Hasil untuk "Microbial ecology"

Ashish Adhikari, Nattapol Kraisitudomsook, Krista L. Bonfantine et al.

Background Wildland fires are increasing in both frequency and severity in many areas globally. Smoke from wildland fires (wildfires and prescribed burns), as well as agricultural burning, releases not only pollutants but also viable microorganisms, including pathogens capable of long-distance dispersal, potentially posing unrecognized risks to human, animal, and plant health. Objectives This scoping review synthesizes knowledge about pathogenic microbial dispersal in smoke from wildland fires, identifies gaps in pathogen ecology and epidemiology, and outlines research priorities in a One Health framework. Methods This review followed the Arksey & O’Malley framework with PRISMA-ScR guidance, using systematic searches in PubMed, Google Scholar, and grey literature sources (USDA Forest Service, World Health Organization, U.S. Environmental Protection Agency). After screening and applying inclusion criteria, 36 studies were retained that addressed microbial transport, viability, and disease associated with wildland fire smoke. Results There is evidence that wildland fire smoke can aerosolize diverse microbial assemblages, including pathogenic fungi such as Coccidioides and Puccinia, and bacteria capable of forming heat-resistant spores. If microbes can remain viable in smoke across greater distances, there would be the possibility of long-distance dispersal while suspended in smoke plumes. However, data about infection outcomes, dose–response relationships, and host susceptibility are lacking. Current wildland fire smoke surveillance focuses almost exclusively on abiotic pollutants, leaving microbial risks largely ignored. Conclusions A One Health approach integrates fire ecology, aerobiology, microbiology, and epidemiology across host species. After determining how important the role of dispersal in smoke is for human, animal, and plant health, priority actions may include improving pathogen viability sampling, incorporating microbial monitoring into smoke surveillance networks, and developing predictive models to assess health and ecological risks.

Kathryn M Ellwood, Ashley E Kramer, Aditya Dutta

Eggs are a globally important food source and integral to optimal poultry production. Understanding the microbial ecology of the hen reproductive tract is essential for improving both food safety and reproductive efficiency. While the oviduct has been shown to harbor a continuous microbial community, this study is the first to demonstrate the presence of microbiota on the hen ovary surface, suggesting that the ovary is an extension of the oviductal microbial continuum. In this study, the ovarian and oviductal microbiomes of white-leghorn hens from mid-lay (high laying) and post-lay (lower laying) cohorts were analyzed. Using 16S rRNA sequencing, we identified significant shifts in reproductive tract microbiota between 9- and 18-month-old hens, coinciding with changes in lay performance. Several differentially abundant genera, including Acinetobacter, Ligilactobacillus, Bacillus, and Akkermansia, are known to modulate steroid hormone metabolism, with age-related abundance changes suggesting potential effects on hormone-driven reproductive processes. Other genera such as Ruminococcus_torques_group, Mucispirillum, and Fusobacterium—not traditionally associated with reproductive hormone pathways—may influence laying efficiency through their roles in mucin degradation, immune modulation, and inflammation. Notably, Turicibacter, newly identified on the ovary, increased with age and negatively correlated with lay performance, raising questions about its role in bile acid metabolism and stress response within the hen reproductive tract. Collectively, these findings highlight the ovary as an active microbial niche influenced by age and suggest that both hormone-associated and mucosal-interactive microbes contribute to lay dynamics. This work opens new avenues for probiotic strategies targeting key genera to support hen fertility and egg production across the productive lifespan.

Marcelle L. Johnson, Dieke Boezen, Alexey A. Grum-Grzhimaylo et al.

