Pesticides Deliver a One-Two Punch to Honey Bees

Phys.Org By Society of Environmental Toxicology and Chemistry August 5, 2019

Researchers conduct semi-field experiments on honey bees. Credit: Lang Chen

Researchers conduct semi-field experiments on honey bees. Credit: Lang Chen

Adjuvants are chemicals that are commonly added to plant protection products, such as pesticides, to help them spread, adhere to targets, disperse appropriately, or prevent drift, among other things. There was a widespread assumption that these additives would not cause a biological reaction after exposure, but a number of recent studies show that adjuvants can be toxic to ecosystems, and specific to this study, honey bees.

Jinzhen Zhang and colleagues studied the effects on honey bees when adjuvants were co-applied at "normal concentration levels" with neonicotinoids. Their research, recently published in Environmental Toxicology and Chemistry, found that the mixture of the pesticide and the adjuvant increased the mortality rate of honey bees in the lab and in semi-field conditions, where it also reduced colony size and brooding.

When applied alone, the three pesticide adjuvants caused no significant, immediate toxicity to honeybees. However, when the pesticide acetamiprid was mixed with adjuvants and applied to honeybees in the laboratory, the toxicity was quite significant and immediate. In groups treated with combined pesticide-adjuvant concentrates, mortality was significantly higher than the control groups, which included a blank control (no pesticide, no adjuvant, only water) and a control with only pesticide (no adjuvant). Further, flight intensity, colony intensity and pupae development continued to deteriorate long after the application comparative to the control groups.

Zhang noted that this study, "contributed to the understanding of the complex relationships between the composition of pesticide formulations and bee harm," and stressed that "further research is required on the environmental safety assessment of adjuvants and their interactions with active ingredients on non-target species."

https://phys.org/news/2019-08-pesticides-one-two-honey-bees.html

Quantum Dots Track Pollinators

The Optical Society By Stewart Wills February 18, 2019

A bee, caught after visiting a flower whose pollen grains had been labelled with quantum dots. The glowing dots show evidence of the bee’s travels, and how the pollen attaches to it, when the insect is examined under the microscope in UV light. [Image: Corneile Minnaar]

A bee, caught after visiting a flower whose pollen grains had been labelled with quantum dots. The glowing dots show evidence of the bee’s travels, and how the pollen attaches to it, when the insect is examined under the microscope in UV light. [Image: Corneile Minnaar]

A particularly sobering aspect of global environmental degradation is the rapid decline of insect populations. One recent study in the journal Biological Conservation estimated that 40 percent of the world’s insect species could go extinct within the next three decades, owing to habitat loss due to agriculture and urbanization, pesticides, climate change and other insults.

Quite apart from playing havoc with the food web, declines in certain insect populations threaten the bugs’ crucial role as pollinators. Humans rely on insects to pollinate more than 30 percent of food crops—a huge service that nature provides free of charge.

That makes it essential to understand which insects are pollinating which plants—even to the point of tracking individual pollen grains from flower to flower via their insect vectors. But a robust, useful system for labeling the tiny grains, which are subject to the vicissitudes of wind and weather in addition to the mazy paths of insects, has been fiendishly difficult to devise. Now, a pollination biologist in South Africa has hit upon a novel answer: tag the pollen with fluorescent quantum dots (Meth. Ecol. Evol., doi: 10.1111/2041-210X.13155).

Dots, flowers and pollen

Quantum dots (QDs) are luminescent semiconductor nanocrystals that, when excited by light of a specific wavelength (such as UV), re-emit at visible wavelengths, with the specific emission wavelength depending on the size of the quantum dot. They’ve found use in a wide variety of contexts including biomedical study (see, “Quantum Dots for Biomedicine,” OPN, April 2017). Indeed, the pollination biologist behind the new study, Corneile Minnaar of Stellenbosch University, South Africa, reportedly got the idea for pollen tracking with QDs from a paper on their potential use in targeting and imaging cancer cells.

