication of the KAT2 locus. KAT1 is found in the Brassicaceae family, including in the genera Arabidopsis, Capsella, Thellungiella (=Eutrema) and Brassica, but not in the more distantly related Caricaceae (order Brassicales), or other plants. Gene expression analysis using qRT-PCR and β-glucuronidase reporter genes showed strong expression of KAT2 during germination and in many plant tissues throughout the life cycle, consistent with its observed dominant function in fatty acid β-oxidation. KAT1 was expressed very weakly while KAT5 was most strongly expressed during flower development and in seedlings after germination. Isoform-specific qRT-PCR analysis and promoter β-glucuronidase reporters revealed that the two splicing variants of KAT5 have similar expression profiles. Alternative splicing of KAT5 to produce cytosolic and peroxisomal proteins is specific to and ubiquitous in the Brassicaceae, and possibly had an earlier origin in the order Brassicales. This implies that an additional function for KAT5 arose between 43 and 115 mybp. We speculate that this KAT5 mutation was recruited for a cytosolic function in secondary metabolism.

Tegus of the genera Tupinambis and Salvator are the largest Neotropical lizards and the most exploited clade of Neotropical reptiles. For three decades more than 34 million tegu skins were in trade, about 1.02 million per year. The genus Tupinambis is distributed in South America east of the Andes, and currently contains four recognized species, three of which are found only in Brazil. However, the type species of the genus, T. teguixin, is known from Bolivia, Brazil, Colombia, Ecuador, French Guyana, Guyana, Peru, Suriname, Trinidad and Tobago, and Venezuela (including the Isla de Margarita). Here we present molecular and morphological evidence that this species is genetically divergent across its range and identify four distinct clades some of which are sympatric. The occurrence of cryptic sympatric species undoubtedly exacerbated the nomenclatural problems of the past. We discuss the species supported by molecular and morphological evidence and increase the number of species in the genus Tupinambis to seven. The four members of the T. teguixin group continue to be confused with Salvator merianae, despite having a distinctly different morphology and reproductive mode. All members of the genus Tupinambis are CITES Appendix II. Yet, they continue to be heavily exploited, under studied, and confused in the minds of the public, conservationists, and scientists.

ABSTRACTStreptococcus uberis forms part of the native microbiota of cattle and is able to opportunistically infect the mammary gland; as such, it is a leading cause of bovine mastitis globally. Here, we report the complete genome sequence of S. uberis NZ01, isolated in New Zealand from a cow with a clinical case of bovine mastitis.

ABSTRACTClostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids.IMPORTANCEClostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is preferred over plasmid-based expression systems since expressing antigens from a chromosomal location confers an advantage to the vaccine strains by eliminating the antibiotic maintenance required for plasmids and negates issues with plasmid instability that would result in loss of the antigen. Lactic acid bacteria, including Lactobacillus acidophilus, have shown potential for mucosal vaccine delivery, as L. acidophilus is bile and acid tolerant, allowing transit through the gastrointestinal tract where cells interact with host epithelial and immune cells, including dendritic cells. In this study, we successfully expressed C. botulinum and B. anthracis antigens in the probiotic L. acidophilus strain NCFM. Both antigens were highly expressed individually or in tandem from the chromosome of L. acidophilus.

Single nucleotide substitution played important role in evolutionary expansion of human neocortex.

Rickettsiosis is a re-emergent infectious disease without epidemiological surveillance in Colombia. This disease is generally undiagnosed and several deadly outbreaks have been reported in the country in the last decade. The aim of this study is to analyze the eco-epidemiological aspects of rickettsial seropositivity in rural areas of Colombia where outbreaks of the disease were previously reported. A cross-sectional study, which included 597 people living in 246 households from nine hamlets in two municipalities of Colombia, was conducted from November 2015 to January 2016. The survey was conducted to collect sociodemographic and household characteristics (exposure) data. Blood samples were collected to determine the rickettsial seropositivity in humans, horses and dogs (IFA, cut-off = 1/128). In addition, infections by rickettsiae were detected in ticks from humans and animals by real-time PCR targeting gltA and ompA genes. Data was analyzed by weighted multilevel clog-log regression model using three levels (person, household and hamlets) and rickettsial seropositivity in humans was the main outcome. Overall prevalence of rickettsial seropositivity in humans was 25.62% (95%CI 22.11–29.12). Age in years (PR = 1.01 95%CI 1.01–1.02) and male sex (PR = 1.65 95%CI 1.43–1.90) were risk markers for rickettsial seropositivity. Working outdoors (PR = 1.20 95%CI 1.02–1.41), deforestation and forest fragmentation for agriculture use (PR = 1.75 95%CI 1.51–2.02), opossum in peridomiciliary area (PR = 1.56 95%CI 1.37–1.79) and a high proportion of seropositive domestic animals in the home (PR20-40% vs <20% = 2.28 95%CI 1.59–3.23 and PR>40% vs <20% = 3.14 95%CI 2.43–4.04) were associated with rickettsial seropositivity in humans. This study showed the presence of Rickettsia antibodies in human populations and domestic animals. In addition, different species of rickettsiae were detected in ticks collected from humans and animals. Our results highlighted the role of domestic animals as sentinels of rickettsial infection to identify areas at risk of transmission, and the importance of preventive measures aimed at curtailing deforestation and the fragmentation of forests as a way of reducing the risk of transmission of emergent and re-emergent pathogens.

BackgroundHop is an economically important crop for the Pacific Northwest USA as well as other regions of the world. It is a perennial crop with rhizomatous or clonal propagation system for varietal distribution. A big concern for growers as well as brewers is variety purity and questions are regularly posed to public agencies concerning the availability of genotype testing. Current means for genotyping are based upon 25 microsatellites that provides relatively accurate genotyping but cannot always differentiate sister-lines. In addition, numerous PCR runs (25) are required to complete this process and only a few laboratories exist that perform this service. A genotyping protocol based upon SNPs would enable rapid accurate genotyping that can be assayed at any laboratory facility set up for SNP-based genotyping. The results of this study arose from a larger project designed for whole genome association studies upon the USDA-ARS hop germplasm collection consisting of approximately 116 distinct hop varieties and germplasm (female lines) from around the world.ResultsThe original dataset that arose from partial sequencing of 121 genotypes resulted in the identification of 374,829 SNPs using TASSEL-UNEAK pipeline. After filtering out genotypes with more than 50 % missing data (5 genotypes) and SNP markers with more than 20 % missing data, 32,206 highly filtered SNP markers across 116 genotypes were identified and considered for this study. Minor allele frequency (MAF) was calculated for each SNP and ranked according to the most informative to least informative. Only those markers without missing data across genotypes as well as 60 % or less heterozygous gamete calls were considered for further analysis. Genetic distances among individuals in the study were calculated using the marker with the highest MAF value, then by using a combination of the two markers with highest MAF values and so on. This process was reiterated until a set of markers was identified that allowed for all genotypes in the study to be genetically differentiated from each other. Next, we compared genetic matrices calculated from the minimal marker sets [(Table 2; 6-, 7-, 8-, 10- and 12-marker set matrices] and that of a matrix calculated from a set of markers with no missing data across all 116 samples (1006 SNP markers). The minimum number of markers required to meet both specifications was a set of 7-markers (Table 3). These seven SNPs were then aligned with a genome assembly, and DNA sequence both upstream and downstream were used to identify primer sequences that can be used to develop seven amplicons for high resolution melting curve PCR detection or other SNP-based PCR detection methods.ConclusionsThis study identifies a set of 7 SNP markers that may prove useful for the identification and validation of hop varieties and accessions. Variety validation of unknown samples assumes that the variety under question has been included a priori in a discovery panel. These results are based upon in silica studies and markers need to be validated using different SNP marker technology upon a differential set of hop genotypes. The marker sequence data and suggested primer sets provide potential means to fingerprint hop varieties in most genetic laboratories utilizing SNP-marker technology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1492-2) contains supplementary material, which is available to authorized users.

BackgroundPuumala virus (PUUV) is the most important hantavirus species in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute renal injury (AKI) with thrombocytopenia and frequently gastrointestinal symptoms.Methods456 patients with serologically and clinically confirmed NE were investigated at time of follow-up in a single clinic. The course of the NE was investigated using medical reports. We identified patients who had endoscopy with intestinal biopsy during acute phase of NE. Histopathological, immunohistochemical and molecular analyses of the biopsies were performed.ResultsThirteen patients underwent colonoscopy or gastroscopy for abdominal pain, diarrhea, nausea and vomiting during acute phase of NE. Immunohistochemistry (IHC) revealed PUUV nucleocapsid antigen in 11 biopsies from 8 patients; 14 biopsies from 5 patients were negative for PUUV nucleocapsid antigen. IHC localized PUUV nucleocapsid antigen in endothelial cells of capillaries or larger vessels in the lamina propria. Rate of AKI was not higher and severity of AKI was not different in the PUUV-positive compared to the PUUV-negative group. All IHC positive biopsies were positive for PUUV RNA using RT-PCR. Phylogenetic reconstruction revealed clustering of all PUUV strains from this study with viruses previously detected from the South-West of Germany. Long-term outcome was favorable in both groups.ConclusionsIn patients with NE, PUUV nucleocapsid antigen and PUUV RNA was detected frequently in the intestine. This finding could explain frequent GI-symptoms in NE patients, thus demonstration of a more generalized PUUV infection. The RT-PCR was an effective and sensitive method to detect PUUV RNA in FFPE tissues. Therefore, it can be used as a diagnostic and phylogenetic approach also for archival materials. AKI was not more often present in patients with PUUV-positive IHC. This last finding should be investigated in larger numbers of patients with PUUV infection.