Background: Multipartite viruses individually package their multiple genome segments into virus particles, necessitating the transmission of multiple virus particles for effective viral spread. This dependence poses a cost in the form of reduced transmission compared to monopartite viruses, which only have a single genome segment. The notable cost of a multipartite genome organization has spurred debate on why multipartite viruses are so common among plant viruses, including a search for benefits associated with this organizational form. Methods: We investigated the costs and benefits of multipartite viruses with three approaches. First, we reanalyzed dose–response data to measure the cost of multipartition to between-host transmission for multipartite viruses. Second, we developed a simulation model to explore when the sharing of viral gene products between cells is beneficial. Third, we tested whether multipartite viruses have a broad host range by estimating the host range for plant viruses using metagenomics data. Results: We find that the observed cost to transmission exceeds theoretical predictions. We predict that a virus gene-product-sharing strategy only confers benefits under limited conditions, suggesting that this strategy may not be common. Our results suggest that multipartite and segmented viruses have broader host ranges than monopartite viruses. Conclusions: Our analyses also suggest there is limited evidence for the costs and benefits of a multipartite organization, and we argue that the diversity of multipartite virus–host systems demands pluralistic explanatory frameworks.

M.-C. Leewis, M.-C. Leewis, M.-C. Leewis et al.

<p>Phytoremediation can be a cost-effective method of restoring contaminated soils using plants and associated microorganisms. Most studies follow the impacts of phytoremediation solely across the treatment period and have not explored long-term ecological effects. In 1995, a phytoremediation study was initiated near Fairbanks, Alaska, to determine how the introduction of annual grasses and/or fertilizer would influence degradation of petroleum hydrocarbons (PHCs). After 1 year, grass and/or fertilizer-treated soils showed greater decreases in PHC concentrations compared to untreated plots. The site was then left for 15 years with no active site management. In 2011, we re-examined the site to explore the legacy of phytoremediation on contaminant disappearance, as well as on plant and soil microbial ecology. We found that the recruited vegetation and the current bulk soil microbial community structure and functioning were all heavily influenced by initial phytoremediation treatment. The number of diesel-degrading microorganisms (DDMs) was positively correlated with the percentage cover of vegetation at the site, which was influenced by initial treatment. Even 15 years later, the initial use of fertilizer had significant effects on microbial biomass, community structure, and activity. We conclude that phytoremediation treatment has long-term, legacy effects on the plant community, which, in turn, impact microbial community structure and functioning. It is therefore important to consider phytoremediation strategies that not only influence site remediation rates in the short-term but also prime the site for the restoration of vegetation over the long-term.</p>

Raman P. Nagarajan, Leigh Sanders, Natalie Kolm et al.

AbstractEnvironmental DNA (eDNA) methods complement traditional aquatic monitoring surveys and are especially advantageous for rare and listed species to detect spatial and temporal distribution patterns. However, improvements in ease of use and portability could increase the utility of eDNA methods, leading to more widespread application, including expanding its role in management decision‐making processes. We describe the development of an eDNA detection assay for delta smelt (Hypomesus transpacificus), an endangered fish in the San Francisco Estuary, using SHERLOCK (Specific High‐Sensitivity Enzymatic Reporter Unlocking). SHERLOCK is a clustered regularly interspaced short palindromic repeats (CRISPR)‐based diagnostic tool with the ability to detect species‐specific genetic variants, making it ideal for genetic‐based taxonomic identification of any organism. Because of its high sensitivity and specificity, SHERLOCK is adaptable to eDNA detection in water samples. Here, we describe adaptation of a delta smelt SHERLOCK assay for use with estuarine water eDNA samples. This version of the assay exhibits increased sensitivity compared to the original delta smelt SHERLOCK protocol (new limit of detection approximately three copies per reaction compared to ~300 in original assay) and successfully detected delta smelt eDNA in both experimental and natural contexts. Overall, our results demonstrate that SHERLOCK eDNA detection offers managers an alternative, isothermal methodology, and highlights some challenges for detection of rare, endangered species at low abundance.

Felipe Werle Vogel, Nicolas Carlotto, Zhongzhong Wang et al.

