Meat-Borne-Parasite: A Nanopore-Based Meta-Barcoding Work-Flow for Parasitic Microbiodiversity Assessment in the Wild Fauna of French Guiana.

French Guiana, located in the Guiana Shield, is a natural reservoir for many zoonotic pathogens that are of considerable medical or veterinary importance. Until now, there has been limited data available on the description of parasites circulating in this area, especially on protozoan belonging to the phylum Apicomplexa; conversely, the neighbouring countries describe a high parasitic prevalence in animals and humans. Epidemiological surveillance is necessary, as new potentially virulent strains may emerge from these forest ecosystems, such as Amazonian toxoplasmosis. However, there is no standard tool for detecting protozoa in wildlife. In this study, we developed Meat-Borne-Parasite, a high-throughput meta-barcoding workflow for detecting Apicomplexa based on the Oxford Nanopore Technologies sequencing platform using the 18S gene of 14 Apicomplexa positive samples collected in French Guiana. Sequencing reads were then analysed with MetONTIIME pipeline. Thanks to a scoring rule, we were able to classify 10 samples out of 14 as Apicomplexa positive and reveal the presence of co-carriages. The same samples were also sequenced with the Illumina platform for validation purposes. For samples identified as Apicomplexa positive by both platforms, a strong positive correlation at up to the genus level was reported. Overall, the presented workflow represents a reliable method for Apicomplexa detection, which may pave the way for more comprehensive biomonitoring of zoonotic pathogens.

A tipping point in carbon storage when forest expands into tundra is related to mycorrhizal recycling of nitrogen.

Tundra ecosystems are global belowground sinks for atmospheric CO2 . Ongoing warming-induced encroachment by shrubs and trees risks turning this sink into a CO2 source, resulting in a positive feedback on climate warming. To advance mechanistic understanding of how shifts in mycorrhizal types affect long-term carbon (C) and nitrogen (N) stocks, we studied small-scale soil depth profiles of fungal communities and C-N dynamics across a subarctic-alpine forest-heath vegetation gradient. Belowground organic stocks decreased abruptly at the transition from heath to forest, linked to the presence of certain tree-associated ectomycorrhizal fungi that contribute to decomposition when mining N from organic matter. In contrast, ericoid mycorrhizal plants and fungi were associated with organic matter accumulation and slow decomposition. If climatic controls on arctic-alpine forest lines are relaxed, increased decomposition will likely outbalance increased plant productivity, decreasing the overall C sink capacity of displaced tundra.

DNA meta-barcoding using rbcL based mini-barcode revealed presence of unspecified plant species in Ayurvedic polyherbal formulations.

INTRODUCTION: Ayurveda takes advantage of the beneficial properties of medicinal plants. High demands in combination with inadequate availability of botanicals and a lack of knowledge with respect to their precise identification lead to adulterations in herbal products. Identification becomes more difficult in complex herbal formulations. Four different polyherbal formulations have been analyzed for the present paper. The targeted plants have different pharmacological properties for various ailments. OBJECTIVE: We aimed to examine the rbcL gene based plant DNA mini-barcode to identify target and non-target plants in polyherbal formulations by using high-throughput next generation sequencing. METHODS: Degenerate primers of the selected mini-barcode region have been identified from the literature. A blend of 30 authentic medicinal plant species was used to examine the species resolution capacity of the mini-barcode. DNA was isolated from herbal formulations, an amplicon library was prepared, and sequencing was performed on an IonS5 system. Data were analyzed using various bioinformatics tools. RESULTS: Analysis of control pooled samples revealed the optimum resolving power of the DNA mini-barcode. Data analysis of the commercial samples revealed that only one herbal formulation contained all plants and matched with listed contents. In two formulations, only 10 out of 21 and 11 out of 20 plants were detected, respectively. Additionally, several non-listed plants were also detected in these formulations. Two formulations contained >20% reads assigned to non-target plants. Overall, 21.98% of the reads were assigned to non-target plants. CONCLUSION: The present study clearly demonstrated the successful application and potential of meta-barcoding in the quality control of complex herbal matrices. The results strongly suggest that this approach can be used in pharmacovigilance of processed herbal products.

