Permutational Multivariate Analysis Research Articles

Impact of Dietary Fiber Intake on Microbiota Diversity and Abundance in the Participants of the Full Plate Diet Trial

Background The gut microbiome is a highly diverse, metabolically active and complex community of microorganisms that has a vast number of biological roles which benefit the host. Diet, in particular fiber, has been shown to be a strong influencer of gut microbiota composition, diversity, and richness. Research conducted to investigate the impact of fiber intake on microbiota has involved mainly supplement interventions. Food interventions have not been shown to have a significant effect on these microbes which could be related to the lower level of fiber intake attained from the whole foods vs supplements. Objective The main aim was to assess if the improvement in daily fiber intake to 40g/day from whole foods, among the participants of the Full Plate Diet (FPD) trial, had a positive impact on the gut microbiota diversity and composition. Design Secondary analysis of the FPD, a randomised clinical trial. Setting Drayson Center, Loma Linda University, CA, USA. Methods Fecal samples were collected from 34 FPD participants (n=18 for intervention group receiving the health education; n=16 for the control group) at baseline and after the termination of the intervention using fecal occult blood. Then, after DNA extraction, 16S rRNA gene sequencing was performed to determine the relative abundance of taxa in a bacterial community, and to compare the changes in the overall microbial profile over time between the 2 study groups. For the statistical analysis, alpha diversity was estimated with the Shannon Diversity Index; the significance of alpha diversity differences was tested with linear mixed model. To estimate beta diversity across samples, we computed Bray-Curtis indices. Variation in community structure was assessed with permutational multivariate analyses of variance (PERMANOVA). For the purpose of measuring abundance, differential abundance testing was conducted. Results No significant differences in the Shannon Diversity Index were observed between baseline and post-intervention (P= 0.63). Furthermore, after summarizing operational taxonomic units (OTUs) abundances into Bray-Curtis dissimilarities, performing a pcoa ordination, and conducting permutational analysis of variance, we determined no significant differences in beta-diversity either (P = 0.99). No significant differences when it comes to the abundance of various taxa of microbiota were observed. Conclusion The increase of dietary fiber intake in the form of whole foods was not associated with a change in the abundance and diversity of microbiota in the participants of the FPD trial.

Diversity and community structure of mosquitoes (Diptera: Culicidae) in suburban, field, and forest habitats in Montréal, Québec, Canada

AbstractUnderstanding the medical and economic impacts of mosquitoes (Diptera: Culicidae) begins with knowing their natural history and distribution, including their association with habitat types, particularly those in which human activity is high. The effects of habitat on shaping the community structure of mosquitoes were studied across periurban habitats on the island of Montréal, Québec, Canada in 2014 and 2015. Mosquitoes were collected from 20 fixed sampling locations in suburban backyards, fields, and forests, using CO2-baited light-emitting diode encephalitis vector survey traps. A total of 184 607 mosquitoes were collected, representing eight genera and 35 species. Suburban, field, and forest sites had different communities of mosquitoes, but differences were not apparent among sites within similar habitat types in nonmetric multidimensional scaling ordinations and permutational multivariate analysis of variance. In both years, the greatest abundance of mosquitoes was collected from field habitat, and the highest species richness, from forests. Suburban sites consistently generated the lowest abundance and diversity. Nearly 75% of the total individuals collected were from three species:Aedes vexans(Meigen), 39%;Coquillettidia perturbans(Walker), 18%; andAedes canadensis(Theobald), 16%. This research shows that diverse communities of mosquitoes can be found in forests, fields, and backyards, yet the communities between forests differ from more open habitats. Our community analysis reveals that medically important species (e.g.,Culexsp.) are more commonly encountered in suburban backyards, yet overall mosquito nuisance potential is greater in forest and field habitats. This information highlights important patterns of mosquito abundance and species occurrence, vital for the development of management programmes.

Clinical Manifestations of Polycystic Ovary Syndrome and Associations With the Vaginal Microbiome: A Cross-Sectional Based Exploratory Study.

BackgroundPrevious studies suggest that the vaginal microbiome is associated with polycystic ovary syndrome (PCOS). However, the clinical manifestations of PCOS are heterogeneous. Whether the vaginal microbiome is related with different clinical symptoms was unknown.Materials and MethodsIn this cross-sectional study, 89 female patients with PCOS admitted to Zhongda Hospital (Nanjing, China) were included. Basic demographic information, health-related behaviors, clinical manifestations and sex hormone levels were comprehensively recorded for all patients. Vaginal swabs were acquired for microbiota sequencing of the V3–V4 region of the 16S rRNA gene.ResultsThe prevalence of bacterial vaginitis and vulvovaginal candidiasis was 15.7% and 13.5%, respectively, within the PCOS patients, which were the most important factors affecting the vaginal microbiome (permutational multivariate analysis of variance test, R2 = 0.108, P = 0.001). The vaginal microbiome was associated with specific clinical manifestations of PCOS, including acanthosis nigricans, intermenstrual bleeding, pregnancy history, testosterone level and anti-müllerian hormone level, with P values < 0.05. The abundance of Lactobacillus crispatus was higher (P = 0.010) while that of Lactobacillus iners was lower (P = 0.036) among PCOS patients with elevated testosterone levels. Other potential bacterial biomarkers were not statistically significant after adjusting for confounding factors. No evidence of associations of other common manifestations of PCOS, such as obesity and acne, with the vaginal microbiome was obtained.ConclusionVaginal bacterial species among PCOS patients with variable clinical manifestations, especially differences in testosterone levels, are distinct. Further studies are essential to investigate the microbiota and molecular mechanisms underpinning this disease.

