Kick, kick! Who's in There?

Aquatic macroinvertebrates are critical species to understand when conducting biological surveys of aquatic ecosystems. Many times, population changes over long term monitoring schemes serve as indicators of water quality. Typically, samples are collected with a kicknet. I follow Frost and Huni’s procedures outlined in their 1971 paper. The authors say that their method effectively collects 90% of the benthic (bottom-dwelling) fauna. Samples are preserved in 70% ethanol and taken back to the laboratory for identification. Aquatic macroinvertebrates are usually identified to the lowest taxonomic unit possible, which in many cases is the genus level identification. Species keys are not developed for most larvae because of the morphological (physical) differences in instars (stages of development). If individuals in a sample are damaged, identification to the genus level may not be possible. In addition, there is room for human error in the identification process.

Therefore, there may be a need for other methods of identifying the specimens in a field collection of aquatic macroinvertebrates. One way of doing this is using Next-generation sequencing (NGS). NGS is a new technology that is starting to be applied to the biological sciences. NGS is similar to other DNA sequencing technologies; however, NGS is able to analyze mixtures of DNA. Therefore, the theory is that biologists can collect aquatic macroinvertebrates in the same manner they have in the past, but can analyze a mixture of DNA to get species level identification. DNA can be sequenced from one of three ways although only two are ideal. The first method is to blend the entire sample and analyze the smoothie of aquatic macroinvertebrates. However, this is the least ideal method because it is quite destructive. The other two methods allow the specimens to have minimal damage done to them. One method is for researcher to take a tissue sample from each individual specimen (usually a leg but not all macro invertebrates have those) and add that to the blend of DNA. The second, and least destructive, method is to use the ethanol the aquatic macroinvertebrates have been preserved in; as specimens sit in ethanol, some of their DNA ends up in the liquid ethanol. This is called “free DNA”.

Without getting too technical, the DNA is amplified (copies are made through chemical processes) and sequenced (think of each DNA strand having its own barcode) and then matched against known sequences (barcodes in a library). These known sequences are species specific. This sounds great; however, there are several biases that may prove to be problematic for at least the near future. The greatest issue is that some strands of DNA may get copied more than others and therefore the abundance of individual species will be skewed. Remember that biomonitoring tends of look at changes in populations over time so if there is no accurate indication of population size, this information may be useless. In addition, it may obscure the presence of some species. Also, this bias makes DNA copying not replicable since there is no consistency in which strands will be amplified more than others. There have been attempts to avoid this bias by using different types of amplifiers (copiers) and different combinations of DNA sources. The highest detection rate has been 91.3% in a combined “free DNA” and tissue DNAs along with multiple types of amplifiers.

It should be noted that this is different than eDNA (environmental DNA), which would involve testing pieces of the environment for traces of DNA. An example, and increasing application of eDNA technology, would be taking a water sample and testing for the DNA of invasive fishes to test for presence/absence data. For NGS it is required to have a collected and preserved specimen.

This type of technology could be applied to the monitoring of terrestrial invertebrates such as a collection from a malaise trap. However, the same biases still apply. NGS technology needs to be refined, and if easy to use, I would be happy to try using it in the monitoring programs I am involved in.

This article this post is based on can be found at: http://www.biomedcentral.com/content/pdf/1472-6785-12-28.pdf