DNA barcoding of phytopathogens for disease diagnostics and bio-surveillance.

DNA barcoding has proven to be a versatile tool for plant disease diagnostics in the genomics era. As the mass parallel and next generation sequencing techniques gained importance, the role of specific barcodes came under immense scrutiny. Identification and accurate classification of phytopathogens need a universal approach which has been the main application area of the concept of barcode. The present review entails a detailed description of the present status of barcode application in plant disease diagnostics. A case study on the application of Internal Transcribed Spacer (ITS) as barcode for Aspergillus and Fusarium spp. sheds light on the requirement of other potential candidates as barcodes for accurate identification. The challenges faced while barcoding novel pathogens have also been discussed with a comprehensive outline of integrating more recent technologies like meta-barcoding and genome skimming for detecting plant pathogens.

Moderate phosphorus additions consistently affect community composition of arbuscular mycorrhizal fungi in tropical montane forests in southern Ecuador.

Anthropogenic atmospheric deposition can increase nutrient supply in the most remote ecosystems, potentially affecting soil biodiversity. Arbuscular mycorrhizal fungal (AMF) communities rapidly respond to simulated soil eutrophication in tropical forests. Yet the limited spatio-temporal extent of such manipulations, together with the often unrealistically high fertilization rates employed, impedes generalization of such responses. We sequenced mixed root AMF communities within a seven year-long fully factorial nitrogen (N) and phosphorus (P) addition experiment, replicated at three tropical montane forests in southern Ecuador with differing environmental characteristics. We hypothesized: strong shifts in community composition and species richness after long-term fertilization, site- and clade-specific responses to N vs P additions depending on local soil fertility and clade life history traits respectively. Fertilization consistently shifted AMF community composition across sites, but only reduced richness of Glomeraceae. Compositional changes were mainly driven by increases in P supply while richness reductions were observed only after combined N and P additions. We conclude that moderate increases of N and P exert a mild but consistent effect on tropical AMF communities. To predict the consequences of these shifts, current results need to be supplemented with experiments that characterize local species-specific AMF functionality.

Can arbuscular mycorrhizal fungi from non-invaded montane ecosystems facilitate the growth of alien trees?

It is generally assumed that recruitment and expansion of alien species along elevation gradients are constrained by climate. But, if plants are not fully constrained by climate, their expansion could be facilitated or hindered by other factors such as biotic interactions. Here, we assessed the composition of arbuscular mycorrhizal fungi (AMF) in soils along an elevation gradient (i.e. 900 m, 1600 m, 2200 m and 2700 m a.s.l.) through a fungal DNA meta-barcoding approach. In addition, we studied in the greenhouse the effects of AMF on growth and phosphorous (P) nutrition of seedlings of the alien trees Gleditsia triacanthos, Ligustrum lucidum and Pyracantha angustifolia cultivated in soils from those elevations, spanning the elevation at which they already form monospecific stands (below 1450 m a.s.l.) and higher elevations, above their current range of distribution in montane ecosystems of Central Argentina. For comparison, we also included in the experiment the dominant native tree Lithraea molleoides that historically occurs below 1300 m a.s.l. Arbuscular mycorrhizal fungal community composition showed strong community turnover with increasing elevation. The effects of these AMF communities on plant growth and nutrition differed among native and alien trees. While P nutrition in alien species' seedlings was generally enhanced by AMF along the whole gradient, the native species benefited only from AMF that occur in soils from the elevation corresponding to its current altitudinal range of distribution. These results suggest that AMF might foster upper range expansion of these invasive trees over non-invaded higher elevations.

Biodiversity of Basidiomycetous Yeasts Associated with Cladonia rei Lichen in Japan, with a Description of Microsporomyces cladoniophilus sp. nov.