Tropical rainforests harbor extraordinary biodiversity. The Amazon basin is thought to hold 30% of all river fish species in the world. Information about the ecology, reproduction, and recruitment of most species is still lacking, thus hampering fisheries management and successful conservation strategies. One of the key understudied issues in the study of population dynamics is recruitment. Fish larval ecology in tropical biomes is still in its infancy owing to identification difficulties. Molecular techniques are very promising tools for the identification of larvae at the species level. However, one of their limits is obtaining individual sequences with large samples of larvae. To facilitate this task, we developed a new method based on the massive parallel sequencing capability of next generation sequencing (NGS) coupled with hybridization capture. We focused on the mitochondrial marker cytochrome oxidase I (COI). The results obtained using the new method were compared with individual larval sequencing. We validated the ability of the method to identify Amazonian catfish larvae at the species level and to estimate the relative abundance of species in batches of larvae. Finally, we applied the method and provided evidence for strong temporal variation in reproductive activity of catfish species in the Ucayalí River in the Peruvian Amazon. This new time and cost effective method enables the acquisition of large datasets, paving the way for a finer understanding of reproductive dynamics and recruitment patterns of tropical fish species, with major implications for fisheries management and conservation.

BackgroundAquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown.ResultsThe screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur.ConclusionThe zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.

Madagascar is one of the world’s top twelve “megadiversity” hot spots hosting unique and threatened flora and fauna. Parasites are a major component of biodiversity but remain largely uncharacterized in wildlife. In this study we combine microscopic and molecular assessment of hemoparasites in endemic reptile species from Madagascar. We detected three distinct parasites: the apicomplexans Hepatozoon and Sarcocystis, and filarial nematodes. The prevalence and intensity of these apicomplexans were low overall, while microfilarial infections in chameleons were relatively high. We detected mixed infections of two Hepatozoon haplotypes in Madagascarophis colubrinus, and of Hepatozoon and microfilariae in a Furcifer sp. Phylogenetic analyses of Hepatozoon showed evidence of prey-predator transmission, with identical sequences found in the snakes M. colubrinus and Ithycyphus oursi, and their prey Furcifer sp. Based on previous studies regarding the life cycle of Hepatozoon domerguei Landau, Chabaud, Michel, and Brygoo, 1970 in these hosts and due to their morphological similarity, we propose that this Hepatozoon haplotype is Hepatozoon domerguei. Future studies, including the examination of invertebrate hosts, are needed to verify this preliminary taxonomic identification. A distinct hemogregarine haplotype was found in Oplurus sp., which displayed morphologically different gametocytes, some of which were apparently inside leukocytes. The Sarcocystis identified from Tracheloptychus petersi was identical to that reported in a North African snake, indicating that the same lineage is found in geographically distinct regions. By combining morphological and genetic information, Foleyella furcata (Linstow, 1899) filarial nematodes were identified in several Furcifer chameleons. This study provides insights into the distribution, diversity and host-parasite interactions of hemoparasites in wild reptile populations from Madagascar.

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.

AbstractUsing morphological, nuclear, and mitochondrial data, we here revise the taxonomy of Apodanthaceae and allocate the 36 names published in the family to ten biological species in two genera, Apodanthes and Pilostyles. All species are endo-parasites that live permanently inside trees or shrubs of the families Salicaceae or Fabaceae and that only emerge to flower. Because of this life history, Apodanthaceae are among the least known families of flowering plants. Nevertheless, the World’s herbaria as of 2013 hold at least 785 collections that, in combination with DNA phylogenies, permit well-founded species circumscriptions and geographic range maps. We also provide a key to all species, discuss the newly accepted or synonymized names, and make available color photos of six of the ten species.

ABSTRACTHere, we report the first complete genome sequence of the Staphylococcus aureus strain Wood 46. Wood 46 has played an important role in understanding the virulence and pathogenesis of S. aureus infections. This report will assist efforts in vaccine development against methicillin-resistant S. aureus (MRSA) infections.

The proteolytic system of Lactobacillus plays an essential role in bacterial growth, contributes to the flavor development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. In this work, a genomic analysis of all genes involved in the proteolytic system of L. delbrueckii subsp. lactis CRL 581 was performed. Genes encoding the cell envelope-associated proteinase, two peptide transport systems, and sixteen peptidases were identified. The influence of the peptide supply on the transcription of 23 genes involved in the proteolytic system of L. delbrueckii subsp. lactis was examined after cell growth in a chemically defined medium (CDM) and CDM supplemented with Casitone. prtL, oppA1, optS, optA genes as well as oppDFBC and optBCDF operons were the most highly expressed genes in CDM; their expression being repressed 6- to 115-fold by the addition of peptides. The transcriptional analysis was confirmed by proteomics; the up-regulation of the PrtL, PepG, OppD and OptF proteins in the absence of peptides was observed while the DNA-binding protein YebC was up-regulated by peptides. Binding of YebC to the promoter region of prtL, oppA1, and optS, demonstrated by electrophoretic mobility shift assays, showed that YebC acts as a transcriptional repressor of key proteolytic genes.

Enterovirus A71 (EV-A71) is an important emerging pathogen causing large epidemics of hand, foot and mouth disease (HFMD) in children. In Malaysia, since the first EV-A71 epidemic in 1997, recurrent cyclical epidemics have occurred every 2–3 years for reasons that remain unclear. We hypothesize that this cyclical pattern is due to changes in population immunity in children (measured as seroprevalence). Neutralizing antibody titers against EV-A71 were measured in 2,141 residual serum samples collected from children ≤12 years old between 1995 and 2012 to determine the seroprevalence of EV-A71. Reported national HFMD incidence was highest in children <2 years, and decreased with age; in support of this, EV-A71 seroprevalence was significantly associated with age, indicating greater susceptibility in younger children. EV-A71 epidemics are also characterized by peaks of increased genetic diversity, often with genotype changes. Cross-sectional time series analysis was used to model the association between EV-A71 epidemic periods and EV-A71 seroprevalence adjusting for age and climatic variables (temperature, rainfall, rain days and ultraviolet radiance). A 10% increase in absolute monthly EV-A71 seroprevalence was associated with a 45% higher odds of an epidemic (adjusted odds ratio, aOR1.45; 95% CI 1.24–1.69; P<0.001). Every 10% decrease in seroprevalence between preceding and current months was associated with a 16% higher odds of an epidemic (aOR = 1.16; CI 1.01–1.34 P<0.034). In summary, the 2–3 year cyclical pattern of EV-A71 epidemics in Malaysia is mainly due to the fall of population immunity accompanying the accumulation of susceptible children between epidemics. This study will impact the future planning, timing and target populations for vaccine programs.

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.

In the 20th century, thirteen distinct human immunodeficiency viruses emerged following independent cross-species transmission events involving simian immunodeficiency viruses (SIV) from African primates. In the late 1900s, pathogenic SIV strains also emerged in the United Sates among captive Asian macaque species following their unintentional infection with SIV from African sooty mangabeys (SIVsmm). Since their discovery in the 1980s, SIVs from rhesus macaques (SIVmac) and pig-tailed macaques (SIVmne) have become invaluable models for studying HIV pathogenesis, vaccine design and the emergence of viruses. SIV isolates from captive crab-eating macaques (SIVmfa) were initially described but lost prior to any detailed molecular and genetic characterization. In order to infer the origins of the lost SIVmfa lineage, we located archived material and colony records, recovered its genomic sequence by PCR, and assessed its phylogenetic relationship to other SIV strains. We conclude that SIVmfa is the product of two cross-species transmission events. The first was the established transmission of SIVsmm to rhesus macaques, which occurred at the California National Primate Research Center in the late 1960s and the virus later emerged as SIVmac. In a second event, SIVmac was transmitted to crab-eating macaques, likely at the Laboratory for Experimental Medicine and Surgery in Primates in the early 1970s, and it was later spread to the New England Primate Research Center colony in 1973 and eventually isolated in 1986. Our analysis suggests that SIVmac had already emerged by the early 1970s and had begun to diverge into distinct lineages. Furthermore, our findings suggest that pathogenic SIV strains may have been more widely distributed than previously appreciated, raising the possibility that additional isolates may await discovery.

