Introduction
In 2016, a primary research paper related to viruses was published in the journal PLOSOne. The study was conducted by a group of researchers from Flinders University and the Research Division at the Commonwealth Scientific and Industrial Research Organization (CSIRO), both of which are located in Adelaide, South Australia. In reviewing this article, it is important to examine the question that was being addressed, the methods that the researchers used to explore the question, and the results of the study. It is also necessary to consider the general significance of this article and how it contributes to the understanding of the field of biology.

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Question Asked
In this study, the authors were exploring the general question of whether or not “microbial patchiness” exists in freshwater systems (Dann et al.). The term “microbial patchiness” refers to differences in the distribution of the abundance of virus-like particles (VLPs) and prokaryotes in aquatic systems (Dann et al.). There were several reasons why the authors asked this question. Most directly, other researchers had recently reported that microbial patchiness exists in marine ecosystems (Dann et al.), so the authors wondered whether the same was true for freshwater systems. Previously, scientists believed that the distributions of microbes in aquatic systems was homogeneous, which affected their approach to sampling (Dann et al.). When researchers studying marine systems found that microbial distributions varied on the micrometer to centimeter scale, it indicated that the question should also be addressed for freshwater systems.

More indirectly, the scientists believed it made sense to explore this question because there is heterogeneity in the spatial distribution of other types of organisms, in both aquatic and terrestrial environments, so it would make sense to test whether the same is true for the microbial community within freshwater aquatic systems (Dann et al.). Specifically, they wanted to know whether there were biodiversity “hotspots” in microbial communities in freshwater ecosystems, as there are in other types of environments.

Methods
In order to address their question, the authors began by collecting samples from a freshwater ecosystem. The samples were collected from Murray River at Murray Bridge in South Australia on June 14, 2012. In total, they took 24 samples. While collecting samples, they monitored the water flow rates. They also made sure that their work would not negatively impact endangered or protected organisms (Dann et al.).

In order to determine the number of VLPs and prokaryotes in their samples, the researchers used flow cytometry (Dann et al.). In flow cytometry, laser technology is used to study the characteristics of cells in a fluid. For instance, it can be used to identify and enumerate different types of cells in a heterogeneous sample (“Introduction to Flow Cytometry”), which is how it was used in this study.

Based on the flow cytometry data, the researchers used computer software to conduct a microscale distribution analysis of the subpopulation of the VLPs and prokaryotes that were present in their samples (Dann et al.). Then, they ran a statistics test to determine whether there were correlations between the subpopulations of VLPs and prokaryotes. From there, they performed a spatial autocorrelation analysis to study spatial dependence within the subpopulations, which involved a series of statistical tests, including Moran’s I, Moran Correlegrams, and Geary’s C (Dann et al.). Again, they used software to conduct these analyses.

Results
The first result reported by the researchers is the abundance of prokaryotes and VLPs in their sample, based on the flow cytometry data. They found that there were four subpopulations in their sample: a high-density nucleic acid prokaryote population (HDNA), a low-density nucleic acid prokaryote population (LDNA), and two different virus-like particles (VLP 1 and VLP 2) (Dann et al.). The authors found that the mean abundance of each subpopulation varied between samples (Dann et al.). They also found that the abundance of VLP 1 was always greater than the abundance of VLP 2, and the abundance of LDNA was always greater than or equal to the abundance of HDNA (Dann et al.).

The authors also reported finding hotspots within their samples. They found that most hotspots were located at the sediment-water interface. For both VLP 1 and VLP 2, the researchers calculated the maximum hotspot value (in particles per milliliter of water), and their results showed that it varied considerably from the background. For VLP 1, there was a 24 fold change in heterogeneity, and for VLP 2, there was a 26 fold change in heterogeneity (Dann et al.). The maximum abundance hotspot values were also calculated for LDNA and HDNA. They were lower than those for the VLPs, but this was due to the fact that LDNA and HDNA were generally less abundant in the samples. When compared to background, there was a 31 fold change in heterogeneity for HDNA and a 15 fold change in heterogeneity for HDNA (Dann et al.).

The previous results refer to changes in heterogeneity across the entire sample area, but the authors found even greater changes in heterogeneity between sample points. When they looked at different sample points (which were spaced 0.9 centimeters apart), the authors found that the maximum change in heterogeneity was 74 fold for VLP 1, 107 fold for VLP 2, 80.5 fold for HDNA, and 41.5 fold for LDNA (Dann et al.).

The results of the correlation tests to examine the relationships between the prokaryotic and VLP subpopulations indicated that that there was a correlation between the two subpopulations. However, the degree of correlation varied between locations, depending on the presence of hotspots (Dann et al.). Also, the spatial autocorrelations tests (Moran’s I and Geary’s C) showed that there were significant autocorrelation values in 29 percent of the subpopulations. Similarly, significant correlograms were only found for about a third of subpopulations in the sediment-water interface environments (Dann et al.).

Based on these results, the authors were able to conclude that microscale microbial patchiness exists in freshwater systems, just like in marine systems (Dann et al.). They also verified the existence of hotspots in freshwater systems and attributed heterogeneity changes between sampling points to their presence (Dann et al.).

Significance
The results of this study are significant because they advance scientific understanding of freshwater systems. They dispute the long-held assumption in the scientific community that microbial communities in freshwater systems are generally homogeneous. Instead, their study indicates that there is heterogeneity in prokaryotic and VLP subpopulations in freshwater systems, and there are hotspots just as there are in other aquatic and terrestrial environments. However, it is important to note that the heterogeneity in this system may be different from other freshwater system, given the variation in flow and shear patterns between streams and rivers (Dann et al.).

This study also advances the field of biology by providing evidence that sampling methods for freshwater systems need to change. In the past, researcher had used bulk phase sampling to study the microbial populations in freshwater systems (Dann et al.). Based on the findings of this study, this method will not be accurate because it does not account for microbial heterogeneity, so different methods will likely need to be developed in the future (Dann et al.). Thus, it is clear that this study has both theoretical and practical value within the field of biology.

    References
  • Dann, Lisa M., Paterson, James S., Newton, Kelly L., Oliver, Rod, and James G. Mitchell. “Distributions of Virus-Like Particles and Prokaryotes within Microenvironments.” PLOSOne, 2016, vol. 11, no. 1, pp. e0146894.
  • “Introduction to Flow Cytometry.” Abcam, 2017, http://www.abcam.com/protocols/introduction-to-flow-cytometry.