Lignocellulosic biomass is a promising substrate for anaerobic digestion (AD) in renewable energy generation but presents a significant challenge during the hydrolysis stage of conventional AD due to the recalcitrant nature of this biomass substrate. Rumen fluid is often employed as a bioaugmentation seed to enhance hydrolysis in the AD of lignocellulosic substrates due to its richness in hydrolytic bacteria. However, using rumen fluid to enhance AD processes presents substantial hurdles, including the procurement difficulties associated with rumen fluid and ethical concerns. In this study, the fecal microbiota of 10 African ruminant species from a large zoological park (Bioparc) in Valencia, Spain, were studied using 16S rRNA gene amplicon sequencing. In this study, the fecal microbiota of 10 African ruminant species from a large zoological park (Bioparc) in Valencia, Spain, were studied using 16S rRNA gene amplicon sequencing. The investigation revealed potential similarities between the fecal microbiota from the African ruminants’ and cows’ rumen fluids, as suggested by theoretical considerations. Although direct comparative analysis with cow rumen fluid was not performed in this study, the theoretical framework and existing literature hint at potential similarities. According to our results, the Impala, Blesbok, Dikdik and Bongo ruminant species stood out as having the greatest potential to be used in bioaugmentation strategies. Key genera such as <i>Fibrobacter</i>, <i>Methanobrevibacter</i>, and <i>Methanosphaera</i> in Impala samples suggested Impala rumen fluid’s involvement in cellulose breakdown and methane production. Blesbok and Dikdik exhibited a high abundance of Bacillus and <i>Atopostipes</i>, potentially contributing to lignin degradation. The richness of <i>Prevotellaceae</i> and <i>Rikenellaceae</i> in the Bongo fecal samples is probably associated with structural carbohydrate degradation. Taken together, our results shed light on the microbial ecology of the gut contents of a whole set of Bovidae ruminants and contribute to the potential application of gut microbiota in AD.

Nize Otaru, Nize Otaru, Anna Greppi et al.

Tryptamine, a neuromodulator derived from tryptophan, has been shown to significantly impact the host gut homeostasis through its production by the gut microbiota. However, the characterization of tryptamine-producing gut bacteria remains limited, the factors regulating tryptamine production largely unexplored, and its effects on the rest of the gut microbial community unknown. In this study, we screened 13 intestinal strains closely related to known tryptamine producers, characterized their production kinetics, and evaluated whether tryptophan decarboxylation to tryptamine contributes to acid stress tolerance, as shown in other amino acid-dependent acid tolerance systems. We also examined the impact of tryptamine on the composition and function of four healthy human gut microbiota by conducting 48-h ex vivo fecal batch fermentations. To complement the ex vivo experiments, we tested the effect of tryptamine exposure (range: 0.5–8 mM) on the growth of 18 intestinal strains. We identified tryptamine production in five taxa, i.e., Enterocloster asparagiformis, Blautia hansenii, Clostridium nexile, Clostridium sporogenes, and Ruminococcus gnavus, with R. gnavus DSM 108212 accumulating up to 3.4 mM tryptamine after 48 h. An increased tryptophan concentration led to higher tryptamine production. However, tryptamine production was not promoted at low pH and may not protect cells from acid-induced cellular damage. Exposing gut microbial communities to 2.4 mM tryptamine caused mild changes in gut microbiota function and composition. All donors showed reduced carbohydrate consumption after 5 h, leading to donor-specific alterations of short-chain fatty acids (SCFAs) (i.e., propionate, acetate, butyrate) and branched-chain fatty acids (BCFAs) (i.e., isobutyrate and isovalerate) after 48 h. Tryptamine also induced a mild change of community structure, with a consistent reduction in the phylum Bacteroidota as well as amplicon sequence variants (ASVs) related to the genera Bacteroides, Blautia, and Faecalibacterium. We confirmed the sensitivity of Bacteroides and Faecalibacterium strains in vitro at concentrations of 2 mM and above. Multiple gut commensals remained unaffected when exposed to 8 mM tryptamine. Taken together, our findings demonstrated that intestinal bacteria-derived tryptamine is a bioactive molecule that not only alters host homeostasis locally but also modulates the physiology of gut microbial communities. The specific mechanism through which tryptamine exerts its inhibitory effects on specific gut microbes while leaving others unaffected remains to be elucidated.

Nina Król, Lidia Chitimia-Dobler, Gerhard Dobler et al.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. <i>Orthoflavivirus encephalitidis</i>), in Europe, it is transmitted by ticks like <i>Ixodes ricinus</i> and <i>Dermacentor reticulatus</i>. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, <i>I. ricinus</i> (<i>n</i> = 4784; 56.9%) was predominant, followed by <i>D. reticulatus</i> (<i>n</i> = 3506; 41.7%), <i>Haemaphysalis concinna</i> (<i>n</i> = 108; 1.3%), and <i>I. frontalis</i> (<i>n</i> = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

Elisavet Stavropoulou, Nikolaos Remmas, Chrysoula (Chrysa) Voidarou et al.