Corneile Minnaar, applying a solution of lipid-tagged quantum dots to the business end of a flower. [Image: Ingrid Minnaar]

Corneile Minnaar, applying a solution of lipid-tagged quantum dots to the business end of a flower. [Image: Ingrid Minnaar]

To use QDs to track individual pollen grains, Minnaar—who began the work as a Ph.D. student at Stellenbosch, where he’s now a postdoc in the lab of pollination biologist Bruce Anderson—first had to figure out how to tie the dots to the pollen. To do so, he began with commercially available, nontoxic CuInSexS2−x/ZnS (core/shell) QDs with four different emission wavelengths: 550 nm (green), 590 nm (yellow), 620 nm (orange) and 650 nm (red). Next, Minnaar chemically tied the QDs to an oleic‐acid ligand molecule that would latch onto the lipid-rich “pollenkitt” that surrounds pollen grains—the same substance that makes pollen stick to the coats of pollinators like honeybees.

Minnaar then took the lipid-doped QDs and dissolved them into a volatile hexane solvent, and micro-pipetted drops of the solvent onto the pollen-rich anthers on flowers of four different plant species. The ligand-bearing QDs quickly stuck to the pollenkitt on the grains, as expected, and the volatile hexane rapidly evaporated away. The result: flowers packed with potentially trackable, QD-labeled pollen.

Building an “excitation box”

The next problem to be solved was how actually to read the signal from the tagged pollen. While Minnaar says he started with a toy pen with a UV LED light to excite the fluorescence in the dots, he clearly needed something a bit more scalable. To get there, he used a 3-D printer to create a black “quantum-dot excitation box” that could fit under a dissection microscope, and that included four commercial UV LEDs, a long-pass UV filter, and supporting housing. In a press release accompanying the work, Minnaar said the UV box could “easily be 3D-printed at a cost of about R5,000 [around US$360], including the required electronic components.”

Minnaar tested the ability of the pollen grains to hold onto the QDs by agitating samples in an ethanol solution, and found that the grip was firm. Also, in a controlled, caged experiment, he trained honeybees to move from tagged to untagged samples of a particular flower species, and found that labeling the grains with the QDs had no effect on the grains’ ability also to stick to the bees.

Robust system

The general robustness of the system suggests it could serve well in tracking pollinators in wild settings, quantifying parameters such as pollen loss and the importance of certain species to sustaining specific kinds of plants. That said, there are still a few limitations, according to the paper. One is that right now, “there are only four commercially available, distinguishable quantum dot colors in the visible range,” which could limit studies to only four plant species at a time. And, while initial tests were encouraging, more work needs to be done to determine whether the labeling and application process has effects on pollen viability that could complicate experiments or affect pollinator behavior.

One other, unavoidable drawback, notwithstanding Minnaar’s clever microscope setup, is the sheer labor of counting and checking the glowing pollen grains to amass experimental data. That's a task likely to while away the hours of grad students for years to come, irrespective of the technique used to label the grains. “I think I've probably counted more than a hundred thousand pollen grains these last three years,” Minnaar said.

Source: British Ecological Society

https://www.osa-opn.org/home/newsroom/2019/february/quantum_dots_track_pollinators/

Honey Bee Parasites Feed on Fatty Organs, Not Blood

Phys.org University of Maryland January 14, 2019

In this electron micrograph, a parasitic mite,  Varroa destructor , is wedged between the abdominal plates of a honey bee's exoskeleton. Credit: UMD/USDA/PNAS

In this electron micrograph, a parasitic mite, Varroa destructor, is wedged between the abdominal plates of a honey bee's exoskeleton. Credit: UMD/USDA/PNAS

Honey bee colonies around the world are at risk from a variety of threats, including pesticides, diseases, poor nutrition and habitat loss. Recent research suggests that one threat stands well above the others: a parasitic mite, Varroa destructor, which specializes in attacking honey bees.

For decades, researchers have assumed that varroa mites feed on blood, like many of their mite and tick cousins. But new University of Maryland-led research suggests that varroa mites instead have a voracious appetite for a honey bee organ called the fat body, which serves many of the same vital functions carried out by the human liver, while also storing food and contributing to bees' immune systems.

The research, published in the Proceedings of the National Academy of Sciences on January 14, 2019, could transform researchers' understanding of the primary threats to honey bees while pointing the way toward more effective mite treatments in the future.

