Ocean ‘Dandruff’ DNA to Better Study Marine Biology

  • DNA Barcoding for all Organisms has Numerous Applications
  • DNA Barcodes from Water Samples Greatly Aide Marine Biologists
  • Aquatic Environmental DNA (eDNA) Proves to be Informative ‘Dandruff’

Human DNA identity analysis is now commonplace methodology that’s frequently featured in newspaper stories, TV crime series, or “who dun it” movies. The same principle (i.e. using a characteristic DNA pattern or signature) applies to identification of all animals, birds, insects, and microbes. Actually, DNA barcoding extends to any organism, whether it is alive or has been dead for hundreds of thousands of years (so long as it’s preserved by fossilization).

Taken from gajitz.com

Marine biologists face a serious challenge with accounting for very diverse forms of marine life that exists in a mindboggling huge volume of water. Consequently, it’s not surprising that analysis of water-borne, marine DNA barcodes—as proxies for going to and counting fish—is rapidly trending in utility and importance. Known formally as environmental DNA (eDNA), the aquatic version has been humorously referred to as ocean ‘dandruff’ by Christopher Jerde of the University of Nevada in Reno (which, ironically, is landlocked and distant from any ocean.) But I digress. Before diving further (pun intended) into ocean dandruff, let’s briefly review the background of DNA barcoding.

DNA Barcodes 101

Prof. Paul Herbert. Taken from uoguelph.ca

In 2003, Prof. Paul Herbert and coworkers in the Department of Zoology at the University of Guelph in Canada published a seminal study titled Barcoding animal life: cytochrome c oxidase subunit 1 (CO1) divergences among closely related species that fundamentally changed the field of taxonomy. In a nutshell, Herbert’s team showed it was feasible to classify millions of species based only on DNA sequence of the mitochondrial gene CO1. In the intervening, relatively short amount of time, there have been thousands of publications dealing with applications and extensions of this concept, which is now recognized to be very powerful and promising albeit with some limitations.

Typically, DNA barcodes are identified by sequencing after PCR amplification of one or more specific genetic loci such as CO1. Following proof that a DNA barcode can differentiate the species of interest, single- or multiplex quantitative PCR (qPCR) can be used to enumerate relative amounts of sample from the field.

The advent of high-throughput sequencing technologies applicable to complex mixtures of individually tagged samples then gave rise to “metabarcoding,” about which interested readers can consult many publications for specific topics.

Craig Venter steers his research yacht, Sorcerer II, under the Sydney Harbour Bridge in his quest to collect microbes from the world’s waters. Photo: Dallas Kilponen. Taken from smh.com.au

BTW, among the many pioneering scientific ventures by uber-famous Craig Venter, is his Global Ocean Sampling Expedition aboard his research yacht, Sorcerer II. The expedition is a quest to unlock the secrets of the oceans by sampling, sequencing and metabarcoding DNA of all (or most) microorganisms living in these waters.

Lest you think this was a well-intended but unproductive journey—some say junket—by Venter and coworkers, here’s a link to peruse 16 resultant publications that I found by searching PubMed. To watch and listen to Venter talk about this work, you can click here for an educational and entertaining—as usual with Venter—TED Talk on Sampling the Ocean’s DNA that’s had over 550,000 views!

Ocean ‘Dandruff’

Now that we’ve covered the basics of DNA barcoding and metabarcoding, let’s turn back to ocean dandruff. Dandruff, simply put, is dead skin cells. Using dandruff as an intended witty metaphor for ocean eDNA is a bit misleading as marine eDNA is comprised of a complex mixture of cellular matter from scales, feces, decomposing tissue, etc. of fish and all other present or past sea creatures. Consequently, the design and specificity of primers for PCR is of paramount importance for obtaining—let alone interpreting—DNA barcodes based on fragment size or sequence.

As reported by Miya et al., monitoring the occurrence of fish species-specific eDNA PCR fragments (~70–300 bp) has traditionally used conventional electrophoretic gel separation and detection. More recently, qPCR using fluorogenic probes has been employed owing to the method’s sensitivity, specificity and potential to quantify the target DNA. For example, it has been possible to accurately estimate the biomass of common carp in a natural freshwater lagoon using qPCR of eDNA concentrations and biomass in aquaria and experimental ponds.

