De novo species identification using 16S rRNA gene nanopore sequencing
Nanopore sequencing is rapidly becoming more popular for use in various microbiota-based applications. Major limitations of current approaches are that they do not enable de novo species identification and that they cannot be used to verify species assignments. This severely limits applicability of the nanopore sequencing technology in taxonomic applications. Here, we demonstrate the possibility of de novo species identification and verification using hexamer frequencies in combination with k-means clustering for nanopore sequencing data. The approach was tested on the human infant gut microbiota of 3-month-old infants. Using the hexamer k-means approach we identified two new low abundant species associated with vaginal delivery. In addition, we confirmed both the vaginal delivery association for two previously identified species and the overall high levels of bifidobacteria. Taxonomic assignments were further verified by mock community analyses. Therefore, we believe our de novo species identification approach will have widespread application in analyzing microbial communities in the future.
Angell et al. (2020) De novo species identification using 16S rRNA gene nanopore sequencing. PeerJ 8:e10029 https://doi.org/10.7717/peerj.10029
Small-scale distribution of microbes and biogeochemistry in the Great Barrier Reef
Microbial communities distribute heterogeneously at small-scales (mm-cm) due to physical, chemical and biological processes. To understand microbial processes and functions it is necessary to appreciate microbes and matter at small scales, however, few studies have determined microbial, viral, and biogeochemical distribution over space and time at these scales. In this study, the small-scale spatial and temporal distribution of microbes (bacteria and chlorophyll a), viruses, dissolved inorganic nutrients and dissolved organic carbon were determined at five locations (spatial) along the Great Barrier Reef (Australia), and over 4 consecutive days (temporal) at a coastal location. Our results show that: (1) the parameters show high small-scale heterogeneity; (2) none of the parameters measured explained the bacterial abundance distributions at these scales spatially or temporally; (3) chemical (ammonium, nitrate/nitrite, phosphate, dissolved organic carbon, and total dissolved nitrogen) and biological (chl a, and bacterial and viral abundances) measurements did not reveal significant relationships at the small scale; and (4) statistically significant differences were found between sites/days for all parameter measured but without a clear pattern.
Carreira et al. (2020) Small-scale distribution of microbes and biogeochemistry in the Great Barrier Reef. PeerJ 8:e10049 https://doi.org/10.7717/peerj.10049
Comparison of the diversity of cultured and total bacterial communities in marine sediment using culture-dependent and sequencing methods
Despite recent great advances in microbial culture, most microbes have not yet been cultured, and the impact of medium composition on the isolation of microbes from natural systems has not been elucidated. To optimize media for culturing marine microbes, microbial communities in three sediment samples were described using high-throughput sequencing (HTS) and culture-dependent techniques. HTS revealed communities dominated by Gammaproteobacteria, and culture-based methods revealed communities dominated by Actinobacteria. Among the total operational taxonomic units (OTUs) from the HTS dataset, 6% were recovered in the culture collection. Four potentially novel bacterial strains belonging to Oceaniovalibus, Psychrobacter and Salegentibacter were isolated. The combination of media cultured more taxa than any single medium. Nutrient-rich and single-carbon/nitrogen-source media supported the growth of relatively few taxa, and the quality of nitrogen strongly influenced the types of bacteria isolated.
Wang et al. (2020) Comparison of the diversity of cultured and total bacterial communities in marine sediment using culture-dependent and sequencing methods. PeerJ 8:e10060 https://doi.org/10.7717/peerj.10060
Functional characterization of quorum sensing LuxR-type transcriptional regulator, EasR in Enterobacter asburiae strain L1
Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N–hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.
Lau et al. (2020) Functional characterization of quorum sensing LuxR-type transcriptional regulator, EasR in Enterobacter asburiae strain L1. PeerJ 8:e10068 https://doi.org/10.7717/peerj.10068
Morphological and molecular systematic review of Marphysa Quatrefages, 1865 (Annelida: Eunicidae) species from South Africa
A vast polychaete fauna is hidden behind complexes of cryptic and pseudo-cryptic species, which has greatly hindered our understanding of species diversity in several regions worldwide. Among the eunicids, Marphysa sanguinea Montagu, 1813 is a typical example, recorded in three oceans and with various species considered its junior synonyms. In South Africa, specimens previously misidentified as M. sanguinea are now known as Marphysa elityeni Lewis & Karageorgopoulos, 2008. Of the six Marphysa Quatrefages, 1865a species recorded from the same region, three have their distributions restricted to South Africa while the others are considered to have worldwide distributions. Here, we evaluated the taxonomic status of the indigenous M. elityeni and investigated the presence of the widespread species Marphysa macintoshi Crossland, 1903 and Marphysa depressa Schmarda, 1861 in South Africa using morphological and molecular data. Our results reveal that M. elityeni is a junior synonym of Marphysa haemasoma, a species previously described from South Africa which is herein reinstated as a valid species. Both M. macintoshi and M. depressa are not present in South Africa and their status as being distributed worldwide deserves further investigation. Marphysa durbanensis Day, 1934 and the new species described here, M. sherlockae n. sp., had been misidentified as M. macintoshi and M. depressa respectively. Thus, the number of Marphysa species with distributions restricted to South Africa increased from three to five. This study reiterates the importance of implementing an integrated taxonomic framework to unravel local biodiversity.