Nitrogen can greatly influence the structure and productivity of microbial communities through its relative availability and form. However, the roles of specific organisms in the uptake of different nitrogen species remain poorly characterized. Most studies seeking to identify agents of assimilation have been correlative, indirectly linking activity measurements (e.g., nitrate uptake) with the presence or absence of biological markers, particularly functional genes and their transcripts. Evidence is accumulating of previously underappreciated functional diversity in major microbial subpopulations, which may confer physiological advantages under certain environmental conditions leading to ecotype divergence. This microdiversity further complicates our view of genetic variation in environmental samples requiring the development of more targeted approaches. Here, next-generation tag sequencing was successfully coupled with stable isotope probing (Tag-SIP) to assess the ability of individual phylotypes to assimilate a specific N source. Our results provide the first direct evidence of nitrate utilization by organisms thought to lack the genes required for this process including the heterotrophic clades SAR11 and the Archaeal Marine Group II. Alternatively, this may suggest the existence of tightly coupled metabolisms with primary assimilators, e.g., symbiosis, or the rapid and efficient scavenging of recently released products by highly active individuals. These results may be connected with global dominance often seen with these clades, likely conferring an advantage over other clades unable to access these resources. We also provide new direct evidence of in situ nitrate utilization by the cyanobacterium Prochlorococcus in support of recent findings. Furthermore, these results revealed widespread functional heterogeneity, i.e., different levels of nitrogen assimilation within clades, likely reflecting niche partitioning by ecotypes.

In alpine Australia, Orthoptera are abundant, dominant herbivores, important prey species, and hosts for parasites and parasitoids. Despite the central role of orthopterans in alpine ecosystems, the impact of parasites on orthopteran populations is under-explored. In this study we describe the relationship between parasite prevalence and host sex, body size and year of collection. We accessed an existing, preserved collection of 640 Kosciuscola tristis collected from across its range between 2007 and 2011. Upon dissection we collected juvenile parasites and used molecular tools to identify them to three families (Nematoda; Mermithidae, and Arthropoda: Diptera: Tachinidae and Sarcophagidae). The prevalence of nematodes ranged from 3.5% to 25.0% and dipterans from 2.4% to 20.0%. Contrary to predictions, we found no associations between parasite prevalence and grasshopper sex or size. Although there was an association between prevalence of both nematodes and dipterans with year of collection, this is likely driven by a small sample size in the first year. Our results provide a foundation for future studies into parasite prevalence within the alpine environment and the abiotic factors that might influence these associations.

For the first time, the phylogenetic relationships between representatives of all 10 copepod orders have been investigated using 28S and 18S rRNA, Histone H3 protein and COI mtDNA. The monophyly of Copepoda (including Platycopioida Fosshagen, 1985) is demonstrated for the first time using molecular data. Maxillopoda is rejected, as it is a polyphyletic group. The monophyly of the major subgroups of Copepoda, including Progymnoplea Lang, 1948 (=Platycopioida); Neocopepoda Huys and Boxshall, 1991; Gymnoplea Giesbrecht, 1892 (=Calanoida Sars, 1903); and Podoplea Giesbrecht, 1892, are supported in this study. Seven copepod orders are monophyletic, including Platycopioida, Calanoida, Misophrioida Gurney, 1933; Monstrilloida Sars, 1901; Siphonostomatoida Burmeister, 1834; Gelyelloida Huys, 1988; and Mormonilloida Boxshall, 1979. Misophrioida (=Propodoplea Lang, 1948) is the most basal Podoplean order. The order Cyclopoida Burmeister, 1835, is paraphyletic and now encompasses Poecilostomatoida Thorell, 1859, as a sister to the family Schminkepinellidae Martinez Arbizu, 2006. Within Harpacticoida Sars, 1903, both sections, Polyarthra Lang, 1948, and Oligoarthra Lang, 1948, are monophyletic, but not sister groups. The order Canuelloida is proposed while maintaining the order Harpacticoida s. str. (Oligoarthra). Cyclopoida, Harpacticoida and Cyclopinidae are redefined, while Canuelloida ordo. nov., Smirnovipinidae fam. nov. and Cyclopicinidae fam. nov are proposed as new taxa.

Aquaporins (AQPs) are tetrameric channel proteins regulating the transmembrane flux of small uncharged solutes and in particular water in living organisms. In plants, members of the plasma membrane intrinsic protein (PIP) AQP subfamily are important for the maintenance of the plant water status through the control of cell and tissue hydraulics. The PIP subfamily is subdivided into two groups: PIP1 and PIP2 that exhibit different water-channel activities when expressed in Xenopus oocytes or yeast cells. Most PIP1 and PIP2 isoforms physically interact and assemble in heterotetramers to modulate their subcellular localization and channel activity when they are co-expressed in oocytes, yeasts, and plants. Whether the interaction between different PIPs is stochastic or controlled by cell regulatory processes is still unknown. Here, we analyzed the water transport activity and the subcellular localization behavior of the complete PIP subfamily (SmPIP1;1, SmPIP2;1, and SmPIP2;2) of the lycophyte Selaginella moellendorffii upon (co-)expression in yeast and Xenopus oocytes. As observed for most of the PIP1 and PIP2 isoforms in other species, SmPIP1;1 was retained in the ER while SmPIP2;1 was found in the plasma membrane but, upon co-expression, both isoforms were found in the plasma membrane, leading to a synergistic effect on the water membrane permeability. SmPIP2;2 behaves as a PIP1, being retained in the endoplasmic reticulum when expressed alone in oocytes or in yeasts. Interestingly, in contrast to the oocyte system, in yeasts no synergistic effect on the membrane permeability was observed upon SmPIP1;1/SmPIP2;1 co-expression. We also demonstrated that SmPIP2;1 is permeable to water and the signaling molecule hydrogen peroxide. Moreover, growth- and complementation assays in the yeast system showed that heteromerization in all possible SmPIP combinations did not modify the substrate specificity of the channels. These results suggest that the characteristics known for angiosperm PIP1 and PIP2 isoforms in terms of their water transport activity, trafficking, and interaction emerged already as early as in non-seed vascular plants. The existence and conservation of these characteristics may argue for the fact that PIP2s are indeed involved in the delivery of PIP1s to the plasma membrane and that the formation of functional heterotetramers is of biological relevance.

Recent studies have renewed interest in sponge ecology by emphasizing the functional importance of sponges in a broad array of ecosystem services. Many critically important habitats occupied by sponges face chronic stressors that might lead to alterations in their diversity, relatedness, and functional attributes. We addressed whether proximity to human activity might be a significant factor in structuring sponge community composition, as well as potential functional roles, by monitoring sponge diversity and abundance at two structurally similar sites that vary in distance to areas of high coastal development in Bocas Del Toro, Panama. We surveyed sponge communities at each site using belt transects and differences between two sites were compared using the following variables: (1) sponge species richness, Shannon diversity, and inverse Simpson’s diversity; (2) phylogenetic diversity; (3) taxonomic and phylogenetic beta diversity; (4) trait diversity and dissimilarity; and (5) phylogenetic and trait patterns in community structure. We observed significantly higher sponge diversity at Punta Caracol, the site most distant from human development (∼5 km). Although phylogenetic diversity was lower at Saigon Bay, the site adjacent to a large village including many houses, businesses, and an airport, the sites did not exhibit significantly different patterns of phylogenetic relatedness in species composition. However, each site had a distinct taxonomic and phylogenetic composition (beta diversity). In addition, the sponge community at Saigon included a higher relative abundance of sponges with high microbial abundance and high chlorophyll a concentration, whereas the community at Punta Caracol had a more even distribution of these traits, yielding a significant difference in functional trait diversity between sites. These results suggest that lower diversity and potentially altered community function might be associated with proximity to human populations. This study highlights the importance of evaluating functional traits and phylogenetic diversity in addition to common diversity metrics when assessing potential environmental impacts on benthic communities.

SummaryNicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.

During acoustic communication, an audible message is transmitted from a sender to a receiver, often producing changes in behavior. In a system where evolutionary changes of the sender do not result in a concomitant adjustment in the receiver, communication and species recognition could fail. However, the possibility of an evolutionary decoupling between sender and receiver has rarely been studied. Frog populations in the Allobates femoralis cryptic species complex are known for their extensive morphological, genetic and acoustic variation. We hypothesized that geographic variation in acoustic signals of A. femoralis was correlated with geographic changes in communication through changes in male-male recognition. To test this hypothesis, we quantified male call recognition using phonotactic responses to playback experiments of advertisement calls with two, three and four notes in eight localities of the Amazonian basin. Then, we reconstructed the ancestral states of call note number in a phylogenetic framework and evaluated whether the character state of the most recent common ancestor predicted current relative responses to two, three and four notes. The probability of a phonotactic response to advertisement calls of A. femoralis males was strongly influenced by the call mid-frequency and the number of notes in most populations. Positive phonotaxis was complete for calls from each individual's population, and in some populations, it was also partial for allotopic calls; however, in two populations, individuals equally recognized calls with two, three or four notes. This evidence, in conjunction with our results from phylogenetic comparative methods, supports the hypothesis of decoupled evolution between sender and receiver in the male-male communication system of the A. femoralis complex. Thus, signal recognition appears to evolve more slowly than the calls.

AbstractThe Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades.