Honey’s antibacterial activity has been recently linked to the inhibitory effects of honey microbiota against a range of foodborne and human pathogens. In the current study, the microbial community structure of honey samples exerting pronounced antimicrobial activity was examined. The honey samples were obtained from different geographical locations in Greece and had diverse pollen origin (fir, cotton, fir–oak, and <i>Arbutus unedo</i> honeys). Identification of honey microbiota was performed by high-throughput amplicon sequencing analysis, detecting 335 distinct taxa in the analyzed samples. Regarding ecological indices, the fir and cotton honeys possessed greater diversity than the fir–oak and <i>Arbutus unedo</i> ones. <i>Lactobacillus kunkeei</i> (basionym of <i>Apilactobacillus kun-keei</i>) was the predominant taxon in the fir honey examined. <i>Lactobacillus</i> spp. appeared to be favored in honey from fir-originated pollen and nectar since lactobacilli were more pronounced in fir compared to fir–oak honey. <i>Pseudomonas</i>, <i>Streptococcus</i>, <i>Lysobacter</i> and <i>Meiothermus</i> were the predominant taxa in cotton honey, whereas <i>Lonsdalea</i>, the causing agent of acute oak decline, and <i>Zymobacter</i>, an osmotolerant facultative anaerobic fermenter, were the dominant taxa in fir–oak honey. Moreover, methylotrophic bacteria represented 1.3–3% of the total relative abundance, independently of the geographical and pollen origin, indicating that methylotrophy plays an important role in honeybee ecology and functionality. A total of 14 taxa were identified in all examined honey samples, including bacilli/anoxybacilli, paracocci, lysobacters, pseudomonads, and sphingomonads. It is concluded that microbial constituents of the honey samples examined were native gut microbiota of melliferous bees and microbiota of their flowering plants, including both beneficial bacteria, such as potential probiotic strains, and animal and plant pathogens, e.g., <i>Staphylococcus</i> spp. and <i>Lonsdalea</i> spp. Further experimentation will elucidate aspects of potential application of microbial bioindicators in identifying the authenticity of honey and honeybee-derived products.

Pascal Laforge, Pascal Laforge, Pascal Laforge et al.

Introduction: A thorough understanding of the microbial ecology within the swine value chain is essential to develop new strategies to optimize the microbiological quality of pork products. To our knowledge, no study to date has followed the microbiota through the value chain from live farm animals to the cuts of meat obtained for market. The objective of this study is to evaluate how the microbiota of pigs and their environment influence the microbial composition of samples collected throughout the value chain, including the meat plant and meat cuts.Method and results: Results from 16S rDNA sequencing, short-chain fatty acid concentrations and metabolomic analysis of pig feces revealed that the microbiota from two farms with differing sanitary statuses were distinctive. The total aerobic mesophilic bacteria and Enterobacteriaceae counts from samples collected at the meat plant after the pre-operation cleaning and disinfection steps were at or around the detection limit and the pigs from the selected farms were the first to be slaughtered on each shipment days. The bacterial counts of individual samples collected at the meat plant did not vary significantly between the farms. Alpha diversity results indicate that as we move through the steps in the value chain, there is a clear reduction in the diversity of the microbiota. A beta diversity analysis revealed a more distinct microbiota at the farms compared to the meat plant which change and became more uniform as samples were taken towards the end of the value chain. The source tracker analysis showed that only 12.92% of the microbiota in shoulder samples originated from the farms and 81% of the bacteria detected on the dressed carcasses were of unknown origin.Discussion: Overall, the results suggest that with the current level of microbial control at farms, it is possible to obtain pork products with similar microbiological quality from different farms. However, broader studies are required to determine the impact of the sanitary status of the herd on the final products.

