The story behind this paper begins...

...years before it was written. In fact, years before the previous paper on the matter was written in our research group. It begins with two elementary school boys in Belgium. One of the boys' fathers is Kai Dallmeier, a colleague from the Rega Institute at the KU Leuven, the other one's father a local bee keeper. While picking up their children from school, the two parents meet and start chatting. Soon they arrive at a common denominator: Can the virologist find out why so many bees are dying every year? Several viruses can decimate honey bee colonies, making them a focus of honey bee virology. But there are other types of viruses whose role is more complicated. More on that later.

The two fathers make a plan. The beekeeper's son takes an envelope with deceased bees to school and hands it over to his classmate who brings it to the scientist's home. The logistics chain continues to the Rega Institute onto the desk of Jelle Matthijnssens, the senior author on our paper, who is leading the laboratory of viral metagenomics. This group focuses on the detection of previously unknown viruses from various sources. Although school children's satchels are not exactly known to be favourable transport vessels for biological samples, a small experiment readily revealed many viruses in the bees the boys had couriered. Together with Dirk de Graaf from the Gent University, who was asked to get involved for his long-standing expertise in honey bee research, this preliminary discovery led to a larger study which was conducted and prominently published by a previous PhD student in our lab, Ward Deboutte. Answers lead to questions, however, and I was lucky enough to be chosen to continue this exciting work as a PhD student under the supervision of Jelle Matthijnssens and Dirk de Graaf.

A triad of uneven scale

This leads us to the paper of this story and viruses that may not be harmful. You see, honey bees (Apis mellifera), like humans, live in symbiosis with bacteria that populate their gut. This bacterial community – the microbiome – influences the animal's immunity, development and behaviour and is transmitted amongst worker bees from generation to generation. Every organism has its viruses, though, and bacteria are no exception. They can be afflicted by a diverse range of bacteriophages (or phages), as bacteria-infecting viruses are called. Some of them are on an eternal infect-takeover-multiply-kill cycle, as you might expect from a virus. But others integrate their genome into that of the bacterium and remain dormant for a while. Since a bacterium consists of only one cell, its fate and that of the phage then become tightly linked. Now it may be more advantageous for the phage to aid its host's survival than to kill it. Indeed, phages sometimes contribute helpful genes to the bacterial repertoire, thereby optimising metabolism, killing competing bacteria or avoiding being killed by them. Phages are studied in many settings and by acting on bacteria – in helping or harming them – they often have a profound impact on the microbiome and beyond. Phages, bacteria and animal then form an interdependent triad, maintaining a complex web of interactions.

Instrumental noise

While previous studies have found a remarkable diversity of honey bee gut phages, we still know very little about who they are and what they do. This is the knowledge gap we addressed in our paper. My work began by painstakingly dissecting several thousand bees amid the white noise of a laminar flow bench. Applying our lab method for virus detection to their squished guts resulted in a few microlitres of clear liquid containing a world of DNA sequence information. Most of the work was done on the computer, though, and much like in the lab, I funnelled hundreds of gigabytes of raw data through an orchestra of software tools to distil them into little more than a handful of spreadsheets. Conducting this orchestra to reveal a symphony hidden in the cacophony of inherently noisy metagenomic data was the greatest scientific challenge of this project – and also the most fun part. Here's the score:

• Movement I­­: We found more than 2000 phages in honey bees from across Europe.
• Movement II: Almost all of these phages were previously undescribed.
• Movement III: Ninety-seven of these phages probably occur in almost all honey bees worldwide.
• Movement IV: Some bee phages carry genes that might help the bacteria that they infect.
• Coda: Phages that carry one of these genes are reduced in areas with intense agricultural land use.

So, do we know why so many bees are dying?

Not quite, but we can ask even better questions now! Bees are dying for many reasons and of course, we can only deliver small pieces to the puzzle. Some pathogens and parasites are spreading like wildfire across bee hives but pesticides, even ones not targeted at insects, can be devastating, too. Some pesticides are only deadly when combined and I was shocked to learn that for licensing, they are only tested individually. When I found phages encoding a gene involved in sulfur metabolism, it was Dirk de Graaf who asked if this could be related to pesticide use. Following this idea led to quite different results than what we had imagined at first but I like how a quick, intuitive thought led us down a path to what I find the most interesting part of our paper. The link between honey bee phages and pesticides is only associative, but it spawns important hypotheses to be tested. To which degree do phages contribute to the bee's health? How and why do phages respond to stressors for the bee, like monoculture, pesticide use, or pathogen burden? These questions are often addressed only in the context of bacteria, leaving phages, the puppet masters of the microbial realm, out of the picture.

Once again, answers lead to questions and I am happy to hand over the torch to a third PhD student, Mariyam Mustajab, to continue a story that started with an envelope of bees, squeezed between school books and lunch boxes.