Data is presented in support of a phylogenetic reconstruction of the species-rich early-divergent angiosperm genus Goniothalamus (Annonaceae) (Tang et al., Mol. Phylogenetic Evol., 2015) [1], inferred using chloroplast DNA (cpDNA) sequences. The data includes a list of primers for amplification and sequencing for nine cpDNA regions: atpB-rbcL, matK, ndhF, psbA-trnH, psbM-trnD, rbcL, trnL-F, trnS-G, and ycf1, the voucher information and molecular data (GenBank accession numbers) of 67 ingroup Goniothalamus accessions and 14 outgroup accessions selected from across the tribe Annoneae, and aligned data matrices for each gene region. We also present our Bayesian phylogenetic reconstructions for Goniothalamus, with information on previous infrageneric classifications superimposed to enable an evaluation of monophyly, together with a taxon-character data matrix (with 15 morphological characters scored for 66 Goniothalamus species and seven other species from the tribe Annoneae that are shown to be phylogenetically correlated).

The focus of this study was to reconstruct a phylogenetic hypothesis for the moth subfamily Arctiinae (tiger moths, woolly bears) to investigate the evolution of larval and adult pharmacophagy of pyrrolizidine alkaloids (PAs) and the pathway to PA chemical specialization in Arctiinae. Pharmacophagy, collection of chemicals for non-nutritive purposes, is well documented in many species, including the model species Utetheisa ornatrix L. A total of 86 exemplar ingroup species representing tiger moth tribes and subtribes (68 genera) and nine outgroup species were selected. Ingroup species included the most species-rich generic groups to represent the diversity of host-plant associations and pharmacophagous behaviors found throughout Arctiinae. Up to nine genetic markers were sequenced: one mitochondrial (COI barcode region), one nuclear rRNA (D2 region, 28S rRNA), and seven nuclear protein-coding gene fragments: elongation factor 1-α protein, wingless, ribosomal protein subunit S5, carbamoylphosphate synthase domain regions, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and cytosolic malate dehydrogenase. A total of 6984 bp was obtained for most species. These data were analyzed using model-based phylogenetic methods: maximum likelihood (ML) and Bayesian inference (BI). Ancestral pharmacophagous behaviors and obligate PA associations were reconstructed using the resulting Bayes topology and Reconstructing Ancestral States in Phylogenies (RASP) software. Our results corroborate earlier studies on the evolution of adult pharmacophagous behaviors, suggesting that this behavior arose multiple times and is concentrated in the phaegopterine-euchromiine-ctenuchine clade (PEC). Our results suggest that PA specialization may have arisen early in the phylogeny of the subfamily and that facultative larval pharmacophagous behaviors are the derived condition.

The increased presence of clinically relevant multidrug resistant bacteria in natural environments is an emerging challenge for global health care. Little is known regarding the occurrence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) from environmental sentinels in Pakistan. The goal of the current study was to gain insights into the prevalence and phylogenetic relationships of ESBL-E. coli recovered from wild birds in Pakistan during winter migration. After initial screening of fecal samples on selective chromogenic agar, ESBL-E.coli were analyzed phenotypically using the Vitek-2 automated system. Genotypic characterization was performed using whole genome sequencing (WGS) followed by an in-depth in silico analysis. Of 150 birds screened, 26 (17.3%) were fecal carriers of ESBL-E. coli. Of these, 88.4% isolates exhibited multidrug resistance (MDR) phenotypes. Resistance to cefotaxime, ceftazidime, ampicillin, doxycycline, tetracycline and sulfamethoxazole/trimethoprim (CTX-CAZ-AM-DC-TE-SXT) represented the most common pattern of MDR (76.9%). WGS data analysis found blaCTX-M-15 as the predominant ESBL genotype (92.3%). Other genes encoding resistance to sulfonamides (sul1/sul2/sul3), aminoglycosides (strA, strB, aadA1, aadA2, aadA5, aac(3)-IId-like, aac(3)-IVa-like and aph(4)-Ia), trimethoprim (dfrA14 or dfrA17), tetracyclines [tet(A)/tet(B)], and fluoroquinolones (qnrS1) were detected commonly, often encoded on IncF-type plasmids (76.9%). ESBL-E. coli were assigned to 17 different sequence types (STs) of which ST10 and ST7097 (4 isolates each) were the most abundant followed by ST4720, ST93, and ST1139 (2 isolates each). Core-genome phylogeny of the isolates found low numbers (0–29) of single nucleotide polymorphisms (SNPs) in isolates belonged to ST7097 originated from two different locations (Chashma barrage and Rasul barrage). Similar trends were found among isolates belong to ST1139. In addition, WGS-based plasmid typing and S1-digestion found plasmids of the same pMLST type (IncF[F-:A-:B53]) and similar sizes in different bacterial and avian hosts suggesting horizontal gene transfer as another possibility for the spread of ESBL-E. coli in avian wildlife in Pakistan.

A new species of the genus Euphlyctis is described from the Barisal district of Bangladesh and compared with its morphologically similar and geographically proximate congeners. The new species is highly divergent in comparison to other congeneric species on basis of sequence divergence in mitochondrial DNA gene sequences (ranging from 5.5% to 17.8% divergence). Euphlyctis kalasgramensis sp. nov. can be readily diagnosed by having the following combination of characters: snout-vent length (SVL) 30.44 – 37.88 mm, absence of mid-dorsal line, nostril–snout length 3% of SVL, nostril much closer to snout tip than eye, nostril–snout length 48% of distance from front of eyes to nostril, relative length of fingers (shortest to longest: 1 = 2 < 4 < 3), tibia length 59% of SVL, foot length 55% of SVL.

Pigs (Sus scrofa) have played an important cultural role in Hawaii since Polynesians first introduced them in approximately AD 1200. Additional varieties of pigs were introduced following Captain Cook's arrival in Hawaii in 1778 and it has been suggested that the current pig population may descend primarily, or even exclusively, from European pigs. Although populations of feral pigs today are an important source of recreational hunting on all of the major islands, they also negatively impact native plants and animals. As a result, understanding the origins of these feral pig populations has significant ramifications for discussions concerning conservation management, identity and cultural continuity on the islands. Here, we analysed a neutral mitochondrial marker and a functional nuclear coat colour marker in 57 feral Hawaiian pigs. Through the identification of a new mutation in the MC1R gene that results in black coloration, we demonstrate that Hawaiian feral pigs are mostly the descendants of those originally introduced during Polynesian settlement, though there is evidence for some admixture. As such, extant Hawaiian pigs represent a unique historical lineage that is not exclusively descended from feral pigs of European origin.

BackgroundComplete mitochondrial genomes are much better suited for the taxonomic identification and phylogenetic studies of nematodes than morphology or traditionally-used molecular markers, but they remain unavailable for the entire Camallanidae family (Chromadorea). As the only published mitogenome in the Camallanina suborder (Dracunculoidea superfamily) exhibited a unique gene order, the other objective of this research was to study the evolution of mitochondrial architecture in the Spirurida order. Thus, we sequenced the complete mitogenome of the Camallanus cotti fish parasite and conducted structural and phylogenomic comparative analyses with all available Spirurida mitogenomes.ResultsThe mitogenome is exceptionally large (17,901 bp) among the Chromadorea and, with 46 (pseudo-) genes, exhibits a unique architecture among nematodes. Six protein-coding genes (PCGs) and six tRNAs are duplicated. An additional (seventh) tRNA (Trp) was probably duplicated by the remolding of tRNA-Ser2 (missing). Two pairs of these duplicated PCGs might be functional; three were incomplete and one contained stop codons. Apart from Ala and Asp, all other duplicated tRNAs are conserved and probably functional. Only 19 unique tRNAs were found. Phylogenomic analysis included Gnathostomatidae (Spirurina) in the Camallanina suborder.ConclusionsWithin the Nematoda, comparable PCG duplications were observed only in the enoplean Mermithidae family, but those result from mitochondrial recombination, whereas characteristics of the studied mitogenome suggest that likely rearrangement mechanisms are either a series of duplications, transpositions and random loss events, or duplication, fragmentation and subsequent reassembly of the mitogenome. We put forward a hypothesis that the evolution of mitogenomic architecture is extremely discontinuous, and that once a long period of stasis in gene order and content has been punctuated by a rearrangement event, such a destabilised mitogenome is much more likely to undergo subsequent rearrangement events, resulting in an exponentially accelerated evolutionary rate of mitogenomic rearrangements. Implications of this model are particularly important for the application of gene order similarity as an additive source of phylogenetic information. Chromadorean nematodes, and particularly Camallanina clade (with C. cotti as an example of extremely accelerated rate of rearrangements), might be a good model to further study this discontinuity in the dynamics of mitogenomic evolution.Electronic supplementary materialThe online version of this article (doi: 10.1186/s12864-017-4237-x) contains supplementary material, which is available to authorized users.

Calocybella is a new genus established to accommodate Rugosomyces pudicus. Phylogenetic analyses based on a LSU-ITS sequence dataset place Calocybella sister to Gerhardtia from which it differs morphologically in the presence of clamp-connections and reddening context. The genus Gerhardtia is emended to also include taxa with smooth spores. According to our morphological analysis of voucher material, Calocybe juncicola s. auct. is shown to be Calocybella pudica.