Challenging the traceability of natural gold by combining geochemical methods: French Guiana example

Considering the high economic importance and worldwide distribution of gold, the ability to identify its provenance is critical and challenging to ensuring a responsible supply chain from deposit to consumer, especially within conflict-affected areas. Here we present an innovative approach to trace the provenance of natural gold from French Guiana through a combination of geochemical and statistical methods. This approach is divided in three steps and allows the identification of illicit gold, the certification of the declared gold origin coming from legal operators and, in some cases, the identification of unknown gold. French Guiana was chosen as a demonstration case of our approach because it is a well-known conflict-affected area where illegal mining is widespread. In the first step, we showed that the use of illegal Hg amalgamation can be easily revealed by looking the gold grain morphology with an optical microscope or by detecting the Hg with laser-induced breakdown spectroscopy that allow direct detection on the field. In the second and principal step, we demonstrated that a declared provenance of gold can be certify with a high degree of confidence by measuring the Ag content of a gold grain population and by checking it against a database with the Kolmogorov-Smirnov statistic. In the final step that allows the identification of the origin of unknown gold, we used (i) the content of minor elements (Cu and Hg), (ii) the identification of mineral inclusions and their relative proportions within samples and (iii) the trace element composition of natural gold grains determined by LA-ICP-MS and coupled with a permutational multivariate analysis of variance and a similarity percentage analysis. This method allows the identification of the provenance of 69% of the gold samples; the provenance of other gold populations (31%) cannot be identified because of their geological similarity with other groups. The traceability of natural gold can be guaranteeing with our innovative approach, in particular by certifying declared gold population provenance. Further challenges to be addressed will be the implementation of such approach in others conflict-affected regions to support the global supply chain of gold.

Stock delineation of striped snakehead, Channa striata using multivariate generalised linear models with otolith shape and chemistry data

Otoliths are commonly used to discriminate between fish stocks, through both elemental composition and otolith shape. Typical studies also have a large number of elemental compositions and shape measures relative to the number of otolith samples, with these measures exhibiting strong mean–variance relationships. These properties make otolith composition and shape data highly suitable for use within a multivariate generalised linear model (MGLM) framework, yet MGLMs have never been applied to otolith data. Here we apply both a traditional distance based permutational multivariate analysis of variance (PERMANOVA) and MGLMs to a case study of striped snakehead (Channa striata) in India. We also introduce the Tweedie and gamma distributions as suitable error structures for the MGLMs, drawing similarities to the properties of Biomass data. We demonstrate that otolith elemental data and combined otolith elemental and shape data violate the assumption of homogeneity of variance of PERMANOVA and may give misleading results, while the assumptions of the MGLM with Tweedie and gamma distributions are shown to be satisfied and are appropriate for both otolith shape and elemental composition data. Consistent differences between three groups of C. striata were identified using otolith shape, otolith chemistry and a combined otolith shape and chemistry dataset. This suggests that future research should be conducted into whether there are demographic differences between these groups which may influence management considerations. The MGLM method is widely applicable and could be applied to any multivariate otolith shape or elemental composition dataset.

Prediction of minimum postmortem submersion interval (PMSImin) based on eukaryotic community succession on skeletal remains recovered from a lentic environment