Miya et al. also describe the development of a set of PCR primers for metabarcoding mitochondrial DNA of 880 species of fish. They sampled eDNA from four tanks with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing. Out of the 180 marine fish species contained in the four tanks, they detected 168 species (93.3%) distributed across 59 families and 123 genera. That’s quite an impressive accomplishment.

Ocean Dandruff Case Studies

Since there are so many fish-related applications of DNA barcodes, I’ve selected several recent examples that are indicative of the utility of ocean ‘dandruff’—and are quite interesting, in my opinion. The first case in point exemplifies how eDNA can be used to deal with rare and endangered species, which are either very hard to find or can be dangerously distressed by catching to obtain samples.

Green SturgeonBergman et al. report that a decline in abundance of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning by these Green Sturgeon are effective at monitoring fish densities in concentrated pool habitats, results do not scale well—pun intended. By contrast, eDNA provides a relatively quick, inexpensive tool to efficiently identify and monitor Green Sturgeon DNA.

Taken from mthsecology.wikispaces.com

These investigators concluded that follow-on work based on this first-ever eDNA study of Green Sturgeon has the potential to provide better knowledge of the spatial extent of Green Sturgeon spawning that could help identify previously unknown spawning habitats and discover factors influencing habitat usage, guiding future conservation efforts.

Monterey Bay—The second case study, by Port et al., involves taking stock of the marine mammals and fish in Monterey Bay using eDNA and, importantly, comparing the results obtained to those from traditional dive surveys.

In brief, this team of researchers from several universities and the Monterey Bay Aquarium Research Institute found that eDNA assessments picked up almost all the organisms scuba divers spied underwater—plus many more that human eyes missed. Here’s some detail on how they did this.

At each scuba survey location as well as at sites offshore, ~1 gallon of water was sampled several feet above the bottom. Four types of habitats were sampled: sea grass beds, Monterey Bay’s unique “Kelp Forest,” sandy areas and rocky reefs. Onshore, in a “clean” (DNA-free) lab, these water samples were filtered to collect cells containing eDNA for storage at −80 °C until eDNA extraction at a university clean lab. A vertebrate‐specific primer set targeting a small region of the mitochondrial DNA 12S rRNA gene was used for PCR followed by gel purification.

Researchers collecting water in Monterey Bay for eDNA analysis. Courtesy Jesse Port. Taken from mercurynews.com

After quantification, pooled amplicons (each having a sample index sequence) were paired-end sequenced on the Illumina MiSeq platform using a 20% PhiX spike‐in control to improve the quality of low‐diversity samples. The conclusions are worth quoting because—in my opinion—the findings represent a new era in marine biology based on nucleic acid analysis:

“We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 meters. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.”

Restated more simply, eDNA analysis of the water picked up 11 of the 12 fish and marine mammals that the divers observed, and—importantly—identified 18 additional animals the divers missed! The efficiency and improvement offered by eDNA analysis compared to traditional seek-and-count methods has been echoed in an editorial I found by Hoffmann et al. titled, tongue-in-cheek, Aquatic biodiversity assessment for the lazy.

Invasive Gobies—The third and final case study deals with detection of invasive, non-native fish to assess whether eDNA can provide a better advanced warning system for detecting these unwanted creatures and implementing eradication steps.

Gobies are an invasive fish species that has colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby (Neogobius melanostomus), pictured below, is among the worst invaders in Europe. Current methods to detect the presence of these gobies are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile.

Taken from animal.memozee.com

To improve monitoring, Swiss and Canadian collaborators developed an assay based on the detection of eDNA in river water, without detecting any native fish species, which is obviously an important assay criterion. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by novices and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s reports and fish community monitoring.

I could go on and on with examples of utility and the many advantages provided by eDNA for marine biology, but I’m sure you get the picture. I hope that you agree with me that eDNA analysis is a very valuable type of trending nucleic acid-based methodology.

As usual, your thoughts or comments are welcomed.