For more than a century, lichens have been used as an example of dual-partner symbiosis. Recently, this has been challenged by the discovery of various basidiomycetous yeasts that coexist in multiple lichen species, among which Cladonia lichens from Europe and the United States were discovered to be highly specifically associated with the basidiomycetous yeast of the family Microsporomycetaceae. To verify this highly specific relationship, we investigated the diversity of basidiomycetous yeasts associated with Cladonia rei, a widely distributed lichen in Japan, by applying two approaches: yeast isolation from the lichen thalli and meta-barcoding analysis. We obtained 42 cultures of Cystobasidiomycetous yeast which were grouped into six lineages within the family Microsporomycetaceae. Unexpectedly, although the cystobasidiomycetes-specific primer was used, not only the cystobasidiomycetous yeasts but species from other classes were also detected via the meta-barcoding dataset; in particular, pucciniomycetous yeasts were found at a high frequency in some samples. Further, Halobasidium xiangyangense, which was detected in every sample with high abundance, is highly likely a generalist epiphytic fungus that has the ability to associate with C. rei. In the pucciniomycetous group, most of the detected species belong to the scale insect-associated yeast Septobasidium genus. In conclusion, even though Microsporomyces species are not the only yeast group associated with Cladonia lichen, our study demonstrated that the thalli of Cladonia rei lichen could be a suitable habit for them.

Can dietary magnesium sources and buffer change the ruminal microbiota composition and fermentation of lactating dairy cows?

Magnesium oxide (MgO) is one of the most used Mg supplements in livestock. However, to avoid relying upon only one Mg source, it is important to have alternative Mg sources. Therefore, the objective of this study was to evaluate the effects of the interaction of two Mg sources with buffer use on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. Twenty lactating Holstein cows were blocked by parity and days in milk into five blocks with four cows each, in a 2 × 2 factorial design. Within blocks, cows were assigned to one of four treatments: 1) MgO; 2) MgO + Na sesquicarbonate (MgO+); 3) calcium-magnesium hydroxide (CaMgOH); 4) CaMgOH + Na sesquicarbonate (CaMgOH+). For 60 d, cows were individually fed a corn silage-based diet, and treatments were top-dressed. Ruminal fluid was collected via an orogastric tube, for analyses of the microbiota composition, volatile fatty acids (VFA), lactate, and ammonia nitrogen (NH3-N). The microbiota composition was analyzed using V4/16S rRNA gene sequencing, and taxonomy was assigned using the Silva database. Statistical analysis was carried out following the procedures of block design analysis, where block and cow were considered random variables. Effects of Mg source, buffer, and the interaction between Mg Source × Buffer were analyzed through orthogonal contrasts. There was no interaction effect of the two factors evaluated. There was a greater concentration of NH3-N, lactate, and butyrate in the ruminal fluid of cows fed with CaMg(OH)2, regardless of the buffer use. The increase in these fermentation intermediates/ end-products can be explained by an increase in abundance of micro-organisms of the genus Prevotella, Lactobacillus, and Butyrivibrio, which are micro-organisms mainly responsible for proteolysis, lactate-production, and butyrate-production in the rumen, respectively. Also, dietary buffer use did not affect the ruminal fermentation metabolites and pH; however, an improvement of the apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral fiber detergent (NDF), and acid fiber detergent (ADF) were found for animals fed with dietary buffer. In summary, there was no interaction effect of buffer use and Mg source, whereas buffer improved total tract apparent digestibility of DM and OM through an increase in NDF and ADF digestibility and CaMg(OH)2 increased ruminal concentration of butyrate and abundance of butyrate-producing bacteria.

Magnesium oxide (MgO) is extensively used as a dietary magnesium (Mg) source in dairy cow diets. However, dairy operations can benefit from other Mg sources. Thus, we evaluated the replacement of dietary MgO with calcium­magnesium hydroxide (CaMg(OH)2) in diets with and without ruminal buffer and their effects on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. The study used 20 lactating Holstein cows that were blocked in groups of four and randomly assigned to one of the four treatments. The ruminal content, feed, feces, and urine were collected for analysis of the microbiota composition, ruminal fermentation, nitrogen metabolism, and apparent nutrient digestibility. There was no interaction effect of dietary buffer use and Mg source, while buffer improved total tract apparent digestibility of the dry matter and fiber components; CaMg(OH)2 increased the ruminal concentration of butyrate and the abundance of butyrate-producing bacteria. In summary, we conclude that using CaMg(OH)2 can improve ruminal fermentation regardless of buffer use, which indicates that we can take advantage of the mineral formulation in the diet to modulate the ruminal microbiota composition.