To examine microbe-mineral interactions in subsurface oceanic crust, we evaluated microbial colonization on crustal minerals that were incubated in borehole fluids for 1 year at the seafloor wellhead of a crustal borehole observatory (IODP Hole U1301A, Juan de Fuca Ridge flank) as compared to an experiment that was not exposed to subsurface crustal fluids (at nearby IODP Hole U1301B). In comparison to previous studies at these same sites, this approach allowed assessment of the effects of temperature, fluid chemistry, and/or mineralogy on colonization patterns of different mineral substrates, and an opportunity to verify the approach of deploying colonization experiments at an observatory wellhead at the seafloor instead of within the borehole. The Hole U1301B deployment did not have biofilm growth, based on microscopy and DNA extraction, thereby confirming the integrity of the colonization design against bottom seawater intrusion. In contrast, the Hole U1301A deployment supported biofilms dominated by Epsilonproteobacteria (43.5% of 370 16S rRNA gene clone sequences) and Gammaproteobacteria (29.3%). Sequence analysis revealed overlap in microbial communities between different minerals incubated at the Hole U1301A wellhead, indicating that mineralogy did not separate biofilm structure within the 1-year colonization experiment. Differences in the Hole U1301A wellhead biofilm community composition relative to previous studies from within the borehole using similar mineral substrates suggest that temperature and the diffusion of dissolved oxygen through plastic components influenced the mineral colonization experiments positioned at the wellhead. This highlights the capacity of low abundance crustal fluid taxa to rapidly establish communities on diverse mineral substrates under changing environmental conditions such as from temperature and oxygen.

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

BackgroundParadoxically, centromeres are known both for their characteristic repeat sequences (satellite DNA) and for being epigenetically defined. Maize (Zea mays mays) is an attractive model for studying centromere positioning because many of its large (~2 Mb) centromeres are not dominated by satellite DNA. These centromeres, which we call complex centromeres, allow for both assembly into reference genomes and for mapping short reads from ChIP-seq with antibodies to centromeric histone H3 (cenH3).ResultsWe found frequent complex centromeres in maize and its wild relatives Z. mays parviglumis, Z. mays mexicana, and particularly Z. mays huehuetenangensis. Analysis of individual plants reveals minor variation in the positions of complex centromeres among siblings. However, such positional shifts are stochastic and not heritable, consistent with prior findings that centromere positioning is stable at the population level. Centromeres are also stable in multiple F1 hybrid contexts. Analysis of repeats in Z. mays and other species (Zea diploperennis, Zea luxurians, and Tripsacum dactyloides) reveals tenfold differences in abundance of the major satellite CentC, but similar high levels of sequence polymorphism in individual CentC copies. Deviation from the CentC consensus has little or no effect on binding of cenH3.ConclusionsThese data indicate that complex centromeres are neither a peculiarity of cultivation nor inbreeding in Z. mays. While extensive arrays of CentC may be the norm for other Zea and Tripsacum species, these data also reveal that a wide diversity of DNA sequences and multiple types of genetic elements in and near centromeres support centromere function and constrain centromere positions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1249-4) contains supplementary material, which is available to authorized users.

The widespread distribution of lentiviruses among African primates, and the lack of severe pathogenesis in many of these natural reservoirs, are taken as evidence for long-term co-evolution between the simian immunodeficiency viruses (SIVs) and their primate hosts. Evidence for positive selection acting on antiviral restriction factors is consistent with virus-host interactions spanning millions of years of primate evolution. However, many restriction mechanisms are not virus-specific, and selection cannot be unambiguously attributed to any one type of virus. We hypothesized that the restriction factor TRIM5, because of its unique specificity for retrovirus capsids, should accumulate adaptive changes in a virus-specific fashion, and therefore, that phylogenetic reconstruction of TRIM5 evolution in African primates should reveal selection by lentiviruses closely related to modern SIVs. We analyzed complete TRIM5 coding sequences of 22 Old World primates and identified a tightly-spaced cluster of branch-specific adaptions appearing in the Cercopithecinae lineage after divergence from the Colobinae around 16 million years ago. Functional assays of both extant TRIM5 orthologs and reconstructed ancestral TRIM5 proteins revealed that this cluster of adaptations in TRIM5 specifically resulted in the ability to restrict Cercopithecine lentiviruses, but had no effect (positive or negative) on restriction of other retroviruses, including lentiviruses of non-Cercopithecine primates. The correlation between lineage-specific adaptations and ability to restrict viruses endemic to the same hosts supports the hypothesis that lentiviruses closely related to modern SIVs were present in Africa and infecting the ancestors of Cercopithecine primates as far back as 16 million years ago, and provides insight into the evolution of TRIM5 specificity.

This study aimed to investigate the circulation of Orthobunyavirus species in thestate of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serumsamples were collected from patients with acute febrile illness with symptoms for upto five days and 387 pools of female Culex quinquefasciatus capturedin 2013 were subjected to nested-reverse transcription-polymerase chain reaction forsegment S of the Simbu serogroup followed by nucleotide sequencing and virusisolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n= 1) and Nova Mutum (n = 1) municipalities were positive for the S segment ofOropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3.Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia,presenting high homology with OROV strains obtained from humans and animals in theBrazilian Amazon. The present paper reports the first detection of anOrthobunyavirus, possibly OROV, in patients and in Cx.quinquefasciatus mosquitoes in MT. This finding reinforces the notionthat arboviruses frequently reported in the Amazon Region circulate sporadically inMT during dengue outbreaks.

AbstractThe western United States is home to numerous narrowly endemic harvestman taxa (Arachnida, Opiliones), including members of the genus Sitalcina Banks, 1911. Sitalcina is comprised of three species groups, including the monospecific Sitalcinacalifornica and Sitalcinalobata groups, and the Sitalcinasura group with eight described species. All species in the Sitalcinasura group have very small geographic distributions, with group members distributed like disjunct “beads on a string” from Monterey south to southern California and southeast to the sky-island mountain ranges of southern Arizona. Here, molecular phylogenetic and species delimitation analyses were conducted for all described species in the Sitalcinasura group, plus several newly discovered populations. Species trees were reconstructed using multispecies coalescent methods implemented in *BEAST, and species delimitation was accomplished using Bayes Factor Delimitation (BFD). Based on quantitative species delimitation results supported by consideration of morphological characters, two new species (Sitalcinaoasiensissp. n., Sitalcinaubickisp. n.) are described. We also provide a description of the previously unknown male of Sitalcinaborregoensis Briggs, 1968. Molecular phylogenetic evidence strongly supports distinctive desert versus coastal clades, with desert canyon taxa from southern California more closely related to Arizona taxa than to geographically proximate California coastal taxa. We hypothesize that southern ancestry and plate tectonics have played a role in the diversification history of this animal lineage, similar to sclerophyllous plant taxa of the Madro-Tertiary Geoflora. Molecular clock analyses for the Sitalcinasura group are generally consistent with these hypotheses. We also propose that additional Sitalcina species await discovery in the desert canyons of southern California and northern Baja, and the mountains of northwestern mainland Mexico.

Reproductive proteins are often observed to be the most rapidly evolving elements within eukaryotic genomes. The major sperm protein (MSP) is unique to the phylum Nematoda and is required for proper sperm locomotion and fertilization. Here, we annotate the MSP gene family and analyze their molecular evolution in 10 representative species across Nematoda. We show that MSPs are hyper-conserved across the phylum, having maintained an amino acid sequence identity of 83.5–97.7% for over 500 million years. This extremely slow rate of evolution makes MSPs some of the most highly conserved genes yet identified. However, at the gene family level, we show hyper-variability in both gene copy number and genomic position within species, suggesting rapid, lineage-specific gene family evolution. Additionally, we find evidence that extensive gene conversion contributes to the maintenance of sequence identity within chromosome-level clusters of MSP genes. Thus, while not conforming to the standard expectation for the evolution of reproductive proteins, our analysis of the molecular evolution of the MSP gene family is nonetheless consistent with the widely repeatable observation that reproductive proteins evolve rapidly, in this case in terms of the genomic properties of gene structure, copy number, and genomic organization. This unusual evolutionary pattern is likely generated by strong pleiotropic constraints acting on these genes at the sequence level, balanced against expansion at the level of the whole gene family.

Genetically diverse RNA viruses like dengue viruses (DENVs) segregate into multiple, genetically distinct, lineages that temporally arise and disappear on a regular basis. Lineage turnover may occur through multiple processes such as, stochastic or due to variations in fitness. To determine the variation of fitness, we measured the distribution of fitness within DENV populations and correlated it with lineage extinction and replacement. The fitness of most members within a population proved lower than the aggregate fitness of populations from which they were drawn, but lineage replacement events were not associated with changes in the distribution of fitness. These data provide insights into variations in fitness of DENV populations, extending our understanding of the complexity between members of individual populations.

