Protections and Interactions in the Beewolf Nursery

by Tobias Engl
 

A note in advance, by Christoph. Since 2006, we have had 144 entries in our 'Symbioses' category, and this post is #145. Do we need say more to reveal our al­most symbiotic relationship with symbioses? Elio introduced the beewolf–Streptomyces symbiosis to STC in his post Quick, Who Discovered Antibiotics? back in 2007 (here's an image of a female beewolf with prey). He then asked me in 2014 if I would like to write the update that was due. I gladly agreed and have been in­fested ever since; see A Snippet: Antibiotics In The Nursery. The beewolves (Philantini), inhabit all continents with more than 130 known species, and always in species-specific symbiosis with Streptomyces philanthri strains, with which they have co-evolved for about 70 million years. This species abundance among the Philantini and their S. philantri symbionts led to research results that I discussed in Even More Antibiotics In The Nursery! in 2018. Last year then, I asked an ex­pert on the beewolf–Streptomyces symbiosis, Tobias Engl, if he would like to bring us up to date on STC. He wanted to, and this is his detailed report. I apologize for keeping his fine manuscript on my desk gathering dust for too long. But finally, and lightly edited, here it goes...
 

 

(click to enlarge)
Figure 1. The beewolf female’s antennal gland secretion (AGS) blocks diffusion of NO⦁ both in vivo and in vitro. (A) Beewolf brood cell in an observation cage, with pa­ralyzed bees at the bottom of the brood cell and the beewolf egg on top of one of the bees. The AGS con­taining Streptomyces bacteria is visible as small white specks at the ceiling of the brood cell, in this case a transparent plastic sheet. Scale bar: 4 mm. Frontispiece: Figure 1, slightly cropped. Source

Beewolves, their larvae, and their Streptomyces symbionts not only use multiple, largely dif­ferent classes of chemical compounds to protect themselves against detrimental microbial competitors. To be most effective, these compounds are produced and deployed in a precisely timed sequence. Beewolves – for the sake of simplicity I will stick with the European beewolf Philanthus triangulum here – are solitary digger wasps that hunt on bees, paralyze them and provision single eggs with one or more bees in subterranean brood cells ("nurseries"). This large stash of nutrients is not only a delicacy for the beewolf larvae, but also for microbes of any sorts! Especially opportunistic fungi that are everywhere in this earthy environment pose a large threat [1,7]. Thus, beewolves had to come with a trick to ensure the undisturbed devel­op­ment of their young. Not one trick, but actually at least three! And to make things a bit more complicated, or interesting for researchers, these defense mechanisms do not work in isola­tion, but rather hand‑in‑hand in real time. So you will hear a bit on hydrocarbons, on nitric oxide, as well as about Streptomyces bacteria and their antibiotic arsenal. But one thing after the other, and an overview to illustrate this time sequence in Figure 2.
 

Honeybees are an attractive food source for the offspring of a specialized predator because they occur seasonally in large numbers. However, once they are overwhelmed and transported – which is a struggle on its own for a single female beewolf – their furry surface poses a challenge when stored in warm and moist brood cells, the nurseries. Such conditions are not only optimal for the growth of insect larvae, but also for the germination of fungal spores. And the furry bees are ripe with spores after being dragged through the subterranean tunnel system the beewolf lays to plant the nursery. For beewolves it is essential to avoid this to happen, as mould fungi grow fast and produce potent toxins that aim on monopolizing the ample supplies of bees and could easily kill the developing beewolf larva too. Bee­wolf females not only clean the bees meticulously, but at the same time impregnate them with a wax-like layer of hydrocarbons, a mix of various long-chain alkanes and alkenes. Beewolves use these compounds to impregnate their own to surface to avoid water condensation, but also for communication. However, on the bees this layer has exactly the opposite effect: the hydrocarbons covering the entire hairy surface of the bees is highly hydrophobic and thereby prevent water condensation. This practically blocks the access of remaining mould spores to water needed for germination and extends the ‘shelf life' of the paralyzed honeybees [3].
 

