Photo: Alison McAfee
VARROA DESTRUCTOR MITES are the number one reason for honey bee colony losses. And yet, we know extremely little about their fundamental biology.

Hunching at my microscope, the sweat in my eyes was making it hard to focus. Ninety-five degrees Farenheit (35 C) is fine for mites, but not for Canadians. After dumping a tube full of mite families on a petri dish, I started sorting them under the lens. Adult males were the first to be gathered – they were easy to recognize, with their tear-dropped, slightly tanned body and spidery legs. They were also the fastest to run away, up and over the sides of my dish. Immobile female deutonymphs were next – some had beautiful black patterns visible through their oval, translucent exoskeleton, pulsing like a heartbeat within the stillness of its shell. Mesmerized, I recalled seeing something similar in the Cyrtarachne spiders of Singapore. The little pudgy-legged protonymphs were my favorite, though. They were so cute, I almost forgot how destructive they can be.

alison-mcafee-varroa-destructor-deutonymph-honey-bee
An eerily beautiful female deutonymph with exquisite black and white patterns visible beyond her translucent exoskeleton.

Varroa destructor mites form their inconspicuous families inside brood cells, where they wreak havoc on the developing honey bees. The mites feed on honey bee hemolymph (blood), simultaneously parasitizing and giving them debilitating viruses to boot. If we’re going to find better ways of combating the enemy, a good place to start is to better understand what we’re dealing with.

What genes turn on and off as the mite develops from embryo to adult? What biological processes underlie these transitions? Broadly speaking, we know that mites have a similar sex-determination system as honey bees, but other than that, what makes male and female mites different? If these seem like trivial questions without any obvious application, you’re right. The questions are so simple, for honey bees they were answered about a decade ago.

This type of basic, fundamental research has lost its charm with the funding agencies, to the detriment of science. After all, it was curiosity about a glowing green jellyfish Aequorea victoria that eventually revolutionized the field of molecular biology and earned Osamu Shimomura a Nobel Prize. My inquiry into a little mite’s life is certainly not going to land on the Nobel committee’s desk, but my point is, you don’t know what you don’t know. It was sheer curiosity that gave me the motivation to pick through thousands of brood cells and sit in a hot room for hours late last September, culminating in a paper I recently submitted to a peer-reviewed journal.

With the help of Queenie Chan  and Jay Evans, I analyzed the proteins within all the major developmental stages of a mite family (Figure 1). Each gene contains the instructions to make a protein, but it’s the proteins that carry out most of the jobs in the cell. The jobs proteins do are important for how cells survive, specialize, and form different organs, tissues, and appendages, so analyzing which ones are produced and at what abundance can tell us a lot about how they’re guiding development.

Fig1
Schematic of the Varroa destructor life cycle. Like honey bees, varroa follow the haplodiploid sex determination system (males have one set of chromosomes and females have two). Normally, the first egg the foundress lays in a brood cell is haploid and becomes a male. While her son is growing up, she lays another egg – this time diploid – which becomes a female, and maybe one or two more after that. When her sons and daughters are adults, they mate (yes, incest!). The daughters then emerge from the brood cell with the adult bee, which marks the beginning of their phoretic life phase outside the brood cell (meanwhile, the male dies). Soon, the daughters will find a brood cell of their own to invade and become a foundress, starting the cycle anew

We looked at three replicates of 50 pooled mites each – about 1,000 mites in total – separating the males from females where possible. By looking at this many mites, our findings should be representative of a normal mite population. Crushing the mites to a pulp within a stabilizing solution, we extracted the proteins from their little arachnid bodies. Using a technology called mass spectrometry, we got our first look at the varroa proteome, which like a snapshot survey of all the mites’ proteins. Well – not quite all of them. Some proteins are simply too tricky to identify. We accurately quantified about 3,000 proteins, around one fifth of all the proteins that are possibly expressed throughout a varroa’s life. This may not seem like an outstanding number, but it is a leap forward compared to what’s been done before us, and it’s certainly enough to see some interesting patterns emerge. Here’s a taste of what we found.

Like most arthropods, mites have an exoskeleton made of chitin (a tough structure of polysaccharides plus proteins), but it transforms from a flimsy, translucent organ container in the protonymph into a tough, tanned exterior in the adult. Despite being similar colors, we found that the mite sons’ and daughters’ exoskeletons are made up of very different chitin structural proteins. In fact, the adult males have remarkably few chitin proteins at all. Foundress mites, on the other hand, have a very high abundance of a different set of chitin proteins. I think these differences between sons, daughters and the foundress reflects the different environmental threats male and female mites face and how their exoskeletons have adapted to meet their needs. Male mites live their entire life in the safety of the brood cell, so they don’t require much of a protective exterior, whereas females must eventually venture out into the hive. There, they are harassed by worker honey bees who bite and claw the mites off their sisters. The female mites, therefore, require a tougher exoskeleton than the males. This is visible to the naked eye, with the exoskeleton hardening into the deep reddish-brown armor we are used to seeing for the phoretic and foundress mites. It is quite satisfying to see these transitions reflected clearly in the types of chitin proteins expressed in the sons, daughters, and foundresses.