Finding Frankfurter Fraud Featuring Famously Familiar PCR

  • While Thousands of PCR-based Tests for Food Authentication Exist, Commercial Adoption is Lax
  • PCR Tests for Halal Frankfurter Products Reported by Malaysian Team
  • PCR-enabled Next Generation Sequencing of USA Frankfurters Exposes Extensive Mislabeling and Adulteration

Regular readers of this blog will know that (a) I favor alliterations, (b) frequently feature PCR-based topics, and (c) I am fond of food facts involving nucleic acids, all of which are found in the title of this posting. While writing style and food are both a matter of taste, so to speak, it’s almost impossible to comment on nucleic acids without involving PCR, in one way or another, as PCR is—in my opinion—the most widely used and important method in molecular biology.

Having said this, and knowing that this summer alone Americans will likely consume an estimated 7 billion (!) hot dogs, (a.k.a. frankfurters or wieners), I thought it apropos to now feature finding frankfurter fraud by PCR. But before getting to that, I thought it’s worth commenting first on a frankfurter vs. wiener vs. hot dog and other “meaty” definitions to get us “linked” up—please pardon the puns, another of my penchants.

Frankfurter, Wiener, Hot Dog Lexicon

Frankfurter sausages and sauerkraut. Taken from tripadvisor.com

While it’s a fact that a resident of Frankfurt, Germany is properly called a Frankfurter, one of Frankfurt’s pork sausage specialties is also called a frankfurter—but spelled with lower case f—and is short for Frankfurter Würstchen, which go back to the 13th century. By the same token, wiener refers to a pork and beef sausage specialty introduced in the 18th century in Vienna, Austria—a city called Wien in German, and hence the word wiener.

Frankfurters and wieners look very similar, with the main culinary distinction being absence of serving with the bun, which by contrast is characteristic of a hot dog. Readers interested in the origin of the hot dog’s name and bun usage will learn at this link—pun intended—that there are various and widely different claims for this name and usage.

Hot dogs in buns. Taken from clowns4kids.com

Dog Factory, a short film by uber-famous Thomas Edison in 1904 poked fun at what went into hot dogs. Taken from wikipedia.org

In one such claim, the term dog is said to have been linked—there I go again—to sausages made from dog meat, as popularized in an old spoof by Thomas Edison (!) pictured below. This issue of what meat(s) hot dogs and similar sausages contain now segues into finding frankfurter fraud using PCR.

Finding Food Fraud by PCR

Before getting to the meat of this matter (oops!) involving frankfurters, I thought it would be informative to provide some larger perspective on finding food fraud, generally, utilizing the power of PCR for specific detection of nucleic acids that are characteristic of a given species of any food whether it be meat, fish, vegetable, etc. My search of Google Scholar for articles with all of the terms “food, fraud, and PCR” gave ~3,900 items. Here are some selected samplings, the first of which was taken from several I found that used TriLink products—hooray!

  • US Food & Drug Administration researchers employed species-specific primers from TriLink for multiplex real-time PCR analysis of salmon and trout species in a range of 80 commercial products in North America. 4 instances (5%) of fraud were found.
  • By contrast, a whopping 40% of commercial pet food products tested by PCR for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) were found to be potentially mislabeled, according to a study by academic researchers.
  • Saffron produced from dried stigma of Crocus sativus is considered to be the most expensive food spice in the world, as ~200,000 (!) flowers must be carefully hand-picked (!) to produce only 1 kg of spice. Combating saffron fraud with PCR has already led to ~150 publications (!).
  • PCR-based food authentication to screen for possible allergens or GMOs is important for prevention of potentially life-threatening food contamination or alleviating consumer perceptions—or perhaps misperceptions, as I’ve commented on previously.

Finding Frankfurter Fraud by PCR

As for finding frankfurter fraud by PCR, the aforementioned ~3,900 items from my Google Search of food, fraud, and PCR was sub-searched for frankfurter, which led to only 3 reports titled as follows:

The third item, which is by a team of Malaysian researchers and is the most recent, offers some interesting introductory perspectives on strict religious, cultural, or geographical restrictions over the consumption of certain meats in the context of commercial frankfurters.

For example, pork is totally unacceptable to Malaysia’s large Muslim population, as well as Jewish and certain Christian denominations. On the other hand, Egyptians prefer buffalo because of their cultural preferences, while some Indians and Europeans avoid beef because of religious requirements and the fear of bovine spongiform encephalopathy (aka “Mad Cow Disease”—click here for an FDA update), respectively.