DNA barcoding in herbal medicine: Retrospective and prospective.

DNA barcoding has been widely used for herb identification in recent decades, enabling safety and innovation in the field of herbal medicine. In this article, we summarize recent progress in DNA barcoding for herbal medicine to provide ideas for the further development and application of this technology. Most importantly, the standard DNA barcode has been extended in two ways. First, while conventional DNA barcodes have been widely promoted for their versatility in the identification of fresh or well-preserved samples, super-barcodes based on plastid genomes have rapidly developed and have shown advantages in species identification at low taxonomic levels. Second, mini-barcodes are attractive because they perform better in cases of degraded DNA from herbal materials. In addition, some molecular techniques, such as high-throughput sequencing and isothermal amplification, are combined with DNA barcodes for species identification, which has expanded the applications of herb identification based on DNA barcoding and brought about the post-DNA-barcoding era. Furthermore, standard and high-species coverage DNA barcode reference libraries have been constructed to provide reference sequences for species identification, which increases the accuracy and credibility of species discrimination based on DNA barcodes. In summary, DNA barcoding should play a key role in the quality control of traditional herbal medicine and in the international herb trade.

Trichoderma spp.-mediated mitigation of heat, drought, and their combination on the Arabidopsis thaliana holobiont: a metabolomics and metabarcoding approach.

Introduction: The use of substances to increase productivity and resource use efficiency is now essential to face the challenge of feeding the rising global population with the less environmental impact on the ecosystems. Trichoderma-based products have been used as biopesticides, to inhibit pathogenic microorganisms, and as biostimulants for crop growth, nutrient uptake promotion, and resistance to abiotic stresses. Methods: In this work, plant metabolomics combined with roots and rhizosphere bacterial metabarcoding were exploited to inspect the performance of Trichoderma spp. biostimulants on Arabidopsis thaliana under drought, heat and their combination and its impact on plant holobiont. Results and discussion: An overall modulation of N-containing compounds, phenylpropanoids, terpenes and hormones could be pointed out by metabolomics. Moreover, metabarcoding outlined an impact on alpha and beta-diversity with an abundance of Proteobacteria, Pseudomonadales, Burkholderiales, Enterobacteriales and Azospirillales. A holobiont approach was applied as an integrated analytical strategy to resolve the coordinated and complex dynamic interactions between the plant and its rhizosphere bacteria using Arabidopsis thaliana as a model host species.

Beating the heat: ecology of desert bobcats.

BACKGROUND: Relative to temperate regions, little is known about bobcats (Lynx rufus) in the Sonoran Desert portion of their range, in part due to the difficulty of sampling an elusive carnivore in harsh desert environments. Here, we quantify habitat selection and evaluate diet of bobcats at Kofa National Wildlife Refuge, Arizona, USA, using multiple sampling techniques including GPS telemetry, camera traps, and DNA metabarcoding. RESULTS: Home ranges during the hot season were smaller than during the cool season. Camera trapping failed to yield a high enough detection rate to identify habitat occupancy trends but third-order resource selection from GPS-collar data showed a preference for higher elevations and rugged terrain at lower elevations. Diet composition consisted of a diverse range of available small prey items, including a higher frequency of avian prey than previously observed in bobcats. CONCLUSIONS: Desert bobcats in our study maintained smaller home ranges and primarily consumed smaller prey than their more northern relatives. This study illustrates the benefit of employing multiple, complementary sampling methods to understand the ecology of elusive species.

Apple endophyte community in relation to location, scion and rootstock genotypes and susceptibility to European canker.

