Honey Bee Social Immunity
by Roberto
Flying insects are key to the functioning of many ecosystems, where they play numerous important roles such as pollination. Thus, the dramatic reduction in their numbers is cause for great concern. From a human perspective, the observed decline of many honey bee (Apis mellifera, Fig. 1) populations is particularly troubling because of their critical role in the food chain. In 2018, Christoph wrote a two-part post (here and here) on the disturbing effects of the widely used herbicide glyphosate on honey bee gut microbiota and its consequent effect on colony health. Today I focus on another threat to the honey bee, the affliction known as American foulbrood, and touch on some encouraging developments regarding its possible control.
Fig. 1. Honey bee, Apis mellifera. Photo credit Roberto Kolter
Let's begin with a bit of background on American foulbrood. This name does not refer to its geographic distribution since it is a worldwide disease. Rather, it's a reflection of where it was first studied. As the name indicates, this disease afflicts the youngest individuals in a colony, one-day-old larvae being the most susceptible. It is the most widespread and destructive of the honey bee brood diseases. The microbial villain behind this disease is the endospore former Paenibacillus larvae. Spores within the larval gut can germinate and then enter rapid vegetative growth, killing the larvae along the way. When bacteria stop growing, they sporulate again. The spores do not grow on adults, but they stick to them. Nurse bees then act as vectors that infect other larvae while feeding them. Thus, the disease can spread quickly to all the brood. There are no good therapies for this disease. Some beekeepers use antibiotics (though, not surprisingly resistance quickly becomes a problem), there's some use of lactic acid bacteria as probiotics, and even some forays into phage therapy. Because the spores are so highly resistant, they are not easily eradicated so the potential for disease persists for very long times. Sadly, at present the most effective method of control is completely destructive. Afflicted beehives and all instruments used in their care are burned. It's a dreadful image (Fig. 2).
Fig. 2. Burning of hives afflicted with American foulbrood. Source
However, there's some hope for a new approach based on recent findings of how honey bees themselves seem to manage the disease. While insects do not produce antibodies, their innate immune response does protect them against pathogens. When exposed to pathogens or pieces of pathogens (so called "pathogen-associated molecular patterns" or PAMPs) insects respond by producing a host of molecules that inhibit pathogen growth, including numerous antimicrobial peptides. These are produced in the "fat body," the insect equivalent of the liver. All of which means that prior exposure to pathogens will render a honey bee more resistant to pathogens. This increased resistance can be transferred to the next generation, a process known as "trans-generational immune priming." For a long time this was believed to be a trait of animals that produced antibodies (think of the immunity gained by babies when they take in the antibodies present in their mother's milk). But over the last couple of decades it's become clear that even without antibodies, insects can prime their young against infections. Maternal exposure to immune elicitors, be they live or dead bacteria or pieces of these, leads to higher immunity in the offspring.
One important player involved in trans-generational immune priming in bees is the egg yolk protein vitellogenin. This protein is produced in large amounts in the bee's fat body and then secreted into the hemolymph and from there into the eggs. In an egg-laying queen, it can be 70% of the hemolymph protein. For long believed to play mainly a nutritional role, a 2015 paper by Salmela et al. pointed to its role in transfer of immunity from the queen mother to its offspring. The authors suspected this new role for vitellogenin because of prior work in fish. They first showed that the protein binds to bacteria and PAMPs. Using extracted bee ovaries, they then showed that purified vitellogenin mediated the uptake of PAMPs into eggs (Fig. 3). Moreover, no other hemolymph protein had this ability. From these results the authors speculated that if a queen bee is exposed to a pathogen, it can transfer immune elicitors, that is PAMPs, to its progeny.
Fig. 3. Freshly detached ovaries were incubated in buffer containing fluorescent (Texas red) E. coli fragments, embedded for cryo-sectioning, and imaged using bright field and fluorescence microscopy. These images are of single eggs in a cryo-sectioned ovary; 20 x magnification, the scale is 50 μm. In the vitellogenin-incubated ovaries, eggs with internalized fluorescent material were observed (left). In the control (right), the bacterial fragments were, typically, found as bright aggregates on the membranes surrounding the eggs. The images represent N = 6 queens. Adapted from source.
If the queen can transfer PAMPs to her eggs, the question immediately arises, where does the queen acquire those PAMPs to begin with? Recall that queen bees spend most of their lives in the hive, venturing out only for their mating flights. The authors reasoned that, if queens are to be exposed, it's likely going to be through eating contaminated food given to them by workers who do spend time outside. And, since the queen bee feeds exclusively on royal jelly, that became a target of investigation. In two papers by Harwood et al. from 2019 and 2021, the authors present evidence that royal jelly made by nurse worker bees may act as a vehicle to transfer immune elicitors. In the first paper they showed that these pathogen fragments were moved from the bees' guts to their hypopharyngeal glands, where royal jelly is made. Using RNA interference to decrease the synthesis of vitellogenin, they showed the requirement of this protein for the transport. In the second paper, they showed that not only did the glands get the PAMPs, the royal jelly itself retained the PAMPs. In addition, royal jelly made by workers exposed to killed pathogen had higher levels of the antimicrobial peptide defensin‑1.
These last results have an important implication for the brood itself. While the queen is fed royal jelly its entire life, all larvae are fed royal jelly during their first three days of development, which happen to be the time when they are most susceptible to the pathogen P. larvae. Thus, exposed workers can provide immunity to the young directly, not only through the mother queen. Ecologists term this social immunity, which bears some similarities to immunization campaigns in humans. This parallel did not escape investigators. If workers immunize the brood through what they feed them, would an intentional oral vaccine be effective? Apparently, yes. Dickel et al. recently published the results of a first oral vaccination trial with inactivated P. larvae, where they showed modest protection. And just in January of this year the United States Department of Agriculture approved the use of this, the first vaccine for honey bees. Time will tell if this provides us with at least a partial solution to the problem of declining honey bee populations.
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