Spill the tea: Who lives in your beverage?

Monitoring eDNA is a relatively recent discipline used to provide a rapid, cost-effective, and standardized collection of data about species distribution and relative abundance in the wild. However, a study focusing on dried samples in tea bags is likely the first of its kind. Scientists usually look for eDNA in soils, aquatic environments, and even air, where animals and insects leave their traces in droppings, saliva, shed skin, carcasses, and eggs.

Study coauthor Henrick Krehenwinkel, an ecological geneticist and assistant professor at the Department for Biogeography at the University of Trier in Germany, has been interested in 'creepy crawlies' ever since he was a kid and compares the scenario to that of a crime scene. "If someone breaks into your house, the police take a DNA sample. Similarly, when an insect sits on the flower and feeds on the nectar, it pollinates the flower, leaving a tiny trace of eDNA, which can be detected," he tells IE in an interview.

In 2017, a publication in Germany reported on the insect decline in the country's nature reserves. The report stated that there had been a 75 percent decline in insect biomass over 30 years. "This was rather shocking. Insects are highly beneficial for our wellbeing and the ecosystem - without bees pollinating the plants, we wouldn't have any fruit. [They are] food for a lot of animals. Thus, insect-plant interaction is very essential," says Krehenwinkel.

At the same time, we know very little about insect-plant interaction, the scientist says. "Which is why we embarked on a mission to learn more about it."

How is the current approach to collecting eDNA different?

Krehenwinkel mentions a significant point. "It allows us to detect insects without having to collect or kill them," he says. "Usually, insect DNA detection is done with traps, like the malaise trap – which is like an invisible window for insects. At the end of this barrier, there is a funnel that ends into a tube with ethanol in which the insect drowns. It’s kind of contradictory because everyone is talking about insect decline but to protect and monitor them, you have to kill them — which is problematic. This is one major reason why we’re developing this approach."

However, there's so much more to the study.

The researcher discusses that studying eDNA can also allow us to "travel back in time" and understand how insect communities have changed. 

"If the drying in plants really preserves the DNA well, then we may still detect the DNA of the insects which interacted with the plant 50 years ago. So, we could take a museum sample, isolate the eDNA from it and enrich it. And that could possibly also allow us to understand how an insect community has changed. At the moment, for example, we're looking at herbarium specimens, which have been collected here in this area 50 years ago. And when this works, we will go back to the same places where those specimens have been collected, collect them again and repeat the same process," he explains.

Why tea?

"Two reasons. Tea, which is very popular, is well-studied. So, we already know the kind of insects that interact with the plants in tea plantations. So, if we test our experiment with tea, we would already know what kind of insects are expected to interact. And from our DNA sequencing, if we get a result showing us those results, then we can say that our approach works," explains Krehenwinkel.

Secondly, when an insect bites into a leaf or a bee pollinates a flower in the wild, the traces left are very unstable. 

"They could be washed in the rain or broken down due to the irradiation from the sun. We can preserve these traces if we dry the sample. So, when we take the leaf samples from nature, we dry them and remove the water from them, making the eDNA very stable. eDNA doesn’t degrade that quickly if we dry it. And tea is nothing else but a dried plant sample. It is harvested, dried, and then stored. This allows us to test two things – does drying really preserve the DNA, and does our protocol work?" he says.

Surely, some insects don't necessarily leave a lot of traces?

"We don't know yet," says Krehenwinkel.

"And we have to experimentally test this in more detail. There are also differences in the way DNA is left on the leaf. For example, if an insect leaves an egg on the leaf, that will contain a lot of DNA. If an insect bites into a leaf, that leaves very little DNA. And insects that walk over the leaf will leave almost no DNA. But we have to test [for] all of these [traces] now," he explains.

The scientists could probably set up an experiment wherein they sterilize a single leaf and remove all traces of insect eDNA on it. "We could then let different insects interact with the leaf and see which are detected and which aren’t. With every scientific method you develop, you need to show that it works pretty well. A lot of additional optimization is needed to [find out] what is detected and not," he tells me.

Why is this specific discovery important to an ordinary person?

Krehenwinkel mentions that a lot of people who read about the discovery may assume that tea is full of insects. That's not the case, he says. It's just a trace amount of insects.