S. Tringe, P. Hugenholtz

B. Koskella

There is longstanding interest in studying microbial communities below ground, while little attention has historically been paid to the above ground portions of plants (the phyllosphere). The phyllosphere has been estimated to make up around 60% of the biomass across all taxa on Earth, making it a key habitat for microbial organisms. The more we study these complex and dynamic communities, the more we come to realize their importance to the health of plant hosts. Overall, the phyllosphere is proving to be both an important microbial habitat and a tractable model system for asking questions in microbial ecology and evolution.

E. Angelakis, F. Armougom, M. Million et al.

Teresita M Porter, Mehrdad Hajibabaei

Multi-marker metabarcoding is increasingly being used to generate biodiversity information across different domains of life from microbes to fungi to animals such as for molecular ecology and biomonitoring applications in different sectors from academic research to regulatory agencies and industry. Current popular bioinformatic pipelines support microbial and fungal marker analysis, while ad hoc methods are often used to process animal metabarcode markers from the same study. MetaWorks provides a harmonized processing environment, pipeline, and taxonomic assignment approach for demultiplexed Illumina reads for all biota using a wide range of metabarcoding markers such as 16S, ITS, and COI. A Conda environment is provided to quickly gather most of the programs and dependencies for the pipeline. Several workflows are provided such as: taxonomically assigning exact sequence variants, provides an option to generate operational taxonomic units, and facilitates single-read processing. Pipelines are automated using Snakemake to minimize user intervention and facilitate scalability. All pipelines use the RDP classifier to provide taxonomic assignments with confidence measures. We extend the functionality of the RDP classifier for taxonomically assigning 16S (bacteria), ITS (fungi), and 28S (fungi), to also support COI (eukaryotes), rbcL (eukaryotes, land plants, diatoms), 12S (fish, vertebrates), 18S (eukaryotes, diatoms) and ITS (fungi, plants). MetaWorks properly handles ITS by trimming flanking conserved rRNA gene regions as well as protein coding genes by providing two options for removing obvious pseudogenes. MetaWorks can be downloaded from https://github.com/terrimporter/MetaWorks and quickstart instructions, pipeline details, and a tutorial for new users can be found at https://terrimporter.github.io/MetaWorksSite.

M. Arias-Andrés, K. Rojas‐Jimenez, H. Grossart

Abstract Microplastics (MP) provide a unique and extensive surface for microbial colonization in aquatic ecosystems. The formation of microorganism-microplastic complexes, such as biofilms, maximizes the degradation of organic matter and horizontal gene transfer. In this context, MP affect the structure and function of microbial communities, which in turn render the physical and chemical fate of MP. This new paradigm generates challenges for microbiology, ecology, and ecotoxicology. Dispersal of MP is concomitant with that of their associated microorganisms and their mobile genetic elements, including antibiotic resistance genes, islands of pathogenicity, and diverse metabolic pathways. Functional changes in aquatic microbiomes can alter carbon metabolism and food webs, with unknown consequences on higher organisms or human microbiomes and hence health. Here, we examine a variety of effects of MP pollution from the microbial ecology perspective, whose repercussions on aquatic ecosystems begin to be unraveled.

F. De Filippis, E. Parente, D. Ercolini

B. Bohannan, R. Lenski

Christina G Warinner, C. Speller, M. Collins

The field of palaeomicrobiology is dramatically expanding thanks to recent advances in high-throughput biomolecular sequencing, which allows unprecedented access to the evolutionary history and ecology of human-associated and environmental microbes. Recently, human dental calculus has been shown to be an abundant, nearly ubiquitous, and long-term reservoir of the ancient oral microbiome, preserving not only microbial and host biomolecules but also dietary and environmental debris. Modern investigations of native human microbiota have demonstrated that the human microbiome plays a central role in health and chronic disease, raising questions about changes in microbial ecology, diversity and function through time. This paper explores the current state of ancient oral microbiome research and discusses successful applications, methodological challenges and future possibilities in elucidating the intimate evolutionary relationship between humans and their microbes.

Natasha Pavlovikj, Joao Carlos Gomes-Neto, Jitender S. Deogun et al.