The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, represents a center of diversification for many organisms. The genetic structure and connectivity of mainland and island microbial populations has been poorly explored, in particular in the case of symbiotic fungi. Here we investigated genetic diversity and spatial structure of the obligate outcrossing lichen-forming fungus Parmelina carporrhizans in the Mediterranean region. Using eight microsatellite and mating-type markers we showed that fungal populations are highly diverse but lack spatial structure. This is likely due to high connectivity and long distance dispersal of fungal spores. Consistent with low levels of linkage disequilibrium and lack of clonality, we detected both mating-type idiomorphs in all populations. Furthermore we showed that the Macaronesian Islands are the result of colonization from the Mediterranean Basin. The unidirectional gene flow, though, seemed not to be sufficient to counterbalance the effects of drift, resulting in comparatively allelic poor peripheral populations. Our study is the first to shed light on the high connectivity and lack of population structure in natural populations of a strictly sexual lichen fungus. Our data further support the view of the Macaronesian Islands as the end of the colonization road for this symbiotic ascomycete.

Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis–as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses–suggesting that HPgV’s protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses–an understudied group of viruses with a high prevalence in the global human population–and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.

Skin disease occurs frequently in many cetacean species across the globe; methods to categorize lesions have relied on photo-identification (photo-id), stranding, and by-catch data. The current study used photo-id data from four sampling months during 2009 to estimate skin lesion prevalence and type occurring on bottlenose dolphins (Tursiops truncatus) from three sites along the southeast United States coast [Sarasota Bay, FL (SSB); near Brunswick and Sapelo Island, GA (BSG); and near Charleston, SC (CHS)]. The prevalence of lesions was highest among BSG dolphins (P = 0.587) and lowest in SSB (P = 0.380), and the overall prevalence was significantly different among all sites (p<0.0167). Logistic regression modeling revealed a significant reduction in the odds of lesion occurrence for increasing water temperatures (OR = 0.92; 95%CI:0.906–0.938) and a significantly increased odds of lesion occurrence for BSG dolphins (OR = 1.39; 95%CI:1.203–1.614). Approximately one-third of the lesioned dolphins from each site presented with multiple types, and population differences in lesion type occurrence were observed (p<0.05). Lesions on stranded dolphins were sampled to determine the etiology of different lesion types, which included three visually distinct samples positive for herpesvirus. Although generally considered non-fatal, skin disease may be indicative of animal health or exposure to anthropogenic or environmental threats, and photo-id data provide an efficient and cost-effective approach to document the occurrence of skin lesions in free-ranging populations.

BackgroundReduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome.ResultsWe found that the MspI-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110–220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40–110 bp, 110–220 bp and 40–220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110–220 bp fragment size appeared to be deeper than in the 40–110 bp and 40–220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions.ConclusionsIn this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.

The aim of this study was to evaluate the potential effects of methyl donor supplementation of pregnant animals in the presence or absence of a concomitant lactation on the methylome of the offspring. Twenty Holstein cows, 10 nulliparous (non-lactating while pregnant) and 10 multiparous (lactating while pregnant) were blocked by parity and randomly assigned to an i.m. weekly injections of a placebo (CTRL) or a solution containing methyl donors (MET). After calving, 5 calves randomly selected from each treatment (two born to non-lactating and three to lactating dams) were blood-sampled to determine their full methylome. There were more than 2,000 CpG differentially methylated between calves born to CTRL and those born to MET, and also between calves born to lactating and non-lactating dams. Most of the differences affected genes involved in immune function, cell growth regulation and differentiation, kinase activity, and ion channeling. We conclude that the coexistence of pregnancy and lactation affects the methylome of the offspring, and that supplementation of methyl donors early in gestation has also consequences on the methylome.

We report locus-specific disintegration of megabase-scale chromosomal conformations in brain after neuronal ablation of Kmt1e/Setdb1 histone H3-lysine 9 methyltransferase, including a large topologically associated 1.2Mb domain conserved in human and mouse and encompassing >70 genes at the clustered Protocadherin (cPcdh) locus. TADcPcdh in mutant neurons showed abnormal accumulations of CTCF transcriptional regulator and 3D genome organizer at cryptic binding sites, converted into permissive state with DNA cytosine hypomethylation and histone hyperacetylation. Broadly upregulated expression across cPcdh included defective S-type Protocadherin single-cell stochastic constraint. Setdb1-dependent loop formations, bypassing 0.2–1Mb of linear genome, radiated from TADPcdh fringes towards cPcdh cis-regulatory sequences, counterbalanced shorter-range facilitative promoter-enhancer contacts and carried loop-bound polymorphisms associated with genetic risk for schizophrenia. We show that KRAB-zinc finger Setdb1 repressor complex, shielding neuronal 3D genomes from excess CTCF binding, is critically required for structural maintenance of TADcPcdh.

BackgroundDifferences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Age-dependent differences in IL-8 production are abolished by treatment with 5-aza-2-deoxycytidine and Trichostatin A (AZA-TSA), suggesting epigenetic regulation of the innate response to LPS. In the current study, we performed reduced representation bisulfite sequencing (RRBS) on fibroblast cultures isolated from the same animals at 5- and 16-months of age to identify genes that exhibit variable methylation with age. To validate the role of methylation in gene expression, six innate response genes that were hyper-methylated in young animals were assessed by RT-qPCR in fibroblasts from animals at different ages and from different breeds.ResultsWe identified 14,094 differentially methylated CpGs (DMCs) that differed between fibroblast cultures at 5- versus 16-months of age. Of the 5065 DMCs that fell within gene regions, 1117 were located within promoters, 1057 were within gene exons and 2891 were within gene introns and 67% were more methylated in young cultures. Transcription factor enrichment of the promoter regions hyper-methylated in young cultures revealed significant regulation by the key pro-inflammatory regulator, NF-κB. Additionally, five out of six chosen genes (PIK3R1, FES, NFATC1, TNFSF13 and RORA) that were more methylated in young cultures showed a significant reduction in expression post-LPS treatment in comparison with older cultures. Two of these genes, FES and NFATC1, were similarly down-regulated in Angus cultures that also exhibit a low LPS response phenotype.ConclusionsOur study has identified immune-related loci regulated by DNA methylation in cattle that may contribute to differential cellular response to LPS, two of which exhibit an identical expression profile in both low-responding age and breed phenotypes. Methylation biomarkers of differential immunity may prove useful in developing selection strategies for replacement cows that are less susceptible to severe infections, such as coliform mastitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3796-1) contains supplementary material, which is available to authorized users.

Humans display structural and functional asymmetries in brain organization, strikingly with respect to language and handedness. The molecular basis of these asymmetries is unknown. We report a genome-wide association study meta-analysis for a quantitative measure of relative hand skill in individuals with dyslexia [reading disability (RD)] (n = 728). The most strongly associated variant, rs7182874 (P = 8.68×10−9), is located in PCSK6, further supporting an association we previously reported. We also confirmed the specificity of this association in individuals with RD; the same locus was not associated with relative hand skill in a general population cohort (n = 2,666). As PCSK6 is known to regulate NODAL in the development of left/right (LR) asymmetry in mice, we developed a novel approach to GWAS pathway analysis, using gene-set enrichment to test for an over-representation of highly associated variants within the orthologs of genes whose disruption in mice yields LR asymmetry phenotypes. Four out of 15 LR asymmetry phenotypes showed an over-representation (FDR≤5%). We replicated three of these phenotypes; situs inversus, heterotaxia, and double outlet right ventricle, in the general population cohort (FDR≤5%). Our findings lead us to propose that handedness is a polygenic trait controlled in part by the molecular mechanisms that establish LR body asymmetry early in development.

Haemophilus influenzae is an important human pathogen involved in invasive disease. Here, we report the whole-genome sequences of 11 nonencapsulated H. influenzae (ncHi) strains isolated from both invasive disease and healthy carriers in Italy. This genomic information will enrich our understanding of the molecular basis of ncHi pathogenesis.

Heroin may have been accidentally contaminated by an animal-derived source along a major drug trafficking route.

Bacillus subtilis PS216, a strain isolated in Slovenia, has been sequenced. PS216 is transformable and forms robust biofilms, making it useful for the study of competence regulation in an undomesticated bacterium.

Alteration of the number of copies of Double Minutes (DMs) with oncogenic EGFR mutations in response to tyrosine kinase inhibitors (TKIs) is a novel adaptive mechanism of glioblastoma. Here we provide evidence that such mutations in DMs, called here Amplification-Linked Extrachromosomal Mutations (ALEMs), originate extrachromosomally and could therefore be completely eliminated from the cancer cells. By exome sequencing of 7 glioblastoma patients we reveal ALEMs in EGFR, PDGFRA and other genes. These mutations together with DMs are lost by cancer cells in culture. We confirm the extrachromosomal origin of such mutations by showing that wild type and mutated DMs may coexist in the same tumor. Analysis of 4198 tumors suggests the presence of ALEMs across different tumor types with the highest prevalence in glioblastomas and low grade gliomas. The extrachromosomal nature of ALEMs explains the observed drastic changes in the amounts of mutated oncogenes (like EGFR or PDGFRA) in glioblastoma in response to environmental changes.