In this Malaysian study, 100% beef, buffalo, or pork frankfurters were prepared as models, and then fully heat-processed in the laboratory to simulate conventional manufacturing procedures. Additional beef, buffalo, or pork frankfurter models were deliberately contaminated by “spiking” in 1%, 0.5%, or 0.1% of buffalo and pork, beef and pork, and beef and buffalo meat, respectively. PCR was then performed using species-specific primer pairs for two genes (cytochrome b and NADH dehydrogenase subunit 5) for cross-validation. Twenty different halal-branded (i.e. pork-free) “beef” frankfurters from Malaysian markets were tested. While no pork was detected in any of the tested “beef” frankfurters, they were all beef- and buffalo-positive, thus revealing that all of the investigated Malaysian commercial “beef” products were buffalo-adulterated.

Halal Certification by PCR

The above mentioned concern for non-pork halal-assurance piqued my interest as to the extent of PCR usage for halal-related certification, and my subsequent Google Scholar search for “halal, certification, PCR” gave nearly 350 items. This relatively large number of reports signals quite widespread adoption of PCR. I encourage those interested to peruse these items later, but will mention here that the following article is the most cited—close to 100 citations as of February 2017—Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

In closing, I think it’s worth noting in the context of halal certification—and increasingly popular “democratization” of technology, about which I’ve previously offered comments—a French start-up company now offers an antibody-based “dip stick” kit (Halal Test) for anyone to use for pork-free halal-assurance at home or even when eating out. Amazing.

Taken with copy write permission from French duo launch HalalTest: ‘We want to democratize analysis’ by Rachel Arthur+Rachel ARTHUR, 05-Nov-2014

Hot Dog! — There’s Now an NGS Food Authentication Service Company

Frankly—pun-to-be intended—I don’t know how “hot dog!” became an exclamatory phrase for good news. It applies, however, to the fact that Clear Labs Inc. (a 2014 start-up in Palo Alto, California) has proven that several issues of concern for consumers of hot dogs can be successfully addressed by PCR/NGS methods.

In a Clear Labs’ poster abstract for the 2016 International Association of Food Protection meeting, results were reported for a study of 345 hot dog products sold by national brands to compare product label information and ingredient lists with the results of NGS analyses. Following DNA extraction, universally accepted regions for animals, plants, and bacteria were PCR-amplified for NGS, which revealed that ~15% of these products had ingredient substitution, unexpected ingredients, or hygienic issues. In addition, 10% of all products labelled as vegetarian contained detectable levels of meat DNA. Vegetarian products also accounted for 67% of hygienic issues, such as human DNA.

At the risk of overly generalizing these findings, I believe that they probably reflect very widespread issues in the food industry, which we as consumers are virtually helpless to deal with, and can only hope that US FDA regulations begin to mandate PCR-based food certification.

Having said this, I didn’t want to end with a “downer.” Therefore, I’ll conclude with some hopefully fun facts about hot dogs, which—if you’re wondering—can be made from beef, pork, turkey, chicken, or a combination (but must be labeled as such, according to FDA information worth reading later).

Fun Frankfurter (aka Hot Dog) Facts

Just for fun, I calculated that the 7 billion frankfurters to-be-consumed by Americans this summer would stretch 56,818 miles (assuming each one were 6 inches long), which would wrap around Earth’s equator 2.3 times if placed end-to-end!

Some other fancinating frankfurter feats:

  • Taken from wikipedia.org

    The world’s longest hot dog was 197 feet and was prepared by Shizuoka Meat Producers and the All-Japan Bread Association for the latter’s 50th anniversary celebration in 2006 at the Akasaka Prince Hotel:

  • The world’s most expensive hot dog is the “California Capitol City Dawg”, served at Capitol Dawg in Sacramento, California and cost $145.49. Proceeds from the sale of each 18-inch long, 3-pound super “Dawg” are donated to the Shriners Hospitals for Children.
  • The annual Nathan’s Hot Dog Eating Contest is held on Independence Day at Nathan’s Famous original, and best-known restaurant in Coney Island, a neighborhood of Brooklyn, New York City. The current champion is Joey “Jaws” Chestnut, who ate a stomach-busting 70 (!) hot dogs and buns in only 10 minutes (!) at the 2016 championship.

Taken from elitedaily.com

As usual, your comments here are welcomed.