An Inside Look at How the Varroa Mite’s True Diet Was Discovered
By Samuel Ramsey, Ph.D., Ron Ochoa, Ph.D., Joe Mowery, Chris Pooley, Connor Gulbronson, Ph.D., and Gary Bauchan, Ph.D.
Varroa destructor, a mite that feeds on honey bees, is the greatest single driver of the global honey bee health decline. For years, scientists and bee keepers were told that the Varroa mite feeds on hemolymph (the bee’s blood) based on scientific research done long ago. However, for our research team led by Samuel Ramsey, Ph.D., this did not make sense, as the mite’s digestive system and mouthparts just did not seem like they were structured properly for blood feeding. Their closest evolutionary relatives were not blood feeders either. As pointed out by insect-rearing expert Allen Cohen, Ph.D., even their excrement was all wrong for them to be blood feeders.
So, how and on what exactly do these mites feed? Our discovery is documented in a paper published in late January in Proceedings of the National Academy of Sciences.
Ramsey, then a Ph.D. student of Dennis vanEngelsdorp, Ph.D., at the University of Maryland, College Park, began his inquiry with a simple question: Are Varroa mites feeding in only one spot? If the mites can feed anywhere on honey bees (Apis mellifera) like a tick can on a person, then they are likely feeding on a tissue available all over the bee’s body (like hemolymph). However, if the mites feed only in one spot, maybe they are feeding on a tissue specific to that location. Out of 104 observations, the mites on adult bees were only found underneath the abdominal plates of their host. Ramsey considered that this might be their feeding site but knew he would need some help to prove it.
This led Ramsey to the office of Ron Ochoa, Ph.D., research entomologist at the Systematic Entomology Lab and the Electron and Confocal Microscopy Unit (ECMU) of the U.S. Department of Agriculture’s Agricultural Research Service (USDA-ARS) in Beltsville, Maryland, and the leading mite expert at the USDA-ARS. In addition, to the idea that Varroa mites only feed on the hemolymph of developing larvae and pupae, researchers were questioning whether the mites feed on adult bees at all. Some had already concluded that feeding only occurs on immature bees because they were never seen feeding on adult bees and because we have long been referring to the life stage of mites found on adult bees as “phoretic,” a term that denotes a nonfeeding stage that uses another organism as a mode of transit.
Gary Bauchan, Ph.D., director of the ECMU, Ochoa, and Ramsey used liquid nitrogen to instantly freeze honey bees that had mites in the region Ramsey proposed feeding was occurring. The exact position of the mites on the honey bees was imaged in a low-temperature scanning electron microscope (LT-SEM). The frozen bees were then removed from the LT-SEM and the mites carefully detached from the bees. During this process, Ramsey monitored Ochoa’s exposure time to the liquid nitrogen to avoid burning himself while he removed the mites from the frozen bees—a manual process that had to be done in approximately 10 seconds. The bees were then returned to the LT-SEM and the location where the mite was located was imaged.
The mite presses itself so tightly to the soft membrane between the bee’s abdominal plates that it leaves a distinct impression of its legs and palps and a mouth-shaped wound in the membrane. Additionally, at this feeding site the ambulacra (foot pads) from the front legs remained stuck to the bees’ membrane, allowing us to quickly orient ourselves to the positioning of the mite’s body relative to the bee’s. We repeated this procedure 10 times to prove this was where the mites were feeding.
A longitudinal section via freeze fracture directly through the bee and its attached mite confirmed in detail that Varroa‘s relatively short mouthparts (200 microns) come to rest just inside of the bee, which is an area surrounded by fat body tissue and hemolymph, leaving us with the conclusion that the mites are feeding on adult bees. It further suggested to us that the mites could be feeding on a nutrient-dense organ called the fat body, which is analogous to the mammalian liver.
To determine if fat body was being consumed, the team had to get a closer look. Joe Mowery, support scientist at the ECMU, and Ramsey collected and prepared specimens by affixing the feeding mite onto the bee with cyanoacrylate glue prior to processing. Then, ultra-thin sections were obtained and studied using a transmission electron microscope (TEM). Clear evidence was discerned that mites were breaking down and removing fat body tissue via extra-oral digestion (and creating an entry point for colonies of microbes).
While observation and electron microscopy had produced clear evidence for fat body feeding, the research turned to Connor Gulbronson, Ph.D., Oak Ridge Institute for Science and Education post-doctoral fellow with the USDA-ARS Floral and Nursery Plants Research Unit (located within the ECMU), and his expertise in fluorescence and confocal microscopy to determine if fat body is the primary tissue consumed. Ramsey generated a diet containing two fluorescent biostains to label the bee’s hemolymph (yellow) and fat body (red). Gulbronson developed protocols to bleach the exoskeletons of the mites, allowing the internal structures to be imaged.
The bees with their glowing innards were then exposed to hungry Varroa. The results were stunning. Gulbronson and Ramsey imaged mites with brilliantly red fluorescing digestive systems with very little discernible fluorescence in the yellow range. To be sure the biostains were staining the correct tissues, each bee was dissected and imaged as well for comparison with the fluorescence inside the mites. This work provided the clearest evidence yet that the mites were primarily consuming fat body tissue.
All facets of this project had lined up pointing to one conclusion, but Ramsey wanted to be sure that this tissue clearly consumed in adult bees was the same tissue consumed in developing larvae and pupae. To determine this relationship, Ramsey developed a novel in vitro rearing system, which mimicked the brood cells in which immature honey bees develop, and constructed decoy pupae to mimic the mite’s host. This allowed Ramsey and lab technicians Judith Joklik and David Lim to undertake the tedious task of feeding the mites diets composed of the different tissues of interest.
Mites fed exclusively honey bee hemolymph starved quickly and produced very few eggs, no different from the control group given no food at all. However, mites fed exclusively fat body survived substantially better than all other treatments and produced far more eggs. This was the first time anyone had was able to rear Varroa mites in vitro without directly feeding on bees. These discoveries provided the study with the very last line of evidence needed to say conclusively that Varroa are not blood-feeders but rather feed on fat.
Collectively, this multi-disciplinary approach of feeding assays, observational analysis, and various microscopy technologies generated a paradigm shift in our understanding of how, where, and what the Varroa mite obtains when parasitizing host honey bees. This research provides a path forward for the development of strategies to controlling this major pest of honey bees worldwide.
Proceedings of the National Academy of Sciences
Samuel Ramsey, Ph.D., is a post-doctoral researcher in the Entomology Department at the University of Maryland, in College Park, Maryland. Email: email@example.com. Ron Ochoa, Ph.D., is a research entomologist at the Systematic Entomology Lab of the U.S. Department of Agriculture’s Agricultural Research Service (USDA-ARS), in Beltsville, Maryland. Email: firstname.lastname@example.org. Joe Mowery is a support scientist at the USDA-ARS Electron and Confocal Microscopy Unit (ECMU) in Beltsville, Maryland. Email: email@example.com. Chris Pooley is an IT specialist at the USDA-ARS ECMU. Email: firstname.lastname@example.org. Connor Gulbronson, Ph.D., is an Oak Ridge Institute for Science and Education post-doctoral fellow at the USDA-ARS Floral and Nursery Plants Research Unit in Beltsville, Maryland. Email: email@example.com. Gary Bauchan, Ph.D., is director of the USDA-ARS ECMU. Email: firstname.lastname@example.org.