Whole Genome Sequence (WGS) data from bacterial species is used for a variety of applications ranging from basic microbiological research, diagnostics, and epidemiological surveillance. The availability of WGS data from hundreds of thousands of individual isolates of individual microbial species poses a tremendous opportunity for discovery and hypothesis-generating research into ecology and evolution of these microorganisms. Flexibility, scalability, and user-friendliness of existing pipelines for population-scale inquiry, however, limit applications of systematic, population-scale approaches. Here, we present ProkEvo, an automated, scalable, reproducible, and open-source framework for bacterial population genomics analyses using WGS data. ProkEvo was specifically developed to achieve the following goals: (1) Automation and scaling of complex combinations of computational analyses for many thousands of bacterial genomes from inputs of raw Illumina paired-end sequence reads; (2) Use of workflow management systems (WMS) such as Pegasus WMS to ensure reproducibility, scalability, modularity, fault-tolerance, and robust file management throughout the process; (3) Use of high-performance and high-throughput computational platforms; (4) Generation of hierarchical-based population structure analysis based on combinations of multi-locus and Bayesian statistical approaches for classification for ecological and epidemiological inquiries; (5) Association of antimicrobial resistance (AMR) genes, putative virulence factors, and plasmids from curated databases with the hierarchically-related genotypic classifications; and (6) Production of pan-genome annotations and data compilation that can be utilized for downstream analysis such as identification of population-specific genomic signatures. The scalability of ProkEvo was measured with two datasets comprising significantly different numbers of input genomes (one with ~2,400 genomes, and the second with ~23,000 genomes). Depending on the dataset and the computational platform used, the running time of ProkEvo varied from ~3-26 days. ProkEvo can be used with virtually any bacterial species, and the Pegasus WMS uniquely facilitates addition or removal of programs from the workflow or modification of options within them. To demonstrate versatility of the ProkEvo platform, we performed a hierarchical-based population structure analyses from available genomes of three distinct pathogenic bacterial species as individual case studies. The specific case studies illustrate how hierarchical analyses of population structures, genotype frequencies, and distribution of specific gene functions can be integrated into an analysis. Collectively, our study shows that ProkEvo presents a practical viable option for scalable, automated analyses of bacterial populations with direct applications for basic microbiology research, clinical microbiological diagnostics, and epidemiological surveillance.

Carolyn Churchland, Per Bengtson, Cindy E. Prescott et al.

As an alternative to clear-cutting, variable-retention harvesting is now standard forest management practice on the coast of British Columbia and in temperate forests globally, due to the benefits associated with maintaining mature forest species and forest structural diversity. Although there is some evidence that variable-retention harvesting, particularly single-tree (dispersed) retention will mitigate the impacts of clear-cutting on soil microbial communities and nutrient cycling, findings have been inconsistent. We examined microbial community structure (phospholipid-fatty acid), and nutrient availability (PRSTM probes) in a large (aggregated) retention patch and over three harvesting treatments: dispersed retention, clear-cut and clear-cut edge 2 years after harvest. Unlike previous studies, we did not observe elevated nitrate in the harvested areas, instead ammonium was elevated. Availability of N and other nutrients were surprisingly similar between the dispersed-retention treatment and the retention patch. The microbial community, however, was different in the clear-cut and dispersed-retention treatments, mostly due to significantly lower abundance of fungi combined with an increase in bacteria, specifically Gram-negative bacteria. This was accompanied by lower δ13CPDB value of the Gram-negative PLFA's in these treatments, suggesting the decline in mycorrhizal fungal abundance may have allowed the dominant Gram-negative bacteria to access more of the recently photosynthesized C. This shift in the microbial community composition in the dispersed-retention treatment did not appear to have a major impact on microbial functioning and nutrient availability, indicating that this harvesting practice is more effective at maintaining generic microbial functions/processes. However, as Mn levels were twice as high in the retention patch compared to the harvested treatments, indicating the other “narrow” processes (i.e., those performed by a small number of specialized microorganisms), such as lignin degradation, catalyzed by Mn peroxidase, which concomitantly removes Mn from solution, may be more sensitive to harvesting regimes. The effect of harvesting on such narrow nutrient cycling processes requires further investigation.