Although recent studies explored using microbial succession during decomposition to estimate the postmortem interval (PMI) and postmortem submersion interval (PMSI), there is currently no published research using aquatic eukaryotic community succession to estimate the minimum postmortem submersion interval (PMSImin). The goals of this study were to determine whether eukaryotic community succession occurs on porcine skeletal remains in a lentic environment, and, if so, to develop a statistical model for PMSImin prediction. Fresh porcine bones (rib N = 100, scapula N = 100) were placed in cages (10′’ x 10′’) attached to floatation devices and submerged in a fresh water lake (Crozet, VA), using waterproof loggers and a YSI Sonde to record temperature and water quality variables, respectively. In addition to baseline samples, one cage, containing five ribs and five scapulae, and water samples (500 mL) were collected approximately every 250 accumulated degree days (ADD). Nineteen sample cohorts were collected over a period of 5200 ADD (579 Days). Variable region nine (V9) of the 18S ribosomal DNA (rDNA) was amplified and sequenced using a dual-index strategy on the MiSeq FGx sequencing platform. Resulting sequences underwent quality control parameters and analysis in mothur v 1.42.3, R v 3.5.3, and R v 3.6.0. Permutational multivariate analysis of variance (PERMANOVA) revealed a significant difference in phylogenetic β-diversity among ribs, scapulae and water (p = 0.001) and among ADD (p ≤ 0.011), which was supported by distinct clustering of samples associated with each ADD in UniFrac distance based non-metric multidimensional scaling (NMDS) ordinations. Using similarity percentage (SIMPER) analysis of class and family level taxa, differences observed between bone types were attributed to Peronosporomycetes_cl, Eukaryota_unclassified, and Intramacronucleata (e.g., Armophorida), however these differences were not statistically significant. Alpha diversity revealed a non-linear increase in phylogenetic diversity with an increase in ADD. Random forest models for ribs and scapulae predicted PMSImin with an error rate within±104 days (937 ADD) and±63 days (564 ADD), respectively. In conclusion, this study suggests that eukaryotic succession is capable of predicting long term PMSImin in lentic systems.

Biopesticide Trunk Injection Into Apple Trees: A Proof of Concept for the Systemic Movement of Mint and Cinnamon Essential Oils.

The use of conventional pesticides is debated because of their multiple potential adverse effects on non-target organisms, human health, pest resistance development and environmental contaminations. In this setting, this study focused on developing alternatives, such as trunk-injected essential oil (EO)-based biopesticides. We analysed the ecophysiology of apple trees (Malus domestica) following the injection of Cinnamomum cassia and Mentha spicata nanoemulsions in the tree’s vascular system. Targeted and untargeted volatile organic compounds (VOCs) analyses were performed on leaf-contained and leaf-emitted VOCs and analysed through dynamic headspace–gas chromatography–mass spectrometry (DHS-GC-MS) and thermal desorption unit (TDU)-GC-MS. Our results showed that carvone, as a major constituent of the M. spicata EO, was contained in the leaves (mean concentrations ranging from 3.39 to 19.7 ng gDW–1) and emitted at a constant rate of approximately 0.2 ng gDW–1 h–1. Trans-cinnamaldehyde, C. cassia’s major component, accumulated in the leaves (mean concentrations of 83.46 and 350.54 ng gDW–1) without being emitted. Furthermore, our results highlighted the increase in various VOCs following EO injection, both in terms of leaf-contained VOCs, such as methyl salicylate, and in terms of leaf-emitted VOCs, such as caryophyllene. Principal component analysis (PCA) highlighted differences in terms of VOC profiles. In addition, an analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA) revealed that the VOC profiles were significantly impacted by the treatment. Maximum yields of photosystem II (Fv/Fm) were within the range of 0.80–0.85, indicating that the trees remained healthy throughout the experiment. Our targeted analysis demonstrated the systemic translocation of EOs through the plant’s vascular system. The untargeted analysis, on the other hand, highlighted the potential systemic acquired resistance (SAR) induction by these EOs. Lastly, C. cassia and M. spicata EOs did not appear phytotoxic to the treated trees, as demonstrated through chlorophyll fluorescence measurements. Hence, this work can be seen as a proof of concept for the use of trunk-injected EOs given the systemic translocation, increased production and release of biogenic VOCs (BVOCs) and absence of phytotoxicity. Further works should focus on the ecological impact of such treatments in orchards, as well as apple quality and production yields.

Recent changes in mountain hay meadows of high conservation value in eastern France

AbstractAimTo assess vegetation changes in mountain hay meadows of high conservation value over 15 years.LocationJura Mountains and Vosges Mountains (France).MethodsIn 2019, we resampled old phytosociological relevés (made from 2005 to 2009) in extensive mountain hay meadows. To detect shifts in composition from old to recent relevés, we performed a permutational multivariate analysis of variance, and visualized observed patterns using non‐metric multidimensional scaling on the Bray–Curtis dissimilarity matrix of the vegetation data. We tested for differences in taxonomic, functional and phylogenetic diversity metrics, CSR strategies and ecological indicator values using paired Wilcoxon–Mann–Whitney tests.ResultsNon‐metric multidimensional scaling paired with a permutational multivariate analysis of variance showed that, overall, old and recent plant communities were not different in the Vosges Mountains, contrary to the situation in the Jura Mountains. Wilcoxon–Mann–Whitney paired tests revealed that, in the Jura Mountains, plant communities underwent a significant decrease in taxonomic, functional and phylogenetic diversity, associated with an increase of ruderal and competitive strategies. Ecological indicator values for nutrient availability and defoliation tolerance both increased, suggesting an intensification of agricultural practices. In the Vosges Mountains, only species richness increased while the inverse Simpson index and Pielou evenness decreased.ConclusionsOur results show that plant diversity is not currently threatened in the studied grasslands in the Vosges Mountains. By contrast, in the studied grasslands in the Jura Mountains, plant diversity is declining. The situation in the Vosges shows that agricultural activities and biodiversity conservation can be reconciled. This situation should be further investigated to help resolve conflicts between agriculture and biodiversity conservation in semi‐natural grasslands.