European apple canker, caused by Neonectria ditissima, is a severe disease of apple. Achieving effective control is difficult with the currently available pesticides. Specific apple endophytes associated with cultivars may partially contribute to the cultivar response to the pathogen and thus could be used for disease management. We sought to determine whether the overall endophyte community differed among cultivars differing in their susceptibility to N. ditissima and to identify specific microbial groups associated with the susceptibility. Using Illumina MiSeq meta-barcoding, we profiled apple tree endophytes in 16 scion-rootstock combinations at two locations and quantified the relative contribution of scion, rootstock and location to the observed variability in the endophyte communities. Endophyte diversity was primarily affected by the orchard location (accounting for 29.4% and 85.9% of the total variation in the PC1 for bacteria and fungi, respectively), followed by the scion genotype (24.3% and 19.5% of PC2), whereas rootstock effects were small (<3% of PC1 and PC2). There were significant differences in the endophyte community between canker-resistant and -susceptible cultivars. Several bacterial and fungal endophyte groups had different relative abundance between susceptible and resistant cultivars. These endophyte groups included putative pathogen antagonists as well as plant pathogens. Their possible ecological roles in the N. ditissima pathosystem are discussed.

ensembleTax: an R package for determinations of ensemble taxonomic assignments of phylogenetically-informative marker gene sequences.

BACKGROUND: High-throughput sequencing of phylogenetically informative marker genes is a widely used method to assess the diversity and composition of microbial communities. Taxonomic assignment of sampled marker gene sequences (referred to as amplicon sequence variants, or ASVs) imparts ecological significance to these genetic data. To assign taxonomy to an ASV, a taxonomic assignment algorithm compares the ASV to a collection of reference sequences (a reference database) with known taxonomic affiliations. However, many taxonomic assignment algorithms and reference databases are available, and the optimal algorithm and database for a particular scientific question is often unclear. Here, we present the ensembleTax R package, which provides an efficient framework for integrating taxonomic assignments predicted with any number of taxonomic assignment algorithms and reference databases to determine ensemble taxonomic assignments for ASVs. METHODS: The ensembleTax R package relies on two core algorithms: taxmapper and assign.ensembleTax. The taxmapper algorithm maps taxonomic assignments derived from one reference database onto the taxonomic nomenclature (a set of taxonomic naming and ranking conventions) of another reference database. The assign.ensembleTax algorithm computes ensemble taxonomic assignments for each ASV in a data set based on any number of taxonomic assignments determined with independent methods. Various parameters allow analysts to prioritize obtaining either more ASVs with more predicted clade names or more robust clade name predictions supported by multiple independent methods in ensemble taxonomic assignments. RESULTS: The ensembleTax R package is used to compute two sets of ensemble taxonomic assignments for a collection of protistan ASVs sampled from the coastal ocean. Comparisons of taxonomic assignments predicted by individual methods with those predicted by ensemble methods show that conservative implementations of the ensembleTax package minimize disagreements between taxonomic assignments predicted by individual and ensemble methods, but result in ASVs with fewer ranks assigned taxonomy. Less conservative implementations of the ensembleTax package result in an increased fraction of ASVs classified at all taxonomic ranks, but increase the number of ASVs for which ensemble assignments disagree with those predicted by individual methods. DISCUSSION: We discuss how implementation of the ensembleTax R package may be optimized to address specific scientific objectives based on the results of the application of the ensembleTax package to marine protist communities. While further work is required to evaluate the accuracy of ensemble taxonomic assignments relative to taxonomic assignments predicted by individual methods, we also discuss scenarios where ensemble methods are expected to improve the accuracy of taxonomy prediction for ASVs.

Relative Weight of Organic Waste Origin on Compost and Digestate 16S rRNA Gene Bacterial Profilings and Related Functional Inferences.