ABSTRACTWe report the complete genome sequence of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from fermented herbage. We have determined the complete genome sequence of strain L. plantarum CGMCC 8198, which consists of genes that are likely to be involved in dairy fermentation and that have probiotic qualities.

AbstractBackgroundSeveral genes have been implicated in a highly variable presentation of developmental delay with psychomotor retardation. Mutations in EMC1 gene have recently been reported. Herein, we describe a proband born of a consanguineous marriage, who presented with early infantile onset epilepsy, scaphocephaly, developmental delay, central hypotonia, muscle wasting, and severe cerebellar and brainstem atrophy.MethodsGenetic testing in the proband was performed using custom clinical exome and targeted next‐generation sequencing. This was followed by segregation analysis of the variant in the parents by Sanger sequencing and evaluation of the splice variant by RNA sequencing.ResultsClinical exome sequencing identified a novel homozygous intronic splice variant in the EMC1 gene (chr1:19564510C>T, c.1212 + 1G>A, NM_015047.2). Neither population databases (ExAC and 1000 genomes) nor our internal database (n = 1,500) had reported this rare variant, predicted to affect the splicing. RNA sequencing data from the proband confirmed aberrant splicing with intron 11 retention, thereby introducing a stop codon in the resultant mRNA. This nonsense mutation is predicted to result in the premature termination of protein synthesis leading to loss of function of the EMC1 protein.ConclusionWe report, for the first time the role of aberrant EMC1RNA splicing as a potential cause of disease pathogenesis. The severe epilepsy observed in our study expands the disease‐associated phenotype and also emphasizes the need for comprehensive screening of intronic splice mutations.

Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals.DOI:http://dx.doi.org/10.7554/eLife.00426.001

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.

Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERβ, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells.

BackgroundThe transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.MethodsImmunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).ResultsGATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells.ConclusionsWe propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.

Tumor suppressor p53 prevents cell transformation by inducing apoptosis and other responses. Homozygous TP53 deletion occurs in various types of human cancers for which no therapeutic strategies have yet been reported. TCGA database analysis shows that the TP53 homozygous deletion locus mostly exhibits co-deletion of the neighboring gene FXR2, which belongs to the Fragile X gene family. Here, we demonstrate that inhibition of the remaining family member FXR1 selectively blocks cell proliferation in human cancer cells containing homozygous deletion of both TP53 and FXR2 in a collateral lethality manner. Mechanistically, in addition to its RNA-binding function, FXR1 recruits transcription factor STAT1 or STAT3 to gene promoters at the chromatin interface and regulates transcription thus, at least partially, mediating cell proliferation. Our study anticipates that inhibition of FXR1 is a potential therapeutic approach to targeting human cancers harboring TP53 homozygous deletion.

Many bacterial regulatory genes appear to be dispensable, as they can be deleted from the genome without loss of bacterial functionalities. In Helicobacter pylori, the hp1043 gene, also known as hsrA, is one of the transcriptional regulator that is essential for cell viability. This gene could not be deleted, nor the amount of protein modulated, supporting the hypothesis that HP1043 could be involved in the regulation of crucial cellular processes. Even though detailed structural data are available for the HP1043 protein, its targets are still ill-defined. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), one of the most powerful approaches to characterize protein-DNA interactions in vivo, we were able to identify genome-wide several new HP1043 binding sites. Moreover, in vitro DNA binding assays enabled precise mapping of the HP1043 binding sites on the new targets, revealing the presence of a conserved nucleotide sequence motif. Intriguingly, a significant fraction of the newly identified binding sites overlaps promoter regions controlling the expression of genes involved in translation. Accordingly, when protein translation was blocked, a significant induction of almost all HP1043 target genes was detected. These observations prompted us to propose HP1043 as a key regulator in H. pylori, likely involved in sensing and in coordinating the response to environmental conditions that provoke an arrest of protein synthesis. The essential role of HP1043 in coordinating central cellular processes is discussed.

Sertoli cells have dual roles during the cells’ lifetime. In the juvenile mammal, Sertoli cells proliferate and create the structure of the testis, and during puberty they cease to proliferate and take on the adult role of supporting germ cells through spermatogenesis. Accordingly, many genes expressed in Sertoli cells during testis formation are repressed during spermatogenesis. 5-Hydroxymethylcytosine (5hmC) is a DNA modification enzymatically generated from 5mC and present in all investigated mammalian tissues at varying levels. Using mass spectrometry and immunofluorescence staining we identified a substantial Sertoli cell-specific global 5hmC increase during rat puberty. Chemical labeling, pull-down and sequencing of 5hmC-containing genomic DNA from juvenile and adult rat Sertoli cells revealed that genes that lose or gain 5hmC belong to different functional pathways and mirror the functions of the cells in the two different states. Loss of 5hmC is associated with genes involved in development and cell structure, whereas gain of 5hmC is associated with genes involved in cellular pathways pertaining to the function of the adult Sertoli cells. This redistribution during maturation shows that 5hmC is a dynamic nucleotide modification, correlated to gene expression.

Subversion of host immune surveillance is a crucial step in viral pathogenesis. Epstein-Barr virus (EBV) encodes two immune evasion gene products, BCRF1 (viral IL-10) and BPLF1 (deubiquitinase/deneddylase); both proteins suppress antiviral immune responses during primary infection. The BCRF1 and BPLF1 genes are expressed during the late phase of the lytic cycle, an essential but poorly understood phase of viral gene expression. Several late gene regulators recently identified in beta and gamma herpesviruses form a viral pre-initiation complex for transcription. Whether each of these late gene regulators is necessary for transcription of all late genes is not known. Here, studying viral gene expression in the absence and presence of siRNAs to individual components of the viral pre-initiation complex, we identified two distinct groups of late genes. One group includes late genes encoding the two immunoevasins, BCRF1 and BPLF1, and is transcribed independently of the viral pre-initiation complex. The second group primarily encodes viral structural proteins and is dependent on the viral pre-initiation complex. The protein kinase BGLF4 is the only known late gene regulator necessary for expression of both groups of late genes. ChIP-seq analysis showed that the transcription activator Rta associates with the promoters of eight late genes including genes encoding the viral immunoevasins. Our results demonstrate that late genes encoding immunomodulatory proteins are transcribed by a mechanism distinct from late genes encoding viral structural proteins. Understanding the mechanisms that specifically regulate expression of the late immunomodulatory proteins could aid the development of antiviral drugs that impair immune evasion by the oncogenic EB virus.

Background: Cancers related to tobacco use and African-American ancestry are under-characterized by genomics. This gap in precision oncology research represents a major challenge in the health disparities in the United States.Methods: The Precision Oncology trial at the Wake Forest Baptist Comprehensive Cancer Center enrolled 431 cancer patients from March 2015 to May 2016. The composition of these patients consists of a high representation of tobacco-related cancers (e.g., lung, colorectal, and bladder) and African-American ancestry (13.5%). Tumors were sequenced to identify mutations to gain insight into genetic alterations associated with smoking and/or African-American ancestry.Results: Tobacco-related cancers exhibit a high mutational load. These tumors are characterized by high-frequency mutations in TP53, DNA damage repair genes (BRCA2 and ATM), and chromatin remodeling genes (the lysine methyltransferases KMT2D or MLL2, and KMT2C or MLL3). These tobacco-related cancers also exhibit augmented tumor heterogeneities. Smoking related genetic mutations were validated by The Cancer Genome Atlas dataset that includes 2,821 cases with known smoking status. The Wake Forest and The Cancer Genome Atlas cohorts (431 and 7,991 cases, respectively) revealed a significantly increased mutation rate in the TP53 gene in the African-American subgroup studied. Both cohorts also revealed 5 genes (e.g. CDK8) significantly amplified in the African-American population.Conclusions: These results provide strong evidence that tobacco is a major cause of genomic instability and heterogeneity in cancer. TP53 mutations and key oncogene amplifications emerge as key factors contributing to cancer outcome disparities among different racial/ethnic groups.

PurposeMicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied.MethodsC57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7).ResultsForm-deprived eyes showed myopic shifts in refractive error (−2.02 ± 0.47 D; P < 0.01). Comparison of the scleral RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed.ConclusionsScleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression.

Background:Mitochondrial disease can present at any age, with dysfunction in almost any tissue making diagnosis a challenge. It can result from inherited or sporadic mutations in either the mitochondrial or the nuclear genome, many of which affect intraorganellar gene expression. The estimated prevalence of 1/4300 indicates these to be amongst the commonest inherited neuromuscular disorders, emphasising the importance of recognition of the diagnostic clinical features.Objective:Despite major advances in our understanding of the molecular basis of mitochondrial diseases, accurate and early diagnoses are critically dependent on the fastidious clinical and biochemical characterisation of patients. Here we describe a patient harbouring a previously reported homozygous mutation in C12orf65, a mitochondrial protein of unknown function, which does not adhere to the proposed distinct genotype-phenotype relationship.Methods:We performed clinical, biochemical and molecular analysis including whole exome sequencing on patient samples and cell lines.Results:We report an extremely rare case of an adult presenting with Leigh-like disease, in intensive care, in the 5th decade of life, harbouring a recessively inherited mutation previously reported in children. A global reduction in intra-mitochondrial protein synthesis was observed despite normal or elevated levels of mt-RNA, leading to an isolated complex IV deficiency.Conclusions:All the reported C12orf65 mutations have shown an autosomal recessive pattern of inheritance. Mitochondrial disease causing mutations inherited in this manner are usually of early onset and associated with a severe, often fatal clinical phenotype. Presentations in adulthood are usually less severe. This patient’s late adulthood presentation is in sharp contrast emphasising the clinical variability that is characteristic of mitochondrial disease and illustrates why making a definitive diagnosis remains a formidable challenge.