Exploring prevalence of potential pathogens and fecal indicators in geographically distinct river systems through comparative metagenomics

Microbial communities are considered as vital members to reflect the health of a riverine system. Among them, pathogenic and fecal indicators imply health risks involved with potability of river water. The present study explores the diverse microbial communities, distribution pattern of potential pathogens, and fecal indicators between the geographically distinct Himalayan and Peninsular river systems of India. It also inquires into the environmental factors associated with community variance and distribution pattern of microbial indicators. The application of high-throughput amplicon sequencing approach unveiled significant demarcation (p < 0.004, Anosim R = 0.62) of samples suggesting unique microbial diversities in these two river sediments. Random forest analysis revealed Desulfobulbulus, PSB_M_3, and Opitutus in Himalayan, while DA101, Bacillus, and Streptomyces in the Peninsular as significant contributors to develop overall dissimilarity between the river systems. Permutational multivariate analysis of variance and co-occurrence network analysis were used to study the relationships between microbial taxa and environmental factors. Amongst the various studied environmental parameters, pH, K, Ca, Mg, Ba, and Al in the Himalayan and salinity, Na, temperature, and Th in the Peninsular significantly influenced shaping of distinct microbial communities. Furthermore, the potential pathogenic genera, including Flavobacterium, Clostridium, Arcobacter, Pseudomonas, and Bacillus were highly prevalent in both the river systems. Arcobacter, Clostridium, Acinetobacter, Bacteroides, and Caloramator were the prominent fecal indicators in these river systems. Our findings provide salient information about the crucial role and interplay between various environmental factors and anthropogenic influences in framing the microbiome of the distinct river systems in India. Moreover, assessing potential pathogenic and fecal indicators suggest the public health risk associated with untreated sewage discharge into these water sources. The detection of various F/S indicators and potentially pathogenic bacteria in Himalayan and Peninsular river systems emphasize the urgent need for future monitoring and management of major riverine systems in India.

Spatial and seasonal variation in physicochemical characteristics and phytoplankton in an estuary of a tropical delta system

The delta of the Parnaíba River has a set of highly dynamic, productive ecosystems located in the Amazon–semiarid interface environment on the coast of Brazil. Phytoplankton is a key element in the evaluation of aquatic environments, as it is extremely sensitive to the broad spatial and seasonal variability of masses of freshwater and the physicochemical conditions of the water. To evaluate spatial–seasonal variation in the hydrological characteristics and the phytoplankton community in relation to the saline gradient, three sampling campaigns were performed at eight points considering seasonal periods (dry, rainy and transition) and spatial distribution (Nearshore Turbid Zone [NTZ], Mixing Zone [MZ] and Tidal River Zone [TRZ]) during neap tides. Variability in the environmental characteristics and phytoplankton was tested using permutational multivariate analysis of variance, dissimilarities and correlations. The data revealed a strong spatial–seasonal​ influence throughout the entire length of the estuary, with a clear spatial gradient related to salinity. Thalassiosira subtilis was the bioindicator for the NTZ, Cyclotella meneghiniana for the MZ and Oocystis parva for the TRZ. Concentrations of chlorophyll a, suspended particulate matter and nitrite were the main variables affected by the two factors combined (seasonality and spatial distribution). Moreover, total dissolved solids, nitrate and nitrite were the variables that promoted favorable ecological conditions for the development of the phytoplankton community associated with seasonality, especially the growth of Cyclotella meneghiniana, which proved to be better adapted to these conditions and was considered a key species in the area studied.

Population structure, pathogenicity, and fungicide sensitivity of Colletotrichum siamense from different hosts in Hainan, China

AbstractAreca palm, rubber tree, and coffee are always planted adjacent to each other or intercropped with each other, and Colletotrichum siamense was found to be the dominant species of Colletotrichum from these crops in Hainan, China. To study the population structure, pathogenicity, and fungicide sensitivity of C. siamense from these three crops in Hainan, isolates were first identified by their morphological characteristics and multilocus phylogeny. Then both permutational multivariate analysis of variance (PERMANOVA) and pairwise FST analysis showed that the original host and geographical origin (counties) had significant effects on genetic variation in the C. siamense population, explaining 5.6% and 16.3% of genetic variation, respectively. There were significant genetic differentiations between coffee and rubber tree populations but the genetic differentiation was small (FST = 0.04), while significant differentiations were observed among all populations from different counties except those between Haikou and Chengmai. Pathogenicity analysis using artificial inoculation showed that isolates had significantly higher disease incidence and diseased lesion diameter on wounded leaves than on nonwounded leaves. However, the original host of an isolate and geographical origin did not significantly affect the pathogenicity of the C. siamense populations from these three hosts. In vitro tests showed that there were no significant differences in the sensitivity of C. siamense populations to carbendazim, prochloraz, difenoconazole, and propiconazole from different original hosts and geographical origins. The present study suggests that host specialization of C. siamense has not occurred yet in coffee, rubber tree, and areca palm.

