Question

How would pH and DO levels affect which organisms are collected for DNA barcoding?

Answer 1

DNA barcoding has become a fast, accurate, accessible and standardized tool for species level identification, by PCR amplification of short DNA sequences and subsequently sequencing it. The method involves collection of sample, DNA extraction, PCR amplification of a short DNA sequence from a standard part of the genome "DNA barcode", and finally sequencing and matching the sequence of the amplicon with the existing library of barcodes worked out for organisms of recognized identity. So DNA barcoding is a must-have tool for species detection and specimen identification.

Specimen collection is the first major step in this process, and probably is the determing step. Spatial and temporal factors, biotic and abiotic factors, trophic state in aquatic ecosystems, and characteristics of the organism's DNA affect the success and sensitivity of DNA barcoding. In absence of biological source, genetic material in the form of environmental DNA (eDNA) is obtained from soils, sediments, water or other environmental samples. eDNA degradation is one of the major drawbacks limiting the use of this technique and it has been demonstrated that pH and dissolved oxygen (DO) levels are among the several abiotic factors that affect eDNA stability (temperature, UV-radiation, BOD, etc.). Degradation rates of eDNA were found to be lower in alkaline conditions ( high pH), accompanied by colder temperatures and low UV-B levels. Higher degradation rates were associated with environments which favored microbial growth, for example , higher temperatures, neutral pH, and moderately high UV-B. This indicates that eDNA integrity will be more in aquatic habitats that have colder temperatures, more protection from UV-B and alkaline conditions and thus allow significant eDNA detection. This is in contrast to those areas that are warmer, sunnier, and neutral or acidic . These findings can enable better characterization of environmental conditions and more robust detection of several aquatic species with eDNA methods.

In a bigger perspective,  DNA barcoding has become even more significant for assessing the global biodiversity pattern. Water quality has been demonstrated to have a direct correlation with biodiversity. Good water quality is characterised by lower nitrates and phosphates, a neutral pH, and increased dissolved oxygen in the water, factors which promote biodiversity and attract organisms that are indicators of good water quality. Buffer zones in so called wetland ecosystems (like pond) can seriously affect the water quality of a wetland by reducing nitrates and phosphates and thus promote higher biodiversity. As previously stated, nitrate, pH, DO, turbidity, and phosphate levels in the water have a major impact on the water quality and on the types of life found within an aquatic ecosystem. Aquatic life is tressed by very high or low pH levels, which eventually reduce the hatching of their young. Algal growth is promoted in wetlands if there are excess nutrients, which will cause eutrophication, a condition in which the DO is depleted in the water, making it less suitable for other aquatic species who thrive on the dissolved oxygen, fishes and other invertebrates. Therefore drastic pH changes may allow collection of adult forms of aquatic life while the juvenile forms are missed. On the contrary, low DO levels will hinder collection of various species of fishes or other invertebrates.

Other reports also suggested that water stratification in sub tropical reservoirs shaped the phyogenetic composition of the microbial communities thriving in the different layers. During stratification, surface waters exhibit higher light intensities, elevated temperatures and increased dissolved oxygen concentrations in comparison to deep waters. But contrary to this, the microorganisms were more active at low temperatures and decreased dissolved oxygen concentrations characterising the deep layers. Infact, diverse bacterial species belonging to nine phyla were isolated from the various strata of water, for example Bacteroidetes bloomed at the surface water layers during summer but decreased in the low dissolved oxygen environment. Similarly Actinobacteria decrease with lower DO levels. While the warmer surface waters are re-circulated during mixing, the colder and dense waters of the deepest layers fail to participate. Moreover, deep waters remain isolated from DO inputs like the surface layers, leading to hypoxic conditions. Cyanobacteria can perform photosynthesis and fix nitrogen, in presence of light and oxygen and are therefore confined to the surface water layers.