Kara et al. (2020) Morphological and molecular systematic review of Marphysa Quatrefages, 1865 (Annelida: Eunicidae) species from South Africa. PeerJ 8:e10076 https://doi.org/10.7717/peerj.10076
Vegetation drives the structure of active microbial communities on an acidogenic mine tailings deposit
Plant-microbe associations are increasingly recognized as an inextricable part of plant biology and biogeochemistry. Microbes play an essential role in the survival and development of plants, allowing them to thrive in diverse environments. The composition of the rhizosphere soil microbial communities is largely influenced by edaphic conditions and plant species. In order to decipher how environmental conditions on a mine site can influence the dynamics of microbial communities, we characterized the rhizosphere soil microbial communities associated with paper birch, speckled alder, and spruce that had naturally colonized an acidogenic mine tailings deposit containing heavy metals. The study site, which had been largely undisturbed for five decades, had highly variable vegetation density; with some areas remaining almost barren, and others having a few stands or large thickets of mature trees. Using Illumina sequencing and ordination analyses (redundancy analysis and principal coordinate analysis), our study showed that soil bacterial and fungal community structures correlated mainly with vegetation density, and plant species. Tailings without any vegetation were the most different in bacterial community structure, compared to all other areas on the mine site, as well as an adjacent natural forest (comparison plot). The bacterial genera Acidiferrobacter and Leptospirillum were more abundant in tailings without vegetation than in any of the other sites, while Bradyrhizobium sp. were more abundant in areas of the tailings deposit having higher vegetation density. Frankia sp. is equally represented in each of the vegetation densities and Pseudomonas sp. present a greater relative abundance in boreal forest. Furthermore, alder rhizosphere showed a greater relative abundance of Bradyrhizobium sp. (in comparison with birch and spruce) as well as Haliangium sp. (in comparison with birch). In contrast, fungal community structures were similar across the tailings deposit regardless of vegetation density, showing a greater relative abundance of Hypocrea sp. Tailings deposit fungal communities were distinct from those found in boreal forest soils. Alder rhizosphere had greater relative abundances of Hypocrea sp. and Thelephora sp., while birch rhizosphere were more often associated with Mollisia sp. Our results indicate that, with increasing vegetation density on the mine site, the bacterial communities associated with the individual deciduous or coniferous species studied were increasingly similar to the bacterial communities found in the adjacent forest. In order to properly assess and restore disturbed sites, it is important to characterize and understand the plant-microbe associations that occur since they likely improve plant fitness in these harsh environments.
Environmental Contamination and Remediation
Gagnon et al. (2020) Vegetation drives the structure of active microbial communities on an acidogenic mine tailings deposit. PeerJ 8:e10109 https://doi.org/10.7717/peerj.10109
Immunoglobulin superfamily member 10 is a novel prognostic biomarker for breast cancer
Background Immunoglobulin superfamily member 10 (IGSF10) is a member of the immunoglobulin superfamily that is expressed at high levels in both the gallbladder and ovary. Currently, the role and possible mechanism of IGSF10 in breast cancer remain unclear. Method By applying real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), the expression of IGSF10 in breast cancer cells and tissues was detected. We collected the clinical information from 700 patients with breast cancer in The Cancer Genome Atlas (TCGA), and analyzed the relationship between IGSF10 expression and the clinicopathological features and survival outcomes of these patients. The potential mechanisms and pathways associated with IGSF10 in breast cancer were explored by performing a gene set enrichment analysis (GSEA). Results According to TCGA data, qRT-PCR and IHC experiments, levels of the IGSF10 mRNA and protein were significantly decreased in breast cancer tissues. IGSF10 expression was significantly correlated with age, tumor size, and tumor stage. Moreover, shorter overall survival (OS) and relapse-free survival (RFS) correlated with lower IGSF10 expression, according to the survival analysis. The multivariate analysis identified that IGSF10 as an independent prognostic factor for the OS (hazard ratio (HR) = 1.793, 95% confidence interval (CI) [1.141–2.815], P = 0.011) and RFS (HR = 2.298, 95% CI [1.317–4.010], P = 0.003) of patients with breast cancer. Based on the GSEA, IGSF10 was involved in DNA repair, cell cycle, and glycolysis. IGSF10 was also associated with the PI3K/Akt/mTOR and mTORC1 signaling pathways. Conclusions This study revealed a clear relationship between IGSF10 expression and the tumorigenesis of breast cancer for the first time. Therefore, further studies are needed to understand the mechanism of IGSF10 in breast cancer.