Soil physicochemical properties shape distinct nematode communities in serpentine ecosystems

Serpentine ecosystems have unique plant communities that have overcome the chemical and physical challenges presented by these soils. However, such a specific soil fauna community has not been identified. It is presumed that such communities are affected directly by the chemical and physical properties of serpentine soils. Here, we focused on nematodes, which comprise a ubiquitous and an abundant group in soil fauna, for comparing their community structure in serpentine and in non-serpentine soils based on morphological approaches. Root biomass, Mg, and Ni were higher in serpentine habitats than non-serpentine ones. In contrast, the soil water content, NO3− and Ca/Mg ratio were higher at non-serpentine habitats than serpentine ones. Among the nematode community indices, the channel index, which indicates the degree of fungal participation in the decomposition channels of soil food webs, was greater in serpentine (94.5 ± 4.8) than in non-serpentine (44.8 ± 3.2) habitats, whereas the enrichment index, which indicates the abundance and activity of primary detrital consumers, was greater in non-serpentine (56.2 ± 3.7) than in serpentine (40.2 ± 2.9) habitats. A non-metric multidimensional scaling scatterplot showed that nematode communities clustered into both serpentine and non-serpentine habitats by a permutational multivariate analysis of variance. Ca/Mg ratio accounted for the most variation in nematode communities: 90 % of the variation between the sites by envfit. Furthermore, relative frequencies of nine taxa (Filenchus, Malenchus, Mesodorylaimus, Monhystera, Paratylenchus, Plectus, Prodesmodora, Teratocephalus, and Tripyla) were greater in serpentine than in non-serpentine habitats, and those of five taxa (Alaimus, Bastiania, Cervidellus, Diptherophora, and Rhabditidae) were greater in non-serpentine than in serpentine ones by indicator species analysis. Although our study was a small sample size and unit, our findings suggest that the structure of nematode communities reflects the physicochemical properties of serpentine soils.

A Comparison of the Colonic Microbiome and Volatile Organic Compound Metabolome of Anoplocephala perfoliata Infected and Non-Infected Horses: A Pilot Study.

Simple SummaryIn horses, tapeworm infection is associated with specific forms of colic (abdominal pain) that can be life-threatening without surgical treatment. There is growing evidence that intestinal parasites interact with the gut bacteria, and the consequences of these interactions may influence the ability of the host to resist infection and parasite-associated disease. We aimed to compare the intestinal bacteria and the gases produced by metabolic processes in the gut between horses that had varying levels of tapeworms and those with no tapeworm present. Overall, the diversity of gut bacteria was similar in horses with and without tapeworms. There were some decreases in beneficial bacteria in horses with tapeworms, indicating a possible negative consequence of infection. Intestinal gases correlated with some bacteria indicating their functionality and use as potential markers of active bacteria. Our study validates further research investigating tapeworm and gut bacteria interactions in the horse.Anoplocephala perfoliata is a common equine tapeworm associated with an increased risk of colic (abdominal pain) in horses. Identification of parasite and intestinal microbiota interactions have consequences for understanding the mechanisms behind parasite-associated colic and potential new methods for parasite control. A. perfoliata was diagnosed by counting of worms in the caecum post-mortem. Bacterial DNA was extracted from colonic contents and sequenced targeting of the 16S rRNA gene (V4 region). The volatile organic compound (VOC) metabolome of colonic contents was characterised using gas chromatography mass spectrometry. Bacterial diversity (alpha and beta) was similar between tapeworm infected and non-infected controls. Some compositional differences were apparent with down-regulation of operational taxonomic units (OTUs) belonging to the symbiotic families of Ruminococcaceae and Lachnospiraceae in the tapeworm-infected group. Overall tapeworm burden accounted for 7–8% of variation in the VOC profile (permutational multivariate analysis of variance). Integration of bacterial OTUs and VOCs demonstrated moderate to strong correlations indicating the potential of VOCs as markers for bacterial OTUs in equine colonic contents. This study has shown potential differences in the intestinal microbiome and metabolome of A. perfoliata infected and non-infected horses. This pilot study did not control for extrinsic factors including diet, disease history and stage of infection.