"And now we have methods that can enrich the tiniest trace amounts and then reconstruct them. I think people should be happy that we detected these amounts of insects because that means that the tea was collected from a field that isn’t dead. Which in turn means that if the tea was constantly sprayed with pesticides, all the insects would have been dead, and we wouldn't have found such a high diversity of living creatures," he says.

Now that is very interesting.

Krehenwinkel continues: "And not only did we find pest species in the tea fields, but we also found other species that are part of a stable and functioning ecosystem. For example, we found spiders that are predators in the tea fields and eat pest species. This shows us that the tea samples we processed are actually indicators of a healthy ecosystem."

It is in all of our interest to preserve insects because they're fundamental to the functioning of ecosystems. "This is why it's very important to develop new approaches to understand how and why communities are changing, and how insects are interacting not only with each other but also with the plants in the ecosystem," he says.

Unending intriguing discoveries

The researchers stress that though they weren't surprised by the discovery, they were astounded to find a variety of different species.

"But, now when I think about it, I realize that it's isn't [as if] some 400 different species were found in a single tea field. I think the tea I bought in a German grocery store was from different places. So they probably imported it from China, India, Sri Lanka, and many other places where tea is produced. And then they probably mix [the tea leaves together]. So, what you have in your tea bag is not usually pure tea from [one plantation in] Darjeeling in India, but it is probably from a lot of different plantations. Which is probably why you have a significant increase in the biodiversity," Krehenwinkel says.

Then, how does he figure out which insects are native to a particular tea plantation?

"Take peppermint tea, for example. It is a mixture from different places, but we can say that at least part of it must have come from the Northwest part of the United States because we found a spider native to the area. Or, in another tea, we found a spider that is only found on the southern slopes of the Himalayas. So, we know at least part of this tea must come from the Himalayas. So, if you have a sample sold as a ‘pure sample’ - with guaranteed purity from Darjeeling, you could very easily use this approach to test if it really was from India," explains Krehenwinkel. 

What is the margin of error?

"This is another thing that we have to explore. So, eDNA approaches are incredibly sensitive, and you have to run a lot of negative controls to standardize your data. You always have to be very careful when you run these protocols and watch out for contamination," says Krehenwinkel.

Recently, eDNA was shown to be transported via air. Thus, finding an eDNA trace in a particular sample does not guarantee that the insect has interacted directly with the plant in that sample; the traces could have been blown onto the field by the wind.

"For example, imagine a bird that has eaten an insect 10 miles away from the field. When it passes the tea field, it probably leaves a few droppings on the leaves, which would still contain DNA from the insect generally found 10 miles away. Also, the eDNA transported via air could be from the adjacent forest and not be native to the tea field," he says.

Further down the line

Krehenwinkel says that the group of researchers is very interested in finding alternative sources for understanding how communities of animals have changed over time.

"Dried plants aren't exactly alternative sources [of eDNA] - we were looking at a lot of different sources, which will allow us to reconstruct communities of animals, especially their attributes over time. For example, we're looking at leaves that have been collected 30 years ago, which will be compared to leaves that have been collected today, to see how an insect community associated with the leaf has changed. This is our focus," he says.

The team is "trying to understand how communities have changed and how human influence on ecosystems is changing the structure and composition of animal communities."

Towards the end of our interview, I ask Krehenwinkel if he has been put off by tea, considering the discovery.

"Well, I don’t drink tea, mostly. I'm usually a coffee drinker," he immediately says. Oh well. "But as I said before, it wouldn't put me off tea because it's not like your tea bag is swimming with pieces of insects; it's really mostly small traces. And that's unavoidable. So, if people would be put off by this result, they couldn't eat anything anymore, because I guarantee you that almost every food item will contain traces of animals," he says.

For example, if you leave a bowl of fruit on your living room table, and then you come home in the evening and eat it, "then I guarantee you that a couple of flies would have interacted with the fruit and landed on it. And then you'll have an eDNA trace of the fruit fly on your apple. As I said before, I'd rather be happy that there’s a high diversity in the tea because it shows that we have a healthy and thriving ecosystem," he adds.