Even though organic waste (OW) recycling via anaerobic digestion (AD) and composting are increasingly used, little is known about the impact of OW origin (fecal matters and food and vegetable wastes) on the end products' bacterial contents. The hypothesis of a predictable bacterial community structure in the end products according to the OW origin was tested. Nine OW treatment plants were selected to assess the genetic structure of bacterial communities found in raw OW according to their content in agricultural and urban wastes and to estimate their modifications through AD and composting. Two main bacterial community structures among raw OWs were observed and matched a differentiation according to the occurrences of urban chemical pollutants. Composting led to similar 16S rRNA gene OTU profiles whatever the OW origin. With a significant shift of about 140 genera (representing 50% of the bacteria), composting was confirmed to largely shape bacterial communities toward similar structures. The enriched taxa were found to be involved in detoxification and bioremediation activities. This process was found to be highly selective and favorable for bacterial specialists. Digestates showed that OTU profiles differentiated into two groups according to their relative content in agricultural (manure) and urban wastes (mainly activated sludge). About one third of the bacterial taxa was significantly affected by AD. In digestates of urban OW, this sorting led to an enrichment of 32 out of the 50 impacted genera, while for those produced from agricultural or mixed urban/agricultural OW (called central OW), a decay of 54 genera over 60 was observed. Bacteria from activated sludge appeared more fit for AD than those of other origins. Functional inferences showed AD enriched genera from all origins to share similar functional traits, e.g., chemoheterotrophy and fermentation, while being often taxonomically distinct. The main functional traits among the dominant genera in activated sludge supported a role in AD. Raw OW content in activated sludge was found to be a critical factor for predicting digestate bacterial contents. Composting generated highly predictable and specialized community patterns whatever the OW origin. AD and composting bacterial changes were driven by functional traits selected by physicochemical factors such as temperature and chemical pollutants.

MinION 16S datasets of a commercially available microbial community enables the evaluation of DNA extractions and data analyses.

New advances in sequencing technology and bioinformatics analysis tools have significantly supported the culture-independent analysis of complex microbial communities associated with environmental, plant, animal and human samples. However, previous work has shown that DNA extraction can have a major influence in the community profile. As such there is a constant need for new methods to efficiently and rapidly prepare and analyze DNA for microbiome research, especially in the case new and emerging technology like the Oxford Nanopore Technologies (ONT) MinION. A commercial standard was used, in triplicate, to evaluate three DNA extraction protocols, including two commercially available and one "in-house" DNA extraction method. All DNA extractions were done as per manufacturer's instructions and prepared with the same commercial ONT 16S sample preparation kit, prior to being analysed using MinION sequencing. Eight MinION 16S datasets of this microbial reference community were obtained. Reads were initially base called and demultiplexed using ONT's Guppy™ sequencing software (version 3.2.4), filtered using NanoFilt and then classified using Usearch. A set of R scripts are presented to process sintax files generated from Usearch and produce an OTU table that can be used for further analyses. All datasets were deposited into the SRA (NCBI) database. These datasets will allow future extraction kit comparisons using MinION sequencing since a standardize laboratory process using commercially available components, such as the MinION 16S sample preparation kit, microbial reference community and extraction kits, were used. The current ONT 16S workflow making use of the Epi2me agent only provides QC metrics and the ID's of the main genera identified and does not provide any tools currently for further downstream community comparison. The analyses scripts provided in the supplementary material will thus further enable the testing of new datasets against these reference sets and provide users the ability to compare their workflows with ours, thus standardizing comparisons and workflows.

Dynamics of marine bacterial biofouling communities after initial Alteromonas genovensis biofilm attachment to anti-fouling paint substrates.

To determine how bacterial communities succeed after the initial attachment of the bacterial biofilm adhesion using 16S rDNA meta-barcoding in plates coated with copper-based anti-fouling (AF) and non-AF (control) coatings as well as ambient seawater, coated plates were submerged in a marine environment in situ. Alteromonas genovensis (Gammaproteobacteria) in AF coating and Pacificibacter sp. (Alphaproteobacteria) in the control plate were initially abundant. In the AF coating, the abundance of A. genovensis decreased rapidly, whereas that of genus Phaeobacter (Alphaproteobacteria), Serratia (Gammaproteobacteria) and Cupriavidus (Betaproteobacteria) increased. Bacterial community in the control plate had a strong connection to pathogenic Vibrio spp. associated with the growth of invertebrates. Therefore, in the in situ AF coating experiment, A. genovensis accumulation was initially and intensively increased, and the bacteria responded to chemical antagonism, induced the proliferation of specific biofilm bacteria and influenced the interactions and recruitment of additional bacterial communities.