Bird Assemblage and Diversity along Different Habitat Types in a Karst Forest Area in Bulacan, Luzon Island, Philippines

Little is known on the effects of degradation and disturbance on bird assemblage in lowland karst forests in the Philippines. In this study, we determined diversity patterns and distribution of birds along the vertical strata in two karst forest fragments adjacent to and one reforestation area within an active limestone quarry area in Bulacan province, Luzon island. Surveys were conducted using mist nets set in the understory (0–3 m) and sub-canopy (4–10 m) from November 2013 to October 2016. A total of 617 individuals belonging to 63 species and 13 feeding guilds were recorded from a mist-netting effort of 654,264.8 mist-net hours (m2∙h); of these, 32 are Philippine endemics and six are threatened species. We recorded the highest number of species in the reforestation area, most of which are generalist and disturbance-tolerant species. Results from permutational multivariate analysis of variance (PERMANOVA) and non-metric multidimensional scaling (NMDS) indicate differences in bird assemblage between the three habitat types and between the two vertical strata within habitat types. Meanwhile, similarity percentage analyses and Mann-Whitney U tests showed that species and guilds that contributed to the observed dissimilarity in the two strata have higher capture rates in the understory. These observations provide insights into the effect of disturbance and habitat alteration in the vertical movement of birds, as evidenced by the shift of some arboreal species to the understory layer in more disturbed habitats. Understanding the behavior and habitat use of birds will, thus, help in identifying appropriate conservation measures to ensure proper resource partitioning among the different bird assemblages in fragmented habitats.

Impacts of mountain forest dieback on aquatic insect communities: A DNA metabarcoding analysis of samples from the Bavarian Forest

Mountain forests are increasingly affected by changes in rainfall and pest outbreaks, and the way forests are managed can have direct consequences for the streams flowing through forests. Aquatic macroinvertebrate communities are great bioindicators and changes to their ecosystem likely translates to changes in their overall composition and abundance. The Bavarian Forest National Park (SE Germany) is dominated by the Norway spruce (Picea abies) which, weakened by storms and other stressors, is susceptible to infestation by the European spruce bark beetle (Ips typographus). The result is large scale forest dieback in some areas, and forest management practices that lead to a predominance of three different forest types (hereafter habitats): Intact forest that is healthy and not impacted by Ips typographus; Disturbed forest that was impacted by Ips typographus, left to regenerate naturally, and from which deadwood was not removed; and Salvaged forest that was heavily impacted by Ips typographus with the same consequences, but from which deadwood was removed, creating a treeless forest meadow. Intact forest that is healthy and not impacted by Ips typographus; Disturbed forest that was impacted by Ips typographus, left to regenerate naturally, and from which deadwood was not removed; and Salvaged forest that was heavily impacted by Ips typographus with the same consequences, but from which deadwood was removed, creating a treeless forest meadow. To analyze the impacts these different forest management strategies have on the aquatic insect communities, 30 samples from 11 different streams were taken using kick-sampling. Operational taxonomic units (OTUs) were identified by bulk metabarcoding of dried, ground samples. A mock community was used to verify the setup and a DNA spike-in with three foreign OTUs was added to each sample to measure the biases introduced by PCR amplification and sequencing. Biases varied across samples, but spike-in OTUs produced a pattern indicating predictable biases which could lead to quantifiable metabarcoding results in the future. In total, 260 macroinvertebrate OTUs were identified. In comparison, a morphological study by Bojková et al. (2018) in the same region with twice the number of sampling sites collected 194 taxa in the same month as our samples. This underlines the potential for metabarcoding in evaluating species richness. Species richness was high across all habitats. A significant difference between the forest conditions was detected: The number of detected Diptera OTUs was lowest in disturbed habitats (55) and highest in salvaged areas (73). A permutational multivariate analysis of variance (PERMANOVA) indicated that habitat (i.e., intact, disturbed, salvaged) had an effect on the observed OTU distribution (9.2%), but that the stream catchment had a much larger effect (39.3%) regardless of the habitat. Our findings indicate that forest management can affect stream macroinvertebrate communities, and that this was most pronounced for the Diptera, a group for which DNA metabarcoding is particularly well suited because of their small size and high diversity.

Extraction kits could affect the interpretation of metabarcoding results of sediment samples taken around salmon farms