(click to enlarge)
Figure 2. Life cycle of the European Beewolf (Philanthus triangulum). (A) Beewolf females hunt Apis mellifera workers and paralyze them by injecting their venom into the bee's thorax. They provide their subterranean brood cells with one to several bees, embalmed in a se­cretion from their postpharyngeal gland. This secretion contains high amounts of long-chain saturated and unsaturated hydrocarbons that prevent infection of the provisions by reducing water condensation. The female deposits an egg on top of the provisions. Before sealing the brood cell, the female applies an antennal gland secretion (AGS) rich in linear unsaturated and saturated hydrocarbons and containing the defensive symbiont Streptomyces philanthi to the brood cell ceiling. (B) The beewolf egg sanitizes the brood cell by releasing high amounts of toxic nitric oxide (NO⦁), with NO⦁ emission peaking at ~14 to 16 h after oviposition. While NO⦁ ef­fectively kills microbial opportunists in the brood cell, S. phi­lanthi withstands NO⦁ via a previously unknown me­chanism. (C) After hatching and feeding on the pro­visioned bees for several days, the larva integrates S. philanthi into its cocoon. On the cocoon surface, the symbionts produce an antibiotic cocktail that provides protection from microbial infestation. (D) After 4 to 6 weeks or in the following summer, the larva undergoes metamorphosis, and the adult ecloses from the cocoon. Source

After the mother beewolf is satisfied with the state of the bees, she smears a secretion of her antennae to the end of a brood cell and finally deposits a single egg into the cell before closing it off. This way no scavengers can disturb her offspring while developing into the next genera­tion, but also the mother has no further possibility to care for them. While she already largely ensured that fungal spores are not able to germinate, actively growing mycelial cells might already be present on the bees or in the soil. These active fungi have no problem with the secreted hydrocarbons, even might use them as a source of nutrition. Now is the time for the beewolf egg to shine, or rather to smell. A few hours after being left on its own, it releases a 2–3 hour long burst of gaseous nitric oxide (NO⦁). The nitric oxide accumulates in the confined space of the brood cell to extremely high concentrations which are lethal to almost all organ­isms [8]. Thereby the beewolf egg basically sterilizes the entire brood cell and gives the em­bryo time to develop. In Figure 3, the nitric oxide (NO⦁) is visualized on a stored egg in a brood cell by spraying the bee with a sensitive, fluorescent dye.
 

After a few days, the developed larvae hatch from the egg and feed for about two weeks on the paralyzed bees. After they consumed all of them, they spin a cocoon in which they lay dormant during winter. Once temperatures rise again, they will moult into an adult wasp and begin the hunt for bees anew. The cocoon is only attached at a single spot to the brood cell wall, thus the dormant larvae kind of hover in the middle of the brood cell to limit their direct contact with anything that could infect them during the hibernation. However, filamentous fungi can breach into the brood cell during the long hibernation time and easily bridge this distance of a few millimetres. To protect themselves, the larvae search for the secretion their mother deposited earlier, scrap it from the brood cell wall and integrate it into the cocoon silk. This secretion contains the symbiotic Streptomyces philanthi bacteria, which start to produce an entire cocktail of chemicals on the cocoon surface. These exhibit potent antifungal activity and form a prophylactic shield for the possibility that fungi might try to infect the larva within [2,6].
 

These three potent mechanisms are highly efficient in safeguarding the beewolf offspring – only 4% of field excavated brood cells showed infestation with mould fungi. However, you might wonder how the symbiotic Streptomyces bacteria that the mother had smeared to the brood cell wall, are able to survive the burst of nitric oxide that killed all other (tested) microorganisms? Researchers from the Kaltenpoth lab at the Max-Planck-Institute for Chemical Ecology in Jena were likewise puzzled by this aspect and addressed in a recent publication. They wondered whether the bacteria themselves were able to deal with the nitric oxide, or whether the beewolf mother had provided them with some sort of protection [4].
 

They first exposed the cultivated Streptomyces philanthi bacteria to nitric oxide concentrations that also occur in beewolf brood cells and com­pared their physiological response to bacteria that were not exposed to nitric oxide as well as to closely related Streptomyces bacteria that life freely in the soil. The beewolf-associated bacteria showed indeed a strong upregulation of enzymes that likely help them to detoxify nitric oxide or mitigate its detrimental effects. The soil bacteria on the other hand did not show this reaction – they died immediately. However, the researchers realized that this could not be the full answer: the S. philanthi bacteria ultimately also died after the exposure to nitric oxide in this artificial setting.
 

(click to enlarge)
Figure 3. Visualization of NO⦁ emission by beewolf eggs using fluorescence imaging. The droplets on the bee with the egg (B) show a bright yellow and orange fluorescence indicating the presence of NO⦁. Images are composites of multiple pictures of the x/y plane and z-axis. Scale bar = 1 mm. Source