The exoskeleton is not the only thing that’s different between male and female mites; overall, about 100 proteins were differentially regulated. Some were more abundant in the males and some in the females, but how do we make sense of all this data? When there are very few proteins to look at, it’s feasible to sift through them one by one, noting their functions and manually making sense of how they might be working together. But one hundred proteins verges on being too many to reasonably interrogate. To overcome this, biologists do what’s called a “functional enrichment analysis,” where we plug the proteins and their biological functions (for example, whether the protein is an enzyme or a structural protein) into a program that tells us if there are more proteins with certain functions than we would expect by chance. That’s precisely what I did, and one thing I found was that many of the proteins differing between males and females are involved in physically unpacking densely twisted DNA to expose new regions, presumably containing male- and female- specific genes. DNA is often condensed in the cell as a kind of physical on/off switch – if enzymes can’t access the DNA sequence, then its genes can never be read and translated. However, special enzymes can remodel the DNA to expose it and the information it encodes. My data suggest that male and female mites have different DNA regions exposed, which contributes to producing two unique sexes. Precisely what regions these are and which genes they contain, though, will require more experiments (which any curious scientist is welcome to pursue).

The foundress mite is the only reproductive female in the family, and it turns out she has very different metabolic requirements than the rest of her progeny. We found that foundresses have very high amounts of lipid (fat) transport proteins and key enzymes that are needed to turn glucose into energy (if you’ve ever taken an intro biochemistry course, these are the enzymes that form the glycolysis pathway and Krebs cycle). These metabolic differences are probably related to her job as a mite mother: laying multiple eggs, as she does in a single stint in a brood cell, is a huge energy investment and is bound to be accompanied by big changes in her metabolism.

These observations made us curious about how the foundress’s diet compares to her sons and daughters. We know that mites feed on honey bee tissue, and thankfully we can distinguish between mite proteins and honey bee proteins. Any honey bee proteins we see in a mite sample are probably there because the mite’s stomach is full of bee juice. So, does the foundress also eat more to meet the energy demands of motherhood? To our surprise, we found that relative to their body size, it’s actually the deutonymphs that eat the most. You can think of them as the teenagers in the mite family, consuming huge amounts of food during their rapid growth phase. Not only is the foundress consuming relatively less honey bee tissue, she also appears to be eating different parts of the bee. The protonymphs, deutonymphs and adult progeny have similar-looking diets (i.e. they consume the same kinds of honey bee proteins), but the foundress also contains a very high abundance of a group of honey bee proteins that aren’t found in the other mites. This is just a hypothesis, but the only feasible way I can think of for this to happen is if the foundress isn’t eating exactly the same thing as the others; for example, if the deutonymphs eat mostly hemolymph and the foundress eats both hemolymph and other tissues.

In addition to honey bee proteins, mass spectrometry also lets us look at the viral proteins within the mites. We only identified two viruses: deformed wing virus (DWV) and another virus that appears to be either Varroa destructor virus (VDV) or a hybrid between VDV and DWV. We couldn’t believe the sheer amount of DWV in these mite samples. There was so much virus, its proteins were within the top 10 highest abundance, including the mites’ own proteins. To put it into perspective, that’s like if you got sick with rhinovirus (the common cold) and the virus replicated inside you so much that its proteins were more abundant than those from your own blood and skin. It’s no wonder why mites are such good vectors for honey bee viruses.

This is just a sampling of the things we have learned from our mite analysis. If you are interested, our full paper is freely available on the preprint server BioRxiv (simply go to http://www.biorxiv.org and search “McAfee varroa”). For full disclosure, this paper is not yet peer reviewed, but we have submitted it to a scientific journal. While it’s provisional, this doesn’t mean that it’s illegitimate science – In the March issue of American Bee Journal I wrote about my unpublished work on the necromones of hygienic behavior, and I am happy to report that in April it was published in Scientific Reports[UPDATE: This varroa study is now peer-reviewed and published in Molecular and Cellular Proteomics.]

This mission to peer in to the varroa developmental cycle had a humble beginning, starting with just a paint brush, petri dish and a cheap dissecting microscope, but modern technology allowed it to be so much more. For each life stage, we only needed about 10 micrograms of protein for the mass spectrometry analysis – that’s one one-hundred-thousandth of a gram. From this tiny sample, we still identified over 3,000 unique proteins. To make all this information as useful as possible, we turned it into an interactive website where anyone can click around to explore the mite proteome (http://foster.nce.ubc.ca/varroa/index.html). The website lists the mite proteins found in each developmental stage with a visual depiction of their relative abundance. If you’re a curious informatics whiz with a passion for biology, I invite you to look for biological patterns for yourself. There is always more to discover, and these days, you don’t have to sit sweating at a microscope to be a scientist.

This article appeared in the July 2017 issue of American Bee Journal.

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