G1P[8] rotaviruses are responsible for the majority of human rotavirus infections worldwide. The effect of universal mass vaccination with rotavirus vaccines on circulating G1P[8] rotaviruses is still poorly understood. Therefore we analyzed the complete genomes of the Rotarix™ vaccine strain, and 70 G1P[8] rotaviruses, detected between 1999 and 2010 in Belgium (36 before and 34 after vaccine introduction) to investigate the impact of rotavirus vaccine introduction on circulating G1P[8] strains. All rotaviruses possessed a complete Wa-like genotype constellation, but frequent intra-genogroup reassortments were observed as well as multiple different cluster constellations circulating in a single season. In addition, identical cluster constellations were found to circulate persistently over multiple seasons. The Rotarix™ vaccine strain possessed a unique cluster constellation that was not present in currently circulating G1P[8] strains. At the nucleotide level, the VP6, VP2 and NSP2 gene segments of Rotarix™ were relatively distantly related to any Belgian G1P[8] strain, but other gene segments of Rotarix™ were found in clusters also containing circulating Belgian strains. At the amino acid level, the genetic distance between Rotarix™ and circulating Belgian strains was considerably lower, except for NSP1. When we compared the Belgian G1P[8] strains collected before and after vaccine introduction a reduction in the proportion of strains that were found in the same cluster as the Rotarix™ vaccine strain was observed for most gene segments. The reduction in the proportion of strains belonging to the same cluster may be the result of the vaccine introduction, although natural fluctuations cannot be ruled out.

ABSTRACTWe report here the 4.19-Mb draft genome sequence of Bacillus licheniformis VSD4, a Gram-positive bacterium of the Bacillaceae family, isolated from leaves of Hedera helix growing at a high-traffic city center in Belgium. Knowledge about its genome will help to evaluate its potential as an inoculant in phylloremediation applications.

Toxoplasma gondii is an important zoonotic pathogen infecting one third of the world population and numerous animals. A key factor to its wide distribution is the ability to interconvert between fast replicating tachyzoites and slowly growing bradyzoites, and to establish lifelong chronic infection in intermediate hosts. Although it is well accepted that stage conversion plays key roles in the pathogenesis and transmission of the parasite, little is known about the molecular mechanisms behind it. Using existing gene expression data from TOXODB and published work, we looked for proteins with novel functional domains and whose expression is up-regulated in the bradyzoite stage, hoping to find molecules that have critical roles in regulating stage conversion and bradyzoite formation. In this study we characterized two such proteins ANK1 and DnaK-TPR, both of which are primarily expressed in bradyzoites and contain novel motifs to mediate protein-protein interactions. Through CRISPR/CAS9 directed gene editing technology, both genes were individually knocked out in type 1 strain TgHB2 and type 2 strain ME49. Disruption of neither of these two genes affected the growth or replication of tachyzoites in vitro, consistent with their minimal expression at this stage. However, mutants lacking ANK1 or DnaK-TPR displayed modest virulence attenuation during mice infection. Surprisingly, inactivation of neither ANK1 nor DnaK-TPR seemed to have a significant impact on bradyzoite differentiation in vitro or cyst formation in vivo. These results suggest that ANK1 and DnaK-TPR probably do not directly contribute to bradyzoite differentiation, but likely affect other aspects of bradyzoite biology.

The xylanase-producing bacterial strain Cellvibrio mixtus J3-8 was isolated from grassland giant snails. The draft genome of strain J3-8 comprises 5,171,890 bp in 152 contigs with a G+C content of 46.66%. This is the first genome report about this bacterial species.

The most important proteins involved in olfaction include odorant binding protein (OBP), chemosensory protein (CSP), olfactory receptor (OR), and gustatory receptor (GR). Despite that the exhaustive genomic analysis has revealed a large number of olfactory genes in a number of model insects, it is still poorly understood for most nonmodel species. This is mostly due to the reason that the small antenna is challenging for collection. We can generally isolate one or few genes at a time by means of the traditional method. Here, we present the large-scale identifying members of the main olfactory genes from the head of Tomicus yunnanensis using Illumina sequencing. In a single run, we obtained over 51.8 million raw reads. These reads were assembled into 57,142 unigenes. Nearly 29,384 of them were functionally annotated in the NCBI nonredundant database. By depth analysis of the data, 11 OBPs, 8 CSPs, 18 ORs, and 8 GRs were retrieved. Sequences encoding full length proteins were further characterised for one OBP and two CSPs. The obtained olfactory genes provide a major resource in further unraveling the molecular mechanisms of T. yunnanensis chemoperception. This study indicates that the next generation sequencing is an attractive approach for efficient identification of olfactory genes from insects, for which the genome sequence is unavailable.

This is the first complete mitochondrial genome sequence for sunflower and the first complete mitochondrial genome for any member of Asteraceae, the largest plant family, which includes over 23,000 named species. The master circle is 300,945-bp long and includes 27 protein-coding sequences, 18 tRNAs, and the 26S, 5S, and 18S rRNAs.

Pontibacter akesuensis strain AKS 1T was found in Akesu, Xinjiang Province, China, and exhibits the extraordinary ability to metabolize various substrates and is resistant to solar radiation. To gain insight into the bacterial genetic determinants for this adaptability, we report the complete genome sequence of strain AKS 1T.

ABSTRACTStaphylococcus aureus PX03 can produce acetoin efficiently. Here, we present a 2.38-Mb assembly of its genome sequence, which might provide further insights into the molecular mechanism of its acetoin biosynthesis to further improve its biotechnological applications.

ABSTRACTStreptococcus parauberis strain SP-llh was isolated from cows with mastitis in western China in 2015. The 2,522,235-bp genome sequence consists of 46 large contigs in 14 scaffolds and contains 2,620 predicted protein-coding genes, with a G+C content of 35.3%.

Arthrobacter sp. strain PAO19 is a polyphosphate-accumulating organism isolated from maize rhizosphere soil. Here we report its genome sequence, which may shed light on its role in phosphate removal from water bodies. To our knowledge, this is the first genome announcement of a polyphosphate-accumulating strain of the genus Arthrobacter.

We report the draft genome assembly of Enterococcus pernyi. The genome sequence is 3.09 Mb in length with a G+C content of 38.35%. It covers 3,153 genes with an average length of 854 bp, and contains 65 tRNAs, 13 small RNAs, and 18 rRNAs. Moreover, it contains 9 genomic islands with an average length of 14,058 bp and 3 prophages with an average length of 37,430 bp.

We report the draft genome sequence of Jiangella alkaliphila KCTC 19222T, isolated from cave soil in Jeju, Republic of Korea. This genome sequence, together with the previously sequenced J. gansuensis strain DSM 44835T, identified from a desert environmental source, will give us a better understanding of the school of “evolutionary taxonomy.”

Clostridium acetobutylicum is an organism involved in the production of acetone and butanol by traditional acetone-butanol-ethanol fermentation (ABE). We report the draft genome sequence of C. acetobutylicum strain GXAS18-1, which can produce ABE directly from cassava flour.

Sphingobium sp. strain YL23, a novel bacterium isolated from sewage sludge of a domestic wastewater treatment plant, has been shown to completely degrade bisphenol A under aerobic conditions. Here, we describe a 3.8-Mb assembly of its genome sequence and major findings from its annotation.

Pseudomonas nitroreducens TX1 ATCC PTA-6168 was isolated from rice field drainage in Taiwan. The bacterium is of special interest because of its capability to use nonionic surfactants (alkylphenol polyethoxylates) and estrogen-like compounds (4-t-octylphenol and 4-nonylphenol) as a sole carbon source. This is the first report on the genome sequence of P. nitroreducens.

Aerococcus urinaeequi strain AV208 was isolated from an ascites sample from a patient with chronic kidney disease. The assembled genome contained 2,227,638 bp with a 39.1% G+C content. The genome harbors a Tn1546 transposon-like structure with a vanA gene causing vancomycin resistance phenotypes of strain AV208.

B lymphopoiesis requires that immunoglobulin genes be accessible to the RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin suggesting that additional mechanisms must restrict RAG-mediated DNA cleavage. Here, we demonstrate developmental downregulation of interleukin 7 (IL-7) receptor signaling in small pre-B cells induced expression of the bromodomain family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-BCR-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5′ to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings provide a paradigm in which, at any particular antigen receptor locus, specialized mechanisms enforce lineage and stage specific recombination.