"Bee researchers often refer to three Ps: parasites, pesticides and poor nutrition. Many studies have shown that varroa is the biggest issue. But when compromised by varroa, colonies are also more susceptible to the other two," said UMD alumnus Samuel Ramsey (Ph.D. '18, entomology), the lead author of the paper. "Now that we know that the fat body is varroa's target, this connection is now much more obvious. Losing fat body tissue impairs a bee's ability to detoxify pesticides and robs them of vital food stores. The fat body is absolutely essential to honey bee survival."

In addition to breaking down toxins and storing nutrients, honey bee fat bodies produce antioxidants and help to manage the immune system. The fatty organs also play an important role in the process of metamorphosis, regulating the timing and activity of key hormones. Fat bodies also produce the wax that covers parts of bees' exoskeletons, keeping water in and diseases out.

According to Ramsey, the assumption that varroa mites consume honey bee blood (more accurately called hemolymph in insects) has persisted since the first paper on the topic was published in the 1960s. Because this paper was written in Russian, Ramsey said, many researchers opted to cite the first English-language papers that cited the original study.

In this cross-section of a honey bee's abdomen, a parasitic varroa mite (orange) can be seen lodged between the bee's abdominal plates, where the mite feeds on honey bee fat body tissue. Credit: UMD/USDA/PNAS

In this cross-section of a honey bee's abdomen, a parasitic varroa mite (orange) can be seen lodged between the bee's abdominal plates, where the mite feeds on honey bee fat body tissue. Credit: UMD/USDA/PNAS

"The initial work was only sufficient to show the total volume of a meal consumed by a mite," Ramsey added. "It can be a lot easier to cite a recent summary instead of the original work. Had the first paper been read more widely, many folks might have questioned these assumptions sooner."

Ramsey noted several observations that led him to question whether varroa mites were feeding on something other than hemolymph. First, insect hemolymph is very low in nutrients. To grow and reproduce at the rates they do, varroa mites would need to consume far more hemolymph than they would be able to acquire from a single bee.

Second, varroa mites' excrement is very dry—contrary to what one would expect from an entirely liquid blood diet. Lastly, varroa mites' mouthparts appear to be adapted for digesting soft tissues with enzymes then consuming the resulting mush. By contrast, blood-feeding mites have very different mouthparts, specifically adapted for piercing membranes and sucking fluid.

The first and most straightforward experiment Ramsey and his collaborators performed was to observe where on the bees' bodies the varroa mites tended to attach themselves for feeding. If the mites grabbed on to random locations, Ramsey reasoned, that would suggest that they were in fact feeding on hemolymph, which is distributed evenly throughout the body. On the other hand, if they had a preferred site on the body, that could provide an important clue to their preferred meal.

"When they feed on immature bees, mites will eat anywhere. But in adult bees, we found a very strong preference for the underside of the bees' abdomen," Ramsey said. "More than 90 percent of mites we found on adults fed there. As it happens, fat body tissue is spread throughout the bodies of immature bees. As the bees mature, the tissue migrates to the underside of the abdomen. The connection was hard to ignore, but we needed more evidence."

Ramsey and his team then directly imaged the wound sites where varroa mites gnawed on the bees' abdomens. Using a technique called freeze fracturing, the researchers used liquid nitrogen to freeze the mites and their bee hosts, essentially taking a physical "snapshot" of the mites' feeding habits in action. Using powerful scanning electron microscopes to visualize the wound sites, Ramsey saw clear evidence that the mites were feeding on fat body tissue.

This microscopic image shows a varroa mite that has consumed honey bee fat body tissue tagged with Nile red, a fat-soluble fluorescent dye. Observing this red fluorescence in the mites' digestive systems helped researchers determine that varroa mites feed on honey bee fat body tissue--not blood, as previously assumed. Credit: UMD/USDA/ PNAS

This microscopic image shows a varroa mite that has consumed honey bee fat body tissue tagged with Nile red, a fat-soluble fluorescent dye. Observing this red fluorescence in the mites' digestive systems helped researchers determine that varroa mites feed on honey bee fat body tissue--not blood, as previously assumed. Credit: UMD/USDA/PNAS

"The images gave us an excellent view into the wound sites and what the mites' mouthparts were doing," Ramsey said. "We could see digested pieces of fat body cells. The mites were turning the bees into 'cream of honey bee soup.' An organism the size of a bee's face is climbing on and eating an organ. It's scary stuff. But we couldn't yet verify that blood wasn't also being consumed."