Differences in PCR strategies and errors, sequencing errors and methods used for extractions affect sequence data and potentially its interpretation. These effects could vary based on the target fragments, which are also influenced by limitations of incomplete databases. In this study, we tested the effects of two different proprietary DNA extraction kits on sediment samples, for the purposes of benthic monitoring of salmon farms. The levels of organic enrichment at farms show a gradient from cage edge to more distant locations. The effects of enrichment on benthic communities can be established with metabarcoding. We collected samples at three salmon farms in Scotland, at varying distances from cage edge. The sediments underneath two of the farms was fine while the sediment under the other was coarse, with a larger mean particle size. We extracted the samples with two different kits, each using a different mass of sediment – Qiagen DNeasy PowerSoil Pro (0.5 g) and Qiagen DNeasy PowerMax (5 g). We then subjected each extract to three independent PCRs targeting 16S (bacterial) and CO1 (eukaryotic) fragments. The PCR products of samples and blanks were sequenced with an Illumina MiSeq instrument on a single run. We denoised the sequenced data using DADA2 and rarefied it before analysis (Callahan et al. 2016). The 16S data was annotated against the seven-level SILVA database (Quast 2012). We collated this data at ‘Family’ level. The CO1 data was filtered to remove Amplicon Sequence Variants (ASVs) present in only one sample and ASVs with a frequency of less than ten reads across all samples. The read count data of family level 16S and ASVs of CO1 were transformed and converted to Bray-Curtis dissimilarity matrices (Bray and Curtis 1957). A permutational multivariate analysis of variance was carried out. We also ordinated these data with non-metric multi-dimensional scaling. 474 bacterial families and 3380 eukaryotic ASVs were included in the analysis. The samples extracted with both kits demonstrated a gradient based on distance from cage edge. This gradient in sampling stations was observed with both the 16S and CO1 markers. Data from both markers and kits showed a greater distinction between cage edge stations and more distant stations in the farms characterised by fine sediment. The two extraction kits showed similar trends but differed in their results. The 16S data showed a separation of samples by extraction kit along the y-axis. PowerMax extractions were associated with higher values on the y-axis (Fig. 1). The multivariate analysis of variance of the 16S data showed that extraction kit contributes to approximately 7% (p&amp;lt;0.001) of variation in data. The CO1 ASV data also showed a grouping of samples of both kits along the x-axis on the basis of distance from the farm (Fig. 1). The CO1 data showed that extraction kits contribute to about 5% (p&amp;lt;0.001) of the variation. The results of the two extraction kits were more similar to each other with the CO1 marker than with 16S. The greater axes values and grouping in the CO1 ordination, indicate that it is able to split farms and distances better than 16S. We show that both extraction kits demonstrated a gradient according to distance from the cage edge. However, there was a systematic difference between the extraction kits. Variability due to kit was greater with the 16S marker despite it including fewer bacterial families than CO1 ASVs. We recommend that the same extraction kit be used to develop protocols for monitoring of fish farms with metabarcoding. Though both kits demonstrate the same major trend, subtle differences may not be distinguished. These variations between the kits could influence the results and interpretation of metabarcoding.

Microbiomics, Metabolomics, Predicted Metagenomics, and Hepatic Steatosis in a Population-Based Study of 1,355 Adults.

Previous small studies have appraised the gut microbiome (GM) in steatosis, but large-scale studies are lacking. We studied the association of the GM diversity and composition, plasma metabolites, predicted functional metagenomics, and steatosis. This is a cross-sectional analysis of the prospective population-based Rotterdam Study. We used 16S ribosomal RNA gene sequencing and determined taxonomy using the SILVA reference database. Alpha diversity and beta diversity were calculated using the Shannon diversity index and Bray-Curtis dissimilarities. Differences were tested across steatosis using permutational multivariate analysis of variance. Hepatic steatosis was diagnosed by ultrasonography. We subsequently selected genera using regularized regression. The functional metagenome was predicted based on the GM using Kyoto Encyclopedia of Genes and Genomes pathways. Serum metabolomics were assessed using high-throughput proton nuclear magnetic resonance. All analyses were adjusted for age, sex, body mass index, alcohol, diet, and proton-pump inhibitors. We included 1,355 participants, of whom 472 had steatosis. Alpha diversity was lower in steatosis (P=1.1∙10-9 ), and beta diversity varied across steatosis strata (P=0.001). Lasso selected 37 genera of which three remained significantly associated after adjustment (Coprococcus3: β=-65; Ruminococcus Gauvreauiigroup: β=62; and Ruminococcus Gnavusgroup: β=45, Q-value=0.037). Predicted metagenome analyses revealed that pathways of secondary bile-acid synthesis and biotin metabolism were present, and D-alanine metabolism was absent in steatosis. Metabolic profiles showed positive associations for aromatic and branched chain amino acids and glycoprotein acetyls with steatosis and R. Gnavusgroup, whereas these metabolites were inversely associated with alpha diversity and Coprococcus3. We confirmed, on a large-scale, the lower microbial diversity and association of Coprococcus and Ruminococcus Gnavus with steatosis. We additionally showed that steatosis and alpha diversity share opposite metabolic profiles.