To clarify what happens in a brood cell, they transferred some of the female-deposited se­cre­tion to a piece of filter paper that was impregnated with an indicator solution and put this secretion sandwich back into the brood cell. Once exposed to nitric oxide (NO⦁) the indicator turns almost black – but where the female secretion was applied, the filter paper stayed white; it prevented the nitric oxide to reach and react with the indicator. This might be due to the bacteria detoxifying all the NO⦁ but could also be a property of the matrix of this secretion. Previous work had already shown that the secretion contains largely hydrocarbons of a very similar composition like the secretions females use to impregnate themselves and the honey­bees. To test whether the hydrocarbons could prevent the diffusion of nitric oxide, they ex­tracted them from the beewolf surface (without the head that contains additional glands) and added a layer of then to a solution of the same indicator and exposed them to nitric oxide. The covered indicator solution showed only very little coloration, indicating that the hydrocarbon extract effectively prevents the diffusion of nitric oxide. Finally, to test whether only hydrocar­bons can also rescue cultures of S. philanthi when exposed to nitric oxide, they overlaid small colonies of the bacteria with one of the components of the beewolf hydrocarbon mixture, which is liquid at room temperature and commercially available. The covered colonies grew visibly after nitric exposure while uncovered ones stopped to grow and presumably died.
 

Over the years, studies of the highly specialized life of beewolves revealed multiple solutions for a single problem: how to prevent fungal infesta­tion of provision and offspring. This fascinating system revealed not only mechanisms of anti‑fungal defence which are intriguing on their own, but also highlights the intertwined nature of biology. The timing of single steps matter and the different 'players' interact with each other: The Streptomyces bacteria defend the host, but also need to be protected against the nitric oxide sterilization. The hydrocarbons are used for multi­ple purposes. They cover the beewolves themselves, embalm the bees and protect, possibly even feed the Streptomyces bacteria. This highlights that single traits are not free to evolve under a single selection pressure. They might rather be constrained by multiple factors that limit their evolvability. However, such details get only evident by comprehensive studies of this particular wasp's facets of life and should serve as a moti­vation to keep studying the secrets of life around us.
 

If you got hooked on the beewolf–Streptomyces symbiosis, there are some excellent short videos for you. For example, the episode A Wasp Mom’s Gift: Blankets of Bacteria from Ed Yong's I Contain Multitudes on Medium (requires signing-in) or YouTube (with ads) featuring Martin Kaltenpoth, from seven years ago. And secondly, several clips as this or this on the YouTube channel of @WhistlingJoe.

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Tobias is Project Group Leader in the Dept. of Insect Symbiosis (Martin Kaltenpoth) at the MPI for Chemi­cal Ecology in Jena, Germany. He studied Biology at Universität Regensburg, Germany and ob­tained his doctorate in 2011. For his postdoc, he joined Martin Kaltenpoth's group – first in Jena, then in Mainz, and again in Jena – and has been working on the beewolf–Streptomyces symbiosis ever since, like Martin.

 

References

[1] Engl T, Bodenstein B, Strohm E. (2016). Mycobiota in the brood cells of the European beewolf, Philanthus triangulum (Hymenoptera: Crabronidae). European Journal of Entomology 113: 271–277. DOI: 10.14411/eje.2016.033

[2] Engl T, Kroiss J, Kai M, Nechitaylo T, Svatoš A, Kaltenpoth M. (2018). Evolutionary stability of antibiotic protection in a defensive symbiosis. Proc Natl Acad Sci USA 115(19): E2020–E2029. PMID: 29444867

[3] Herzner G, Strohm E. (2007). Fighting fungi with physics: Food wrapping by a solitary wasp prevents water condensation. Current Biology 17(2): R46–R47. PMID: 17240324

[4] Ingham CS, Engl T, Matarrita-Carranza B, Vogler P, Huettel B, Wielsch N, Svatoš A, Martin Kaltenpoth M. (2023). Host hydrocarbons protect symbiont transmission from a radical host defense. Proc Natl Acad Sci USA 120(31). PMID: 37487102

[5] Ingham CS, Engl T, Kaltenpoth M. (2023). Protection of a defensive symbiont does not constrain the composition of the multifunctional hydrocarbon profile in digger wasps. Biology Letters 19(11). PMID: 37909057

[6] Kroiss J, Kaltenpoth M, Schneider B, Schwinger M-G, Hertweck C, Maddula RK, Strohm E, Ales Svatoš A. (2010). Symbiotic Streptomycetes provide antibiotic combination prophylaxis for wasp offspring. Nat Chem Biol 6(4):261–263. DOI: 10.1038/nchembio.331

[7] Strohm E, Linsenmair KE. (2001). Females of the European beewolf preserve their honeybee prey against competing fungi. Ecological Entomology 26(2): 198–203. DOI: 10.1046/j.1365-2311.2001.00300.x

[8] Strohm E, Herzner G, Ruther J, Martin Kaltenpoth M, Engl T. (2019). Nitric oxide radicals are emitted by wasp eggs to kill mold fungi. eLife 8: e43718. PMID: 31182189