To further shore up their case, Ramsey and his colleagues fed bees with one of two fluorescent dyes: uranine, a water-soluble dye that glows yellow, and Nile red, a fat-soluble dye that glows red. If the mites were consuming hemolymph, Ramsey expected to see a bright yellow glow in the mites' bellies after feeding. If they were feeding on fat bodies, on the other hand, Ramsey predicted a telltale red glow.

"When we saw the first mite's gut, it was glowing bright red like the sun. This was proof positive that the fat body was being consumed," Ramsey said. "We've been talking about these mites like they're vampires, but they're not. They're more like werewolves. We've been trying to drive a stake through them, but turns out we needed a silver bullet."

To drive the proverbial final nail into the coffin of the idea that mites feed on hemolymph, Ramsey performed one last experiment. First, he painstakingly perfected the ability to raise varroa mites on an artificial dietary regimen—hardly an easy task for a parasite that prefers meals from a live host. Then, he fed them diets composed of hemolymph or fat body tissue, with a few mixtures of the two for good measure.

The results were striking: mites fed a diet of pure hemolymph starved, while those fed fat body tissue thrived and even produced eggs.

"These results have the potential to revolutionize our understanding of the damage done to bees by mites," said Dennis vanEngelsdorp, a professor of entomology at UMD and a co-author of the study, who also served as Ramsey's advisor. "Fat bodies serve so many crucial functions for bees. It makes so much more sense now to see how the harm to individual bees plays out in the ways that we already know varroa does damage to honey bee colonies. Importantly, it also opens up so many new opportunities for more effective treatments and targeted approaches to control mites."

Read more at: https://phys.org/news/2019-01-honey-bee-parasites-fatty-blood.html#jCp

More information: Samuel D. Ramsey el al., "Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph," PNAS (2018). www.pnas.org/cgi/doi/10.1073/pnas.1818371116 

Journal reference: Proceedings of the National Academy of Sciences 

Provided by: University of Maryland

Shedding New Light on Honey Bee Chromosomes

Bug Squad Author: Kathy Keatley Garvey Published on: December 3, 2018

Honey bee, Apis mellifera. (Photo by Kathy Keatley Garvey)

Honey bee, Apis mellifera. (Photo by Kathy Keatley Garvey)

Honey bee geneticists with long ties to UC Davis are putting together those missing pieces of the puzzle involving bee chromosomes.

Newly published research by a team of Germany-based honey bee geneticists, collaborating with Robert Eugene (“Rob”) Page Jr., of Arizona State University/University of California, Davis, offers new insights in the ability to modify and study the chromosomes of honey bees.

Martin Beye, a professor at the University of Düsseldorf, Germany and a former postdoctoral fellow in Page's lab at UC Davis, served as the lead author of the research, “Improving Genetic Transformation Rates in Honeybees,” published in Scientific Reports in the journal Nature.

The researchers accomplished the work in Beye's lab in Germany and the Page labs.

“The significance of this paper lies in the ability to modify the chromosomes of honey bees and study the effects of individual genes,” said Page, former professor and chair of the UC Davis entomology department before capping his academic career as the Arizona State University provost.

“The honey bee genome,” Page explained, “is composed of about 15,000 genes, each of which operates within a complex network of genes, doing its small, or large, share of work in building the bee, keeping its internal functions operating, or helping it function and behave in its environment. The ability to transform, change, genes, or add or delete genes from chromosomes of bees, has been exceptionally challenging and the effort spans decades. Martin tackles problems such as this. He takes on the most challenging genetic problems and solves them.”

Beye was the first to map the major sex-determining gene for honey bees, considered one of the most important papers ever published on honey bee genetics. He “then moved on and developed a way to implement gene editing, being able to alter single genes within the genome,” Page related. “Now he has developed a method to introduce new genetic material into the honey bee.”

In their abstract, the six-member team wrote that “Functional genetic studies in honeybees have been limited by transformation tools that lead to a high rate of transposon integration into the germline of the queens. A high transformation rate is required to reduce screening efforts because each treated queen needs to be maintained in a separate honeybee colony. Here, we report on further improvement of the transformation rate in honeybees by using a combination of different procedures.”

Specifically, the geneticists employed a hyperactive transposase protein (hyPBaseapis), tripling the amount of injected transposase mRNAs. They injected embryos into the first third (anterior part) of the embryo. These three improvements together doubled the transformation rate from 19 percent to 44 percent.