Wang et al. (2020) Immunoglobulin superfamily member 10 is a novel prognostic biomarker for breast cancer. PeerJ 8:e10128 https://doi.org/10.7717/peerj.10128
Genomics versus mtDNA for resolving stock structure in the silky shark (Carcharhinus falciformis)
Conservation genetic approaches for elasmobranchs have focused on regions of the mitochondrial genome or a handful of nuclear microsatellites. High-throughput sequencing offers a powerful alternative for examining population structure using many loci distributed across the nuclear and mitochondrial genomes. These single nucleotide polymorphisms are expected to provide finer scale and more accurate population level data; however, there have been few genomic studies applied to elasmobranch species. The desire to apply next-generation sequencing approaches is often tempered by the costs, which can be offset by pooling specimens prior to sequencing (pool-seq). In this study, we assess the utility of pool-seq by applying this method to the same individual silky sharks, Carcharhinus falciformis, previously surveyed with the mtDNA control region in the Atlantic and Indian Oceans. Pool-seq methods were able to recover the entire mitochondrial genome as well as thousands of nuclear markers. This volume of sequence data enabled the detection of population structure between regions of the Atlantic Ocean populations, undetected in the previous study (inter-Atlantic mitochondrial SNPs FST values comparison ranging from 0.029 to 0.135 and nuclear SNPs from 0.015 to 0.025). Our results reinforce the conclusion that sampling the mitochondrial control region alone may fail to detect fine-scale population structure, and additional sampling across the genome may increase resolution for some species. Additionally, this study shows that the costs of analyzing 4,988 loci using pool-seq methods are equivalent to the standard Sanger-sequenced markers and become less expensive when large numbers of individuals (>300) are analyzed.
Kraft et al. (2020) Genomics versus mtDNA for resolving stock structure in the silky shark (Carcharhinus falciformis). PeerJ 8:e10186 https://doi.org/10.7717/peerj.10186
DNA barcoding for identification of anuran species in the central region of South America
The use of COI barcodes for specimen identification and species discovery has been a useful molecular approach for the study of Anura. Here, we establish a comprehensive amphibian barcode reference database in a central area of South America, in particular for specimens collected in Mato Grosso do Sul state (Brazil), and to evaluate the applicability of the COI gene for species-level identification. Both distance- and tree-based methods were applied for assessing species boundaries and the accuracy of specimen identification was evaluated. A total of 204 mitochondrial COI barcode sequences were evaluated from 22 genera and 59 species (19 newly barcoded species). Our results indicate that morphological and molecular identifications converge for most species, however, some species may present cryptic species due to high intraspecific variation, and there is a high efficiency of specimen identification. Thus, we show that COI sequencing can be used to identify anuran species present in this region.
Koroiva et al. (2020) DNA barcoding for identification of anuran species in the central region of South America. PeerJ 8:e10189 https://doi.org/10.7717/peerj.10189
Structure and functions of Yellow-breasted Boubou (Laniarius atroflavus) solos and duets
Background Birds have extremely well-developed acoustic communication and have become popular in bioacoustics research. The majority of studies on bird song have been conducted in the temperate zones where usually males of birds sing to attract females and defend territories. In over 360 bird species mostly inhabiting the tropics both males and females sing together in duets. Avian duets are usually formed when a male and female coordinate their songs. We focused on a species with relatively weakly coordinated duets, with male solo as the prevailing vocalisation type. Methods Instead of analysing a set of recordings spread over a long time, we analysed whole day microphone-array recordings of the Yellow-breasted Boubou (Laniarius atroflavus), a species endemic to West African montane rainforests. We described the structure of the solo and duet vocalisations and temporal characteristics of daily activity based on 5,934 vocal bouts of 18 focal pairs and their neighbours. Results Birds had small, sex specific repertoires. All males shared three types of loud whistles functioning as song type repertoires in both solos and duets. Females vocalised with five types of harsh, atonal notes with a more variable and usually lower amplitude. Three of them were produced both as solos and in duets, while two seem to function as alarm and excitement calls given almost exclusively as a solo. Solos were the most common vocalisation mode (75.4%), with males being more vocally active than females. Duets accounted for 24.6% of all vocalisations and in most cases were initiated by males (81%). The majority of duets were simple (85.1%) consisting of a single male and female song type, but altogether 38 unique duet combinations were described. Males usually initiated singing at dawn and for this used one particular song type more often than expected by chance. Male solo and duet activities peaked around dawn, while female solos were produced evenly throughout the day. Discussion Yellow-breasted Boubou is a duetting species in which males are much more vocal than females and duetting is not a dominating type of vocal activity. Duet structure, context and timing of daily production support the joint resource defence hypothesis and mate guarding/prevention hypotheses, however maintaining pair contact also seems to be important. This study provides for the first time the basic quantitative data describing calls, solos and duet songs in the Yellow-breasted Boubou.
Wheeldon et al. (2020) Structure and functions of Yellow-breasted Boubou (Laniarius atroflavus) solos and duets. PeerJ 8:e10214 https://doi.org/10.7717/peerj.10214