Maternal gut microbiota reflecting poor diet quality is associated with spontaneous preterm birth in a prospective cohort study

A processed diet, high in fat and low in fiber, is associated with differences in the gut microbiota and adverse health outcomes in humans; however, little is known about the diet-microbiota relation and its impact on pregnancy. Spontaneous preterm birth (SPTB), a pregnancy outcome with serious short- and long-term consequences, occurs more frequently in black and in obese women in the United States. In a prospective, case-control sample matched for race and obesity (cases=16, controls=32), we compared the fecal gut microbiota, fecal and plasma metabolites, and diet in the late second trimester. We hypothesized that a Western diet would be associated with reduced microbiota richness and a metabolic signature predicting incidence of SPTB. The fecal microbiota was characterized by 16S-tagged sequencing and untargeted metabolomics was used to analyze both plasma and fecal metabolites. Wilcoxon's rank-sum test was used for the comparison of microbiota genera, α-diversity, fecal and plasma metabolites, and dietary variables between term and SPTB. β-Diversity was analyzed using permutational multivariate ANOVA, and metabolite associations were assessed by module analysis. A decrease in α-diversity was strongly associated with the development of SPTB, especially in the taxonomic class of Betaproteobacteria. Of 824 fecal metabolites, 22 metabolites (mostly lipids) differed between cases and controls (P<0.01), with greater DHA (22:6n-3) and EPA (20:5n-3) in cases [false discovery rate (FDR)<0.2]. The most significant fecal metabolite module (FDR-adjusted P=0.008) was dominated by DHA and EPA. Dietary saturated fat (primarily palmitate) intake was greater in cases (31.38±7.37 compared with 26.08±8.62 g, P=0.045) and was positively correlated with fecal DHA and EPA (P<0.05). Reduced α-diversity of the gut microbiota and higher excretion of omega-3 (n-3) fatty acids in stool may provide a novel biomarker signature predicting SPTB in women with a low-fiber, high-fat diet. Further investigation of these markers in a larger sample is needed for validation.

Parametric fitting and morphometric analysis of 3D open curves based on discrete cosine transform

Fourier transform methods are usually adopted to fit 2D closed curves representing samples’ profiles to be studied in morphometric analysis. As for problems concerning 3D open curves, landmark-based methods are widely used. In this paper, a parametric method based on discrete cosine transform (DCT) is proposed to serve as a morphometric tool for 3D open curves. DCT transforms real signal (coordinates) into a combination of cosine functions. DCT describes the shape of a curve with coefficients generated from the fitting curve. Four examples are introduced to be fitted with DCT. The first example is 3D spiral curves with different shapes, added random disturbances to make this model more general. A curve alignment is also utilized to eliminate the non-shape effect. The other three examples of suture curves abstracted from 3D human skulls on which semilandmarks and landmarks are aligned with General Procrustes Analysis (GPA) to eliminate the effect brought by location, size, and orientation. These 3D curves with different diagnoses are matched with DCT. Coefficients generated in the fitting result are analyzed with between-group principal component analysis (bgPCA) and one-way permutational multivariate analysis of variance (PERMANOVA). Different groups of four examples are separated and present significant differences in the results of one-way PERMANOVA. Statistical analyses demonstrate that DCT is promising in morphometric analysis of 3D open curves.

Antidepressants fluoxetine and amitriptyline induce alterations in intestinal microbiota and gut microbiome function in rats exposed to chronic unpredictable mild stress

Antidepressant medications are known to modulate the central nervous system, and gut microbiota can play a role in depression via microbiota–gut–brain axis. But the impact of antidepressants on gut microbiota function and composition remains poorly understood. Thus this study assessed the effect of serotonin reuptake inhibitor antidepressant fluoxetine (Flu) and tricyclic antidepressant amitriptyline (Ami) administration on gut microbiota composition, diversity, and species abundance, along with microbial function in a chronic unpredictable mild stress (CUMS)-induced depression rat model. Oral administration of Ami and Flu significantly altered the overall gut microbiota profile of CUMS-induced rats, as assessed using the permutational multivariate analysis of variance test. At the phylum level, 6-week of antidepressant treatment led to a decreased Firmicutes/Bacteroidetes ratio due to an enhanced Bacteroidetes and reduced Firmicutes relative abundance. Flu was more potent than Ami at altering the Firmicutes and Bacteroidetes levels in the CUMS rats. At the family level, both antidepressants significantly increased the abundance of Porphyromonadaceae. However, an increased Bacteroidaceae level was significantly associated with Ami, not Flu treatment. Furthermore, at the genus level, an increase in the relative abundance of Parabacteroides, Butyricimonas, and Alistipes was observed following Ami and Flu treatment. Subsequent metagenomics and bioinformatics analysis further indicated that Ami and Flu likely also modulated metabolic pathways, such as those involved in carbohydrate metabolism, membrane transport, and signal transduction. Additionally, both antidepressants affected antibiotic resistome, such as for aminoglycoside (aph3iiiA), multidrug (mdtK, mdtP, mdtH, mdtG, acrA), and tetracycline (tetM) resistance in CUMS rats. These data clearly illustrated the direct impact of oral administration of Flu and Ami on the gut microbiome, thus set up the foundation to reveal more insights on the therapeutic function of the antidepressants and their overall contribution to host health.