“We propose that the hyperactive transposase (hyPBaseapis) and the other steps used may also help to improve the transformation rates in other species in which screening and crossing procedures are laborious,” they wrote in their abstract.

For their research, the scientists chose feral Carniolan or carnica colonies. Carniolans, a darker bee, are a subspecies of the Western honey bee, Apis mellifera.

Beye joined the Page lab in 1999 as the recipient of a Feodor Lynen Research Fellowship, an award given to the brightest young German Ph.Ds to provide an opportunity for them to work in the laboratories of U.S. recipients of the Alexander von Humboldt Research Prize. Page, who won the Humboldt Prize in 1995, continues to focus his research on honey bee behavior and population genetics, particularly the evolution of complex social behavior.

Following his postdoctoral fellowship, Beye returned to the Page labs at UC Davis and ASU as a visiting scientist. (link to https://www.ucdavis.edu/news/honeybee-gene-find-ends-150-year-search ) Beye spoke at UC Davis this spring as part of his Humboldt-funded mini sabbatical, the guest of Page and hosted by the Department of Entomology and Nematology. During his visit, he and UC Davis bee scientist Brian Johnson developed collaborative projects that they will begin in the spring of 2019. “This is exactly what the Alexander von Humboldt foundation wants – to build and extend interactive networks of researchers,” Page commented.

About Robert Page Jr.
Noted honey bee geneticist Robert Page Jr., author of The Spirit of the Hive: The Mechanisms of Social Evolution, published by Harvard University Press in 2013, recently received the Thomas and Nina Leigh Distinguished Alumni Award, UC Davis Department of Entomology and Nematology.

Page received his doctorate in entomology from UC Davis and served as a professor and chair of the UC Davis entomology department before capping his academic career as the Arizona State University (ASU) provost. He maintained a honey bee breeding program managed by bee breeder-geneticist Kim Fondrk at the Harry H. Laidlaw Jr. Honey Bee Research Facility, UC Davis, for 24 years, from 1989 to 2015.

Now provost emeritus of ASU and Regents Professor since 2015, he continues his research, teaching and public service in both Arizona and California and has residences in both states. He plans to move to California in December.

Page focuses his research on honey bee behavior and population genetics, particularly the evolution of complex social behavior. One of his most salient contributions to science was to construct the first genomic map of the honey bee, which sparked a variety of pioneering contributions not only to insect biology but to genetics at large.

Resources:

UC Davis Behind the Groundbreaking Discovery of Honey Bee Sex Determination

About Robert E. Page Jr., Recipient of UC Davis Alumni Award

Bee Mite Arrival in Hawaii Causes Pathogen Changes in Honeybee Predators

UC Riverside By Iqbal Pittalwala January 8, 2019

bee mite arrival in Hawaii.jpg

UC Riverside-led research, done on the Big Island, shows effects of mite introduction have cascaded through entire pathogen communities

The reddish-brown varroa mite, a parasite of honeybees and accidentally introduced in the Big Island of Hawaii in 2007-08, is about the size of a pinhead. Yet, its effects there are concerning to entomologists because the mite is found nearly everywhere honeybees are present.

A team led by entomologists at the University of California, Riverside, performed a study on the Big Island and found viruses associated with the mite have spilled over into the western yellowjacket, a honeybee predator and honey raider. The result is a hidden, yet remarkable, change in the genetic diversity of viruses associated with the larger pathogen community of the mite and wasp, with repercussions yet to be understood.

Erin Wilson Rankin examines a western yellowjacket. (I. Pittalwala/UC Riverside)

Erin Wilson Rankin examines a western yellowjacket. (I. Pittalwala/UC Riverside)

“Already, we are seeing that the arrival of the varroa mite in honeybee populations in Hawaii has favored a few virulent strains,” said Erin E. Wilson Rankin, an assistant professor of entomologyand lead investigator of the study published Jan. 9 in the Proceedings of the Royal Society B. “We do not know what the effects of these strains will be. What we know is that the effects of the varroa mite have cascaded through entire communities in Hawaii and probably around the world.”

In particular, the researchers saw a loss in the diversity of deformed wing virus, or DWV, variants, resulting in new strains whose impact is hard to predict. DWV, widespread in honeybee populations globally and made up of several variants, is thought to be partly responsible for global die-off of honeybee colonies. DWV infects bumblebees and has been detected in other insects. The yellowjacket wasps can acquire this virus directly or indirectly from honeybees.

The western honey bee.

The western honey bee.

By a stroke of luck, the researchers had the benefit of studying the honeybee and yellowjacket populations on the Big Island both before and after the varroa mite was introduced there. They saw more association of honeybees with pathogens after the appearance of the mite. Indeed, some pathogens were detected in the honeybee and wasp populations only after the mite was introduced to the island.

“This is one of the first descriptions of pathogens in the western yellowjacket,” Wilson Rankin said. “Evidently, pathogens known to be associated with varroa have spread into non-bee species, despite the mite itself being a bee specialist. We suspect the spread in yellowjackets is partly due to the wasp’s propensity to prey upon bees, which is one way the wasps may be exposed to the pathogens.”

Wilson Rankin noted the pathogens are often incorrectly called “bee pathogens” because they were first found in bees. The pathogens, however, are found in a wide variety of insects.

“We are seeing entirely different predators being affected,” she said. “The mite is not vectoring viruses to the wasps. The viral spread is happening through predation and through flowers. Predators may be passing on pathogens to other species. The yellowjacket, for example, preys on both honeybees and native bees, and may explain why both bee populations are showing the same viruses.”

Wilson Rankin explained wasps have been overlooked by researchers because these arthropods do not have “warm, fuzzy, and furry connotations.”

The western yellowjacket is a honey bee predator and honey-raider.

The western yellowjacket is a honey bee predator and honey-raider.

“They look scary,” she added. “People also get stung by them. People are more afraid of wasps than bees. But our work shows we can examine the health of the arthropod community by using species other than bees. We show for the first time that a predator is being affected by a parasite that does not even infect it.”

The researchers sampled 25-45 bees and wasps for one part of the study, and then about 100 individuals, analyzed in groups, for each of the species during the period before and after the mite was introduced to the Big Island. The researchers did not study native bees, focusing instead on honeybees and yellowjacket wasps, neither of which is native to Hawaii. 

“Our findings suggest that pathogen transmission from domesticated bees, such as honeybees, may be important even for non-bee insects like the wasps we studied,” said Kevin J. Loope, the research paper’s first author, who worked as a postdoctoral scholar in the Wilson Rankin lab during the study. “The impacts may be more subtle than previously observed: we found changes in the genetic variation of viruses found in the wasps, but not changes in the amount of virus. These findings suggest we should look more broadly and in greater detail to figure out how moving domesticated bees for agriculture may influence wild populations of insects.

Loope, now a research assistant professor in the Department of Biology at Georgia Southern University, explained that finding overlap in the pathogens of yellowjacket wasps and domesticated bees also means that using pathogens to control undesirable wasp populations is risky.

“Any biological control efforts using pathogens should be carefully evaluated to prevent inadvertent harm to beneficial bees,” he said.

Kevin Loope excavates a yellowjacket nest in Volcano, Hawaii. (Jessica Purcell/UC Riverside)

Kevin Loope excavates a yellowjacket nest in Volcano, Hawaii. (Jessica Purcell/UC Riverside)

He added that the research team was surprised to find a dramatic difference in the viral genetic diversity between the wasp samples from the two periods — before and after the varroa mite was detected on the Big Island.

“We had predicted we would observe a decline in wasp viral diversity matching the decline described in honeybees in Hawaii, but we were still surprised to see this borne out in the data,” he said. “It’s not so often that you see what you’ve predicted in biology.”

Wilson Rankin and Loope were joined in the research by Philip J. Lester of Victoria University of Wellington, New Zealand; and James W. Baty of Malaghan Institute of Medical Research, New Zealand. Genetic analyses on the bee and wasp samples were performed at UCR and in New Zealand.

Wilson Rankin was supported by grants from the National Science Foundation and the Hellman Fellows Fund. Loope was supported by a postdoctoral fellowship from the National Institute of Food and Agriculture of the U.S. Department of Agriculture.

https://news.ucr.edu/articles/2019/01/08/bee-mite-arrival-hawaii-causes-pathogen-changes-honeybee-predators