Molecular Screening of Microorganisms Associated with Discolored Wood in Dead European Beech Trees Suffered from Extreme Drought Event Using Next Generation Sequencing.

Drought events weaken trees and make them vulnerable to attacks by diverse plant pathogens. Here, we propose a molecular method for fast screening of microorganisms associated with European beech decline after an extreme drought period (2018) in a forest of Thuringia, Germany. We used Illumina sequencing with a recent bioinformatics approach based on DADA2 to identify archaeal, bacterial, and fungal ASVs (amplicon sequence variants) based on bacterial and archaeal 16S and fungal ITS genes. We show that symptomatic beech trees are associated with both bacterial and fungal plant pathogens. Although the plant pathogen sequences were detected in both discolored and non-discolored wood areas, they were highly enriched in the discolored wood areas. We show that almost each individual tree was associated with a different combination of pathogens. Cytospora spp. and Neonectria coccinea were among the most frequently detected fungal pathogens, whereas Erwinia spp. and Pseudomonas spp. were the dominant bacterial plant pathogens. We demonstrate that bacterial plant pathogens may be of major importance in beech decline.

Investigation of the piroplasm diversity circulating in wildlife and cattle of the greater Kafue ecosystem, Zambia.

BACKGROUND: Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. METHOD: To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. RESULTS: A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. CONCLUSION: We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.

First Gut Content Analysis of 4th Instar Midge Larvae (Diptera: Chronomidae) In Large-Scale Weirs Using a DNA Meta-Barcoding Approach.

Chironomidae larvae play an important role in the food chain of river ecosystems in Korea, where it is dominant. However, detailed information on the diet of Chironomidae larvae are still lacking. The purpose of this study was to identify the gut contents of 4th instar larvae of a Chironomidae inhabiting four large-scale weirs (Sejong Weir, Juksan Weir, Gangjeong-Goryeong Weir, and Dalseong Weir) using a DNA meta-barcoding approach. We found that dominant Operational Taxonomic Unit (OUT) was assigned to Paractinolaimus sp. (Nematoda), and the sub-dominant OTU was assigned to Dicrotendipes fumidus (Chironomidae). The most common OTUs among the individuals included phytoplankton, such as Tetrahymena sp., D. armatus, Pseudopediastrum sp., Tetradesmus dimorphus, Biddulphia tridens, and Desmodesmus spp. We calculated the selectivity index (E') and provided scientific evidence that Chironomidae larvae have a significant preference (E' > 0.5) for Desmodesmus armatus, E. minima, and T. dimorphus, while it does not show preference for other species found in its gut. Differences in physico-chemical factors, such as water quality, nutrients, Chl-a, and carbon concentrations, resulting from anthropogenic impacts (i.e., construction of large-scale weirs) as well as the particle size of prey organisms (small-sized single cell) and effects of chemicals (chemokinesis) could affect the feeding behavior of Chironomidae larvae.

Dark Ophiuroid Biodiversity in a Prospective Abyssal Mine Field.

The seafloor contains valuable mineral resources, including polymetallic (or manganese) nodules that form on offshore abyssal plains. The largest and most commercially attractive deposits are located in the Clarion Clipperton Fracture Zone (CCZ), in the eastern Pacific Ocean (EP) between Hawaii and Mexico, where testing of a mineral collection system is set to start soon [1]. The requirement to establish pre-mining environmental management plans has prompted numerous recent biodiversity and DNA barcoding surveys across these remote regions. Here we map DNA sequences from sampled ophiuroids (brittle stars, including post-larvae) of the CCZ and Peru Basin onto a substantial tree of life to show unprecedented levels of abyssal ophiuroid phylogenetic diversity including at least three ancient (>70 Ma), previously unknown clades. While substantial dark (unobserved) biodiversity has been reported from various microbial meta-barcoding projects [2, 3], our data show that we have considerably under-estimated the biodiversity of even the most conspicuous mega-faunal invertebrates [